Mar Drugs. 2015 Dec; 13(12): 7150–7249.
Published online 2015 Dec 3. doi: 10.3390/md13127059
PMCID: PMC4698589
RuAngelie Edrada-Ebel1,* and Marcel Jaspars2
Preface
The
9th European Conference on Marine Natural Products (ECMNP) in Glasgow
follows its predecessors in La Toja (2013), Tjärnö (2011), Porto (2009),
Ischia (2007), Paris (2005), Elmau (2002), Santiago de Compostela
(1999), and Athens (1997). The first event in Athens in 1997 was
supported by the 5th European Framework Program and the Training and
Mobility of Researchers funding more than 50 fellowships which continued
to the 4th Conference. From the 4th Conference in Paris to the 8th
Conference in La Toja, the meetings were self-financed but equally
successful with the preceding events. The 9th ECMNP in Glasgow held on
the 30th of August to the 3rd of September 2015 was partly supported by
three EU-FP7 consortia: SeaBioTech, BlueGenics, and PharmaSea.
The
first European Conference on Marine Natural Products (ECMNP) was
initiated in 1997 from Athens and organised by Professor Vassilious
Roussis. After realising that European young scientists were not able to
attend its major international counterpart which was mostly graced by
more senior scientists, the organisers of the ECMNP have focused their
attentions on stimulating the younger generation of scientists in the
areas of marine chemistry to promote active interaction and future
collaboration around the globe. In the European conference series, young
scientists presented their work to get the experience of an
international scientific atmosphere by sharing the stage with
outstanding senior experts in the field who delivered the plenary
lectures. It is worth mentioning that in this year’s conference, we had
the participation of 94 PhD student researchers which is 50% of the
entire attendance, 37 postdoc scientists; 36 senior researchers; as well
as the participation of 21 industry experts from around Europe and the
US which included PharmaMar SA (Spain), Ingenza Ltd (UK), Bio-Prodict BV
(The Netherlands), Marine Biopolymers Limited (UK), Matís ohf
(Iceland), Biosortia Pharmaceuticals (USA), Axxam SpA (Italy), Apivita
SA (Greece), Pharmaq AS (Norway), NanotecMarin GmbH (Germany), and
SeaLife Pharma GmbH (Austria).
A one-day free
pre-conference workshop was offered for the first time to 40 early
career researchers. The workshop was hosted by the Strathclyde Institute
of Pharmacy and Biomedical Sciences, organised and led by SeaBioTech
with support from BIOCOM AG, Berlin and PharmaSea. The aim of the
workshop is to integrate and disseminate the project objectives and
methodologies of five marine biotechnology FP7 consortia (MicroB3,
MaCumBa, BlueGenics, PharmaSea and Seabiotech) to younger scientists
involved in marine research. This was in line with the objectives of the
ECMNP in promoting Marine Natural Products Research to a younger
generation of scientists.
After 18 years and
eight events, it is most inspiring and uplifting to perceive that many
of the young scientists in the initial events are now established and
leading active groups in the European marine research community. The
European conference is organized every two years alternating with the
Gordon Conferences on Marine Natural Products while the international
MaNaPro Symposia, which remains the most important appointment for all
scientists interested in Marine Chemistry, are held every three years.
The conference in Glasgow welcomed 188 participants from around Europe
but was also very international with scientists from US, Brazil, Chile,
Costa Rica, Australia, New Zealand, Kenya, South Africa, Egypt, Saudi
Arabia, Indonesia, Malaysia, Thailand, Myanmar, South Korea, and China.
Under the theme: “The sea as sustainable source of new medicine and
renewable energy”, our target audience included scientists both from
academia and industry working not only in the field of marine natural
product chemistry but also pharmacology, microbiology, biotechnology,
and ecology. The conference highlighted the evolution of marine natural
products chemistry to a multi-disciplinary field of research. Together
with the three EU-FP7 consortia, this conference in Glasgow was led by
the University of Strathclyde and jointly organized with three Scottish
Universities: the University of Aberdeen, Heriot-Watt University and
University of Highlands and Islands; along with the Phytochemical
Society of Europe. The three industry-focused EU-FP7 consortia being
under the same marine biotechnology umbrella aimed to increase the
involvement and participation of SME in this conference through oral and
poster presentations. There were five plenary lectures, 11 invited
speakers, 43 competitively chosen oral presentations, and 123 posters.
We had plenary and invited speakers from both the Industry and Academia.
There were ten sessions chaired by leading scientists in their
respective fields: Professor Peter Proksch on Chemical Ecology for
session 1; Professor William Fenical on Deep Sea and Polar Research for
session 2; Dr. Efstathia Ioannou on Marine Toxins and Bioassays for
session 3; Professor Ronald Quinn on Dereplication Metabolomics, and
Rational Approaches to Bioprospecting for session 4; Professor Anake
Kijjoa on Isolation and Structure Elucidation for session 5; Professor
Linda Harvey on Industrial Biotechnology, Polymers and Biomolecules for
session 6; Dr. Carmen Cuevas on Organic Synthesis for session 7;
Professor Gabriel Koenig on Biosynthesis of Marine Natural Products in
Microbes for session 8; Professor Hartmut Laatsch on Marine
Microbes/Fungi on session 9; and Professor Marcel Jaspars on Marine
Policy for session 10. The session on Marine Policy was put together and
was delivered by experts to provide greater awareness on international
laws of bioprospecting covered by UNCLOS (United Nations Convention on
the Law of the Sea) and the Nagoya Protocol. Each day of the conference
was opened by a plenary lecture while every session started with an
invited lecture from an established scientist and progressed on with
five 10-min oral presentations from early career researchers.
Professor
Dr. Joern Piel from ETH Zurich gave the opening plenary which
appropriately provided the marine natural products research community
with insights into the function of bacteria in various sponges. He
presented examples of biosynthetic studies on selected sponge natural
products and their practical implications for the generation of
sustainable production systems. The key word “sustainability” led to the
ecology topics for the first day of the conference. Dr. Joanne Porter
from Heriot-Watt University steered the marine ecology session pointing
out the marine ecological perspective on future exploitation of marine
natural products. Dr. Porter focused on the under-investigated phylum
Bryozoa with the discovery and development of bryostatin from uncultured
Endobugula sertula bacterium associated with larvae of Bugula neritina.
The ecological interaction ultimately leads to the identification of a
wider range of novel compounds by investigating the organisms’ growth
strategies. Close working relationships between chemists and marine
ecologists is likely to lead to a deeper understanding of the functional
role of compounds in the marine environment. Topics for short oral
presentations followed the trend for discussion on the role of secondary
metabolites in defence and evolution, chemical interactions between
toxic microalgae and Mediterranean benthic diatoms, as well as dual
induction of microbial secondary metabolites by fungal/bacterial
co-cultivation. Professor Conxita Avila from the University of Barcelona
expanded the topic on chemical ecology to the Antarctic region by
demonstrating the high biological and chemical diversity found in the
extreme and unique marine environment. This was complemented by the oral
presentations on the isolation and bioactivity screening of unique
compounds and extracts from both deep sea and Polar Regions. Chemical
diversity can be bio-prospected through innovative bioassays like using
zebrafish as a biomedically relevant model for functional genomics and in vivo drug discovery as introduced by Dr. Camilla Esguerra from the University of Oslo. Dr. Esguerra described the in vivo,
microgram-scale, high-throughput bioassays based on zebrafish embryos
and larvae for a rapid, systematic identification, and pharmacological
characterization of bioactive natural products. Short oral presentations
on the session covered mechanism of action of potential novel
anti-cancer agents and microalgal toxins. The first day ended with a
plenary lecture by Professor Heinz Schröder from the Johannes Gutenberg
University of Mainz. Professor Schröder introduced their pioneering work
with Professor Werner Mueller on the utilisation of bio-silica and the
enzyme silicatein in the synthesis of polymeric silica for the
development of novel bioinspired materials for diverse applications in
nano-optics and nano-biotechnology.
The second
day of the conference focused on the chemistry of new bioactive natural
products and the rational approach of bioprospecting them. It commenced
with the plenary lecture by Dr. Guy Carter from Biosortia
Pharmaceuticals which presented the industry’s view on drug discovery
from marine natural products (MNP). Screening of MNP for pharmaceutical
discovery became the focus of many academic groups, but only a few
Pharma companies have taken the plunge. Dr. Carter highlighted
remarkable developments and success stories of MNP going into the
pharmaceutical industrial pipeline. Professor Kristian Fog Nielsen from
the Technical University of Denmark opened the session on dereplication
and metabolomics with the application of high resolution mass
spectrometry and mass fragmentation libraries to assist in the discovery
of novel compounds which is vital for strain prioritization for further
isolation work. Target-directed dereplication approaches that
efficiently accelerate the isolation of bioactive compounds were
introduced during the session. This included metabolomics tools
utilising both NMR spectroscopy and mass spectrometry that could be
further visualised through molecular networking of the mass
fragmentation data. Genome sequencing techniques was also shown to
evaluate the potential of isolates to produce bioactive compounds by
screening the presence of genes encoding for non-ribosomal peptide
synthetases (NRPSs) and polyketide synthases (PKSs). Further on to the
isolation and structure elucidation of bioactive secondary metabolites,
Dr. Teodor Parella from the Autonomous University of Barcelona presented
exciting novel 2D NMR techniques that increased signal resolution to
elucidate complex structures by collapsing typical proton multiplet
resonances. NMR, mass spectrometry, and circular dichroism techniques to
solve stereochemistry and absolute configuration of small molecules
were further delved into through the short oral presentations. The
subsequent sessions dealt with scale-up production of bioactive
molecules through industrial biotechnology and organic synthesis.
Marine
natural products do not only consist of small molecules but as well as
larger polymers (e.g., polysaccharides) and biomolecules which were
themes under industrial biotechnology. The session was preceded by the
lecture of Professor Guðmundur Óli Hreggviðsson from the University of
Iceland on the application of novel enzymes from thermophilic
microorganism in macroalgal biorefineries. The session was dominated by
collaborative work with industry. The session covered scale-up
production of small molecules in microbes and protein engineering of
novel enzymes through sophisticated software platforms to develop faster
biosynthetic routes for bio- and small molecules. Parallel to
bioprocessing technology, organic synthesis is to date still widely used
to increase the yield of bioactive metabolites as well as foremost to
optimise the bioactivity of a molecule. Dr. Gordon Florence from the
University of St Andrews commenced the session on organic synthesis by
presenting his work on the synthesis of oxazole-containing natural
products and the development of natural product inspired inhibitors of T. brucei.
The session exemplified the organic synthesis of some bioactive
analogues for structure-activity-relationship (SAR) studies as well as
the further application of metabolomics in targeting drug lead cryptic
analogues. One oral contribution presented the use of synthetic
standards to carry out J-based configurational analysis which plays an important role in SAR analysis.
The
theme for the third day of the conference was on natural products from
marine microbes. The final day of the conference was opened with a
plenary lecture by Professor William Gerwick from University of
California San Diego, highlighting his work on cyanobacteria. Professor
Gerwick introduced an innovative approach to discover new drug leads
which involves a combination of technologies i.e., genome-based
information, mass spectrometry-based Molecular Networks, and synthetic
medicinal chemistry. He has described this as a “Top Down versus Bottom
Up” approach. For the session on biosynthesis, Professor Angelo Fontana
from the National Research Council Italy described his work on the
biosynthesis of secondary metabolites in marine diatoms and
dinoflagellates, use of phytoplankton for development of new drugs as
well as the elucidation of biochemical pathways related to the
production of renewable energy which was very appropriate to the theme
of the conference. The session continued with the introduction of
metagenomics approach to bioprospect microorganisms possessing large
number of genes from metabolic pathways relevant for biotechnology,
while further techniques were put together to identify novel
biosynthetic pathways in uncultivated sponge symbionts. The last
scientific session explored on bioactive bacterial and fungal secondary
metabolites from extreme marine environments. Professor Alan Dobson from
University of Cork led the session with his short lecture on mining
novel bioactive compounds from sponge-associated microorganisms for
biopharmaceutical applications by utilising genomic, metagenomic, and
culture-based approaches. The short oral presentations looked into the
chemical diversity of bioactive natural products from marine microbes
isolated from various marine sources.
The final
session of the conference highlighted the importance of recent changes
in marine policy and the potential impact on carrying out natural
products discovery and exploitation. The session was structured into
three invited presentations followed by an extended open discussion. Dr.
Thomas Vanagt from eCoast Belgium introduced the topic of the Nagoya
Protocol and explained the obligations on scientists to ensure they have
legal certainty over the materials collected from within national
jurisdictions. This was complemented with a presentation by Professor
Jaspars from the University of Aberdeen on the recent decision by the UN
to develop a new implementing agreement to the UN Convention on Laws of
the Sea covering the fair and equitable use of biodiversity from areas
beyond natural jurisdiction. He gave three different scenarios where
such an agreement might be employed and a scientific rationale
justifying the preferred option presented. Dr. Olga Genilloud from
Fundacion Medina Spain presented an industrial view of access and
benefit sharing gained from a number of years working in the
pharmaceutical industry. This session ended with a vigorous discussion
in which some points were clarified while others raised further
discussion with the relevant policy advisory committees.
The
conference culminated with a plenary lecture by the Phytochemical
Society of Europe’s Bruker award prize winner, Professor Roussis of the
University of Athens. He gave a fabulous presentation on the search for
algal metabolites with biomedical-biotechnological potential from the
eastern Mediterranean. The presentation perfectly encapsulated many of
the themes addressed by the conference: how an understanding of the
taxonomy and chemical ecology of an organism may assist in the directed
search for new products and processes; how sophisticated analytical and
bioinformatic techniques can speed the process of discovery and finally
how such materials may find applications in a range of fields from
pharmaceuticals through antifoulants to biomedically important
materials.
The 9th ECMNP in Glasgow was
successful and met its objective to promote marine natural products
research to younger generation of scientists who will lead the future
research in marine chemistry and its adjunct fields in ecology,
molecular biology, pharmacology, microbiology, and biotechnology. In
this conference, we have witnessed early career researchers developing
innovative approaches in the discovery of new marine drugs and these new
generation of scientists will lead marine natural products research in
the future.
Acknowledgments
This
work was supported by grants from the European Commission within its
FP7 Programme, under the thematic area KBBE.2012.3.2-01 with Grant
Number Nos. 311932 “SeaBioTech”, 311848 “BlueGenics”, and 312184
PharmaSea.
Session 1: Plenary Speaker Abstracts
Natural Product Pathways from Sponge Symbionts
Jörn Piel
Institute of Microbiology, ETH Zurich, Switzerland
Sponges
are among the richest sources of bioactive natural products from marine
habitats. Since many sponges harbor diverse bacterial communities, it
has long been suspected that many sponge-derived compounds are of
microbial origin. For a diverse range of complex polyketides [1],
ribosomally modified peptides, and nonribosomal peptides from the sponge
Theonella swinhoei, “Entotheonella” bacteria were identified
as source using metagenomic and single-bacterial analysis.
“Entotheonella” belong to a new, uncultivated candidate phylum termed
“Tectomicrobia” and exhibit a rich and variable specialized metabolism
involving unusual biosynthetic steps. Functional characterization of
enzymes suggests that “Entotheonella” spp. offer interesting
biotechnological opportunities in addition to their high drug discovery
potential. The talk presents methods to study “Entotheonella” and
provides insights into the function of these bacteria in various
sponges. In addition, biosynthetic studies on selected sponge natural
products are discussed, as well as practical implications for the
generation of sustainable production systems [2,3].
Polytheonamides from “Entotheonella factor”, a symbiont of the sponge Theonella swinhoei
References
- Ueoka, R.; Uria, A.R.; Reiter, S.; Mori, T.; Karbaum, P.; Peters, E.E.; Helfrich, E.J.N.; Morinaka, B.I.; Gugger, M.; Takeyama, H.; et al. Metabolic and evolutionary origin of actin-binding polyketides from diverse organisms. Nat. Chem. Biol. 2015, 11, 705–712.
- Wilson, M.C.; Mori, T.; Rückert, C.; Uria, A.R.; Helf, M.J.; Takada, K.; Gernert, C.; Steffens, U.A.E.; Heycke, N.; Schmitt, S.; et al. An environmental bacterial taxon with a large and distinct metabolic repertoire. Nature 2014, 506, 58–62.
- Freeman, M.F.; Gurgui, C.; Helf, M.J.; Morinaka, B.I.; Uria, A.R.; Oldham, N.J.; Sahl, H.-G.; Matsunaga, S.; Piel, J. Metagenome Mining Reveals Polytheonamides as Posttranslationally Modified Ribosomal Peptides. Science 2012, 338, 387–390.
The Promise and Perils of Marine Natural Products as Leads for Drug Discovery
Guy Carter
Biosortia Pharmaceuticals, Dublin, OH, USA
For
natural product research scientists, the discovery of elaborate and
often exotic metabolites provides sufficient intellectual challenge to
drive the exploration of the biosphere for novel products. For several
decades marine natural products (MNP) have served as an important new
source of chemical diversity. In the early days of MNP research the
promise was that the oceans would be the next great source of new
medicines—replacing much explored terrestrial plants and microorganisms.
The screening of MNP for pharmaceutical discovery became the focus of
many academic groups, however only a few Pharma companies took the
plunge. The rate of discovery of new MNP continues to increase on an
annual basis yielding over 1000 new compounds per year, most of which
have been reported as biologically active [1]. Now nearly 50 years since
the first Drugs from the Sea conference [2], the expected cornucopia of
new marine drugs is scarcely populated [3]. Where is the disconnect? In
this presentation I will take a critical look at the reasons why the
Promise has not yet been fulfilled as well as highlight notable
developments and success stories.
References
- Blunt, J.W.; Copp, B.R.; Keyzers, R.A.; Munro, M.H.G.; Prinsep, M.R. Marine natural products. Nat. Prod. Rep. 2014, 31, 160–258.
- Freudenthal, H.D. (Ed.) Proceedings of “Drugs from the Sea” Conference; Marine Technology Society of the United States: Kingston, RI, USA, 1967.
- Newman, D.J.; Cragg, G.M. Marine-sourced anti-cancer and cancer pain control agents in clinical and late pre-clinical development. Mar. Drugs 2014, 12, 255–278.
Top Down versus Bottom Up: Orthogonal Approaches for the Discovery of New Bioactive Natural Products from Marine Cyanobacteria
Alban Pereira 1, Jehad Almaliti 1, Karin Kleigrewe 1, Robin Kinnel 1,2, Anton Korobeynikov 3, Vincenzo Di Marzo 4, Lena Gerwick 1 and William Gerwick 1
1
Scripps Institution of Oceanography and Skaggs School of Pharmacy and
Pharmaceutical Sciences, University of California San Diego, 9500 Gilman
Drive MC 0212, La Jolla, CA 92093, USA
2 Hamilton College, Clinton, NY, USA
3
Center for Algorithmic Biotechnology, Saint Petersburg State University
and Algorithmic Biology Laboratory, Saint Petersburg Academic
University, Russia
4 Institute of Biomolecular Chemistry, National Research Council, Pozzuoli, Italy
The
unique organisms living in the world’s oceans are an inspiring source
of new pharmaceutical leads. To date, some 13 drugs of marine derivation
or inspiration have reached the clinic in the US, Europe or Asia, and
many more are on the horizon. Our laboratory has focused on the
bioactive metabolites available from marine cyanobacteria and algae,
both of which are extraordinarily rich in structurally diverse natural
products. Moreover, we are exploring the integrated use of several
different technologies, such as genome-based information, mass
spectrometry-based Molecular Networks, and synthetic medicinal
chemistry, to innovatively discover and develop new drug leads from
these marine organisms. For example, a Curaçao collection of a
tuft-forming cyanobacterium, Symploca sp., possessed an extract
that was highly cytotoxic to several different cancer cell lines. A
combination of a bioassay- and NMR-guided isolation process yielded two
compounds, named carmaphycin A and B, which were responsible for the
potent activity. We are exploring their utility as potential anticancer
agents through a variety of orthogonal approaches which include
synthetic organic chemistry, genome sequencing, and metabolomics.
Another recent project involves a new approach that integrates genomic
and metabolomic information to identify structurally novel and
potentially bioactive metabolites. This latter work identified a series
of novel chlorinated acyl amides that have potent binding properties to
cannabinoid receptors. By integrating several different and contemporary
approaches, new vistas in the natural products sciences are being
revealed.
Sustainable Oceans—Our Treasure in the Past and in the Future: Case Study of Sponges
Werner E.G. Müller and Xiaohong Wang
ERC
Advanced Investigator Grant Research Group, Institute for Physiological
Chemistry, University Medical Center of the Johannes Gutenberg
University Mainz, Duesbergweg 6, D-55128 Mainz, Germany
In
the last decade the phylogenetically oldest metazoan phylum, the
Porifera (sponges) gained special interest. Mainly due to the
introduction of molecular biological techniques solid evidence was
elaborated which indicated that this phylum provides a cornucopia of new
information which allows a grasping for the understanding of the
dynamics of evolutionary processes occurring during the Earth period of
Ediacara until today. Furthermore, the species of this phylum are rich
and valuable sources for bioprospecting, the translation of life-science
discoveries into practical products or processes for the benefit of the
society. Bioprospecting: The field of bioprospecting of Porifera may be
of tremendous potential benefit for humans from the applied point of
view. Taking into account that the chemical diversity of the natural
bioactive compounds obtained from the marine biota is much higher than
the one of those compounds, synthesized in standard combinatorial
chemistry approaches, and also that natural compounds display an
impressively high selectivity, the high value of the secondary
metabolites from natural resources in general and from sponges in
particular can only be roughly imagined. Until now, a bioactive compound
from sponges has been applied in clinics only in one case:
arabinofuranosyladenine (ara-A) as antiviral drug; ara-A is a derivative
of a lead structure isolated from a sponge. The future—evochemistry:
Thanks to the progress initiated by the pressure of the society for a
sustainable use of natural resources for human benefit, the exploitation
of natural biodiversity became possible through the application of the
techniques of molecular biology and modern cell biology [1–3]. Novel
directions: Biomaterials. There is an increasing need for novel
materials to be used as scaffolds in biomaterials in general and in
tissue engineering (bone and cartilage) in particular. Siliceous sponges
are unique in their ability to synthesize their silica skeleton
enzymatically. The responsible enzymes, the silicateins which have been
isolated from demosponges, polymerize alkoxide substrates to silica.
Silica is an important component of materials such as bioactive glasses
and composites based on glasses, ceramics and (organic) polymers. New
strategies for the structure-directed synthesis of amorphous silica
(biosilica) can now be envisaged [4–6]. Conclusion: It is fortunate
that, according to the fossil records, the phylogenetic oldest metazoan
phylum, the Porifera did not become extinct during the last 800 million
years. Considerable impact in biotechnology can be expected from studies
on the recombinant preparation of bioactive, low-molecular weight
compounds and of the development of new biomaterials [biosilica] from
marine sources.
Acknowledgments
W.E.G.
M. is a holder of an ERC Advanced Investigator Grant (no. 268476
BIOSILICA). This work was supported by grants from the European
Commission (Grant no. 311848 “BlueGenics” and Grant no. 286059
“CoreShell”), as well as the European Commission/EUREKA (EUROSTARS Grant
no. 4289 “SILIBACTS”).
References
- Müller, W.E.G.; Wang, X.H.; Proksch, P.; Perry, C.C.; Osinga, R.; Gardères, J.; Schröder, H.C. Principles of biofouling protection in marine sponges: A model for the design of novel biomimetic and bio-inspired coatings in the marine environment? Mar. Biotechnol. 2013, 15, 375–398.
- Natalio, F.; Corrales, T.P.; Panthöfer, M.; Schollmeyer, D.; Müller, W.E.G.; Kappl, M.; Butt, H.J.; Tremel, W. Flexible minerals: Self-assembled calcite spicules with extreme bending strength. Science 2013, 339, 1298–1302.
- Wang, X.H.; Schröder, H.C.; Müller, W.E.G. Enzyme-based biosilica and biocalcite: Biomaterials for the future in regenerative medicine. Trends Biotechnol. 2014, 32, 441–447.
- Wang, X.H.; Schröder. H.C.; Wang, K.; Kaandorp, J.A.; Müller, W.E.G. Genetic, biological and structural hierarchies during sponge spicule formation: From soft sol-gels to solid 3D silica composite structures. Soft Matter 2012, 8, 9501–9518.
- Schröder, H.C.; Wang, X.H.; Tremel, W.; Ushijima, H.; Müller, W.E.G. Biofabrication of biosilica-glass by living organisms. Nat. Prod. Rep. 2008, 25, 455–474.
- Müller, W.E.G.; Schröder, H.C.; Schlossmacher, U.; Grebenjuk, V.A.; Ushijima, H.; Wang, X.H. Induction of carbonic anhydrase in SaOS-2 cells, exposed to bicarbonate and consequences for calcium phosphate crystal formation. Biomaterials 2013, 34, 8671–8680.
In Search of Algal Metabolites with Biomedical-Biotechnological Potential from the East Mediterranean
Vassilios Roussis
Department of Pharmacognosy and Chemistry of Natural Products, Faculty of Pharmacy, University of Athens, Greece
The
biodiversity of the Mediterranean ecosystem hosts an immense number of
indigenous species, as well as organisms that have migrated from the
Atlantic Ocean, the Red and the Black Seas. Although the Mediterranean
basin occupies only 0.8% of the world’s ocean area, it accounts for 7.5%
of all described marine species. Many of these organisms have been
proven a prolific source of interesting metabolites with a broad
spectrum of bioactivities. As part of our studies on the chemical
composition and biological activity of marine organisms, our group has
investigated a significant number of algal species found along the Greek
coastline. In search of a fast and reliable screening tool for the
chemical profiling of Laurencia algal extracts and the
detection of new secondary metabolites, we have developed a high
throughput fingerprinting methodology based on the complementary
application of LC-HRMS-DAD and 2D NMR. The preliminary results of this
study point out the potential for the direct screening of crude algal
extracts in order to detect new compounds, as well as to trace
biomarkers and/or monitor the presence of targeted metabolites.
Brominated diterpenes isolated from the red alga Sphaerococcus coronopifolius, as well as a panel of synthetic analogues have been evaluated for their settlement inhibitory potential on the cyprids of Balanus amphitrite
resulting in the detection of compounds that exhibit high levels of
activity without toxic effects on non-target organisms. The active
compounds were loaded in copper oxide nanospheres and were incorporated
in CNT-enriched self-healing/self-polishing anticorrosive/antifouling
marine paints. Novel fibrous biocomposites comprising ulvan, a sulfated
polysaccharide extracted from the green seaweed Ulva rigida,
and a number of copolymers were successfully prepared using the
electrospinning technique. Such nanofibrous matrices represent
potentially useful materials in the biomedical sector as tissue
engineering scaffolds, wound dressings, or drug delivery systems.
Session 2. Invited Speakers Abstracts
Chemical Ecology
Future Exploitation of Marine Natural Products: A Marine Ecological Perspective
Joanne S Porter
School of Life Sciences, Heriot Watt University, Edinburgh, UK
The
downsizing of natural product chemistry by big Pharma, and the
associated emphasis of combinatorial chemistry approaches to develop new
molecules have not borne fruit to novel pharmaceuticals to the extent
originally envisaged. In recent years, interest in natural products has
increased particularly in the marine environment as many of the
terrestrial niches have been exhausted or rendered inefficient by the
rediscovery problem. In order to maximize the chances of developing
novel bioactives for a variety of applications in the future, it is
critical that innovative approaches are considered. For many of the
marine phyla that have been investigated there is some understanding of
the roles that natural products play. However, the potential functions
of small molecules in other phyla are less well understood. A relatively
under-investigated phylum is the Bryozoa; studies on the more abundant,
particularly foliose species, have shown that bryozoans have good
potential as a source of novel bioactive compounds. However, a large
number of bryozoans are surface-encrusting species and are difficult to
study. In these cases, where there is little biomass available to
analyze, an alternative approach is to investigate the ecological
relationships between the bryozoan and other organisms. Specialist
predators (for example, sea slugs) that live in close association with
marine invertebrates may concentrate or modify bryozoan-derived
molecules. Microbial symbionts are also a potential source of novel
compounds in bryozoa, the best example being the production of
bryostatin by the uncultured Endobugula sertula bacterium associated with larvae of Bugula neritina.
Isolation of the primary producers and cultivation of these microbes
would be expected to overcome some of the issues of availability and
scaling typically encountered in biotechnology development pipelines. In
the future the development of close working relationships between
chemists and marine ecologists to develop innovative approaches is
likely to lead to a deeper understanding of the functional role of
compounds in the marine environment. A more complete knowledge of
ecological relationships will guide investigation of related organisms
that mirror growth strategies or share habitats, ultimately leading to
the identification of a wider range of novel molecules.
Deep Sea and Polar
Research Living in the Cold: Chemical Ecology in Antarctica
Conxita Avila
Universitat de Barcelona, Facultat de Biologia, Department Animal Biology (Invertebrates), Catalonia, Spain
During many years it was believed that very few animals would live in higher latitudes (i.e., low biodiversity) and that they would have low levels of chemical defenses (i.e.,
low chemical diversity). However, this has been proven not to be true,
with many evidences accumulating on the knowledge of both polar (mostly
Antarctic) biodiversity and chemical ecology. The extreme and unique
marine environments surrounding Antarctica, along with their
evolutionary history and the abundant unusual interactions taking place
in its benthic communities are a fantastic natural laboratory for
finding new natural products. In general, many bioactive compounds have
been described from the oceans of our planet, being probably essential
for species’ survival. Surprisingly, only a small fraction of these
molecules has been tested for ecological significance, and even less if
we look at compounds from polar organisms. Some Antarctic animals are
now known to be prolific producers of chemicals, with wide potential
applications. But in Antarctica, as in other areas of the planet that
are difficult to reach (i.e., deep sea), a huge number of
organisms remain to be studied yet, both at biological and at chemical
levels. I will review here some of the known compounds found in
Antarctic animals as well as their role in the communities where the
animals live.
Marine Toxins and Bioassays
Fishing for Neuroactive Small Molecules: Zebrafish as a Platform for Marine Biodiscovery
Camilla Esguerra
Chemical Neuroscience, Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway
Emerging
challenges within the current drug discovery paradigm are prompting
renewed interest in secondary metabolites as an attractive source of
novel, structurally diverse small molecules that have been
evolutionarily “pre-selected” for bioactivity. With the recent
validation of zebrafish as a biomedically relevant model for functional
genomics and in vivo drug discovery, the zebrafish
bioassay-guided identification of natural products is an attractive
strategy to generate new lead compounds in a number of indication areas.
We have recently developed a number of in vivo,
microgram-scale, high-throughput bioassays based on zebrafish embryos
and larvae for the systematic identification and pharmacological
characterization of bioactive natural products. Zebrafish offer the
ability to rapidly evaluate—at a very early stage in the drug discovery
process—not only the therapeutic potential of natural products, but also
their potential hepato-, cardio-, and neurotoxicities. Due to the
requirement for only microgram quantities of compounds to be tested, in vivo
assays based on zebrafish are useful not only for bioassay-guided
isolation, but also for the subsequent derivatization of bioactive
natural products prioritized for further development as drug discovery
leads. Within the EU marine biodiscovery project PharmaSea (www.pharma-sea.eu),
for example, zebrafish-based CNS assays have been used to screen
thousands of crude extracts of marine microorganisms, and are being used
for the bioactivity-guided isolation of neuroactive secondary
metabolites from these extracts. These findings underscore the potential
utility of zebrafish disease and toxicity models for marine
biodiscovery.
Dereplication Metabolomics, and Rational Approaches to Bioprospecting
Dereplication and Metabolomics in Microbial Bioprospecting
Kristian Fog Nielsen
Technical University of Denmark, Department for Systems Biology, Lyngby, Denmark
For
discovery of novel compounds, fast dereplication of known compounds is
vital for prioritization of which strains and extracts to explore
further. Usually UHPLC with high-resolution mass spectrometry (HRMS) is
used as the number of candidate returned from a database search is
reduced 5–20 fold compared to nominal mass MS. For exploring the major
peaks in an UHPLC-HRMS data-file from a relative clean extract, a simple
peak picking algorithm followed by adduct pattern interpretation is
fast. However not all natural products ionize well and if combined with
many strongly ionizing compounds, media components, contaminants from
filters plastic-ware etc. the simple task get extremely complicated and time-consuming. For this 3 approaches are used:
- (i) A metabolomics approach where 4-6 replicates per condition, along with 4-6 replicates of media blanks are analyzed in a single sequence. However a single data-file may contain 10000-20000 features (ion clusters) including many random false features to be discarded using the replicates.
- (ii) A targeted approach using lists (up to many thousands) of elemental compositions of possible candidates (aggressive dereplication), where isotopic patterns, known adducts, retention time windows can be exploited along with reference standards and known blanks. These can then be combined with a peak picking approach for novel compounds.
- (iii) MS/HRMS library approach where known and tentatively identified compounds are identified by auto-MS/MS (data dependent MS/MS), and if combined ion-trap MSn can be used for identification of known substructures in larger unknown structures.
For
dereplication fungal extracts we have found a combination of II and III
to suit our applications as we can employee taxonomic knowledge, 25
years of HPLC-DAD data, and bioinformatics. Upcoming technologies in the
field include 2D LC and ion-mobility.
Advances in Isolation and Structure Elucidation
Modern NMR Methods for Structure Elucidation of Natural Products
Teodar Parella
Servei de Ressonància Magnètica Nuclear, Universitat Autònoma de Barcelona, Catalonia, Spain
High-resolution
NMR spectroscopy is a fundamental tool for characterizing the structure
and dynamics of natural products. Here, a general overview of novel NMR
techniques will be presented and exemplified on several compounds. New
versions of the HSQMBC experiment (LR-HSQMBC, HSQMBC-TOCSY,
edited-HSQMBC, etc.) will be shown as complementary tools of
the classical experiments [1–3]. On the other hand, the advantages to
collapse typical proton multiplet resonances to singlet lines will be
highlighted, allowing an unprecedented increase of signal resolution and
an easy chemical shift determination and differentiation, even in the
analysis of highly overlapped regions and complex mixtures [4].
Application of these pure-shift techniques to resolve typical chemical
problems and to determine NMR parameters will be discussed. As an
example, an improved homodecoupled variant of the ADEQUATE experiment
has been useful to resolve long-standing structural questions associated
with the structure of cryptospirolepine, a complex cryptolepis alkaloid
[5].
References
- Williamson, R.T.; Buevich, A.; Martin G.E.; Parella, T. LR-HSQMBC: A sensitivity NMR technique to probe very long-range heteronuclear coupling pathways. J. Org. Chem. 2014, 79, 3887–3894.
- Saurí, J.; Marcó, N.; Williamson, R.T.; Martin G.E.; Parella, T. Extending long-range heteronuclear NMR connectivities by HSQMBC-TOCSY and HSQMBC-COSY experiments. J. Magn. Reson. 2015, 258, 25–32.
- Saurí, J.; Sistaré, E.; Williamson, R.T.; Martin, G.E.; Parella, T. Implementing multiplicity editing in selective HSQMBC experiments. J. Magn. Reson. 2015, 252, 170–175.
- Castañar, L.; Parella, T. Broadband 1H homodecoupled NMR experiments: Recent developments, methods and applications. Magn. Reson. Chem. 2015, 53, doi:10.1002/mrc.4238.
- Saurí, J.; Bermel, W.; Buevich, A.V.; Sherer, E.C.; Joyce, L.A.; Sharaf, M.H.M.; Schiff, P.L.; Parella, T.; Williamson, R.T.; Martin, G.E. Homodecoupled 1,1- and 1,n-ADEQUATE: Pivotal NMR Experiments for the structure revision of Cryptospirolepine. Angew. Chem. Int. Ed. 2015, 54, 10160–10164.
Industrial Biotechnology, Polymers and Biomolecules
Novel Enzymes and Organisms for Processing Polysaccharides from Brown Algae
Gudmundur Oli Hreggvidsson
Matis ohf. Vinlandsleid & Faculty of Life and Environmental Studies, University of Iceland, Iceland
Brown
algae are abundant in coastal areas of the North Atlantic. Their growth
rates and productivities far exceed those of terrestrial plants and
they accumulate high levels of carbohydrates (up to 60%). The abundance
and the high carbohydrate content make macroalgae a potential highly
valuable feedstock for bioconversion to platform-, specialty-chemicals,
and advanced energy carriers. The possible product range from macroalgae
far surpasses other biomass of comparable bulk and ease of cultivation.
Brown algae are an under-utilized resource, but of special significance
for Northern Europe with abundant growth along long shorelines and the
access to vast sea areas for potential off-shore cultivation. The
polysaccharide components of macroalgae are of potential high
biotechnological value, but because of structural complexity and
“unconventional” and heterogeneous sugar composition, sulfatation and
other modifications they are also challenging as a biorefinery
feedstock. A number of processing problems need to be resolved in order
to make a marine biorefinery an economically feasible option. Matis is
developing enzymes and organisms for use in emerging macroalgal
biorefineries with a special focus on bioconversion of Brown algae
polysaccharides. This includes enzymes for degrading the
polysaccharides, alginate and laminarin, to fermentable mono sugars,
enzymes for use as aids in fractionation of macroalgal biomass and
enzymes for modification or conversion of polysaccharides to added value
derivatives. Matis is, furthermore, involved in developing robust
thermophilic microorganism for use as cell factories in macroalgal
biorefineries. In this work Matis looks somewhat beyond current markets
towards sustainable bulk production of platform chemicals, energy
carries and higher value products for specialized markets. Some examples
will be given in the talk on our recent research in this field.
Organic Synthesis
Bioactive Natural Product Synthesis: From Structure Elucidation to Drug Discovery Platforms
Gordon Florence
School of Chemistry and Biomedical Science Research Complex, University of St Andrews, North Haugh, St Andrews, UK
Nature
provides an armada of structurally diverse secondary metabolites with
unique and often unexplored biological modes of action. Combined with
their molecular architectures natural products continue to provide the
inspiration to develop practical synthetic routes and methods not only
to provide confirmation of their complete structure but to serve as
blueprints for drug discovery This talk will discuss our recent
endeavours encompassing approaches to oxazole containing natural
products and the development of natural product inspired inhibitors of
T. brucei the parasite responsible for African sleeping sickness.
Biosynthesis of Marine Natural Products in Microbes
Sailing the Uncharted Seas of Marine Phytoplankton
Angelo Fontana, Carmen Gallo, Adele Cutignano, Genoveffa Nuzzo, Angela Sardo, Emiliano Manzo and Giuliana d’Ippolito
CNR—Institute of Biomolecular Chemistry, Via Campi Flegrei 34, Pozzuoli, 80078 Naples, Italy
Phytoplankton
is an essential biological component of marine environment. The group
is composed of photosynthetic microorganisms that are central to
ecological and biogeochemical functions. As primary producers, these
single-celled organisms provide nourishment to many marine species and
are also major drivers in the cycling of elements. Carbon uptake by
phytoplankton, and its export as organic matter to the ocean interior (a
mechanism known as the “biological pump”) facilitates the diffusive
drawdown of atmospheric CO2. There are two main types of the
larger phytoplankton species: Diatoms and Dinoflagellates. Diatoms are
the most productive phytoplankton group in the world oceans accounting
for about 40 percent of the marine primary production. These unicellular
photosynthetic organisms form the basis of food webs in coastal and
upwelling systems, support important fisheries and have a major role in
silica cycling. On the other hand, in addition to be important marine
primary producers and grazers, dinoflagellates are the major causative
agents of harmful algal blooms since many species can produce various
toxins that pose a health danger to human populations and negatively
affect economic activities. While the impact of these organisms is very
clear, the most fundamental questions about their physiology and
biochemistry remain to be solved. Thus, despite the central role in
marine habitats and the fascinating chemistry of their secondary
metabolites, our knowledge of the biosynthetic process in these protists
is still at an early stage, providing unique opportunities to make new
and exciting discoveries. Starting from the study of the
chemo-ecological interactions between diatoms and their main grazers,
the copepods, our scientific interest for phytoplankton has over time
expanded to embrace general aspects concerning potential application and
metabolism of these organisms, including regulation of physiological
and ecological mediators. This contribution will discuss these topics
and recent results on biosynthesis of secondary metabolites in marine
diatoms and dinoflagellates.
Marine Microbes
Genomic and Metagenomic Approaches to Mine Marine Sponge Associated Microorganisms for Novel Bioactive Compounds with Potential Biopharmaceutical Applications
Stephen Jackson 1, Stefano Romano 1,2, Lisa Crossman 3, Claire Adams 2, Fergal O’Gara 1,2,4 and Alan Dobson 1
1 Marine Biotechnology Centre, School of Microbiology, University College Cork, Cork, Ireland
2 BIOMERIT Research Centre, School of Microbiology, University College Cork–National University of Ireland, Cork, Ireland
3 School of Biological Sciences, University of East Anglia, Norwich, UK
4 School of Biomedical Sciences, Curtin University, Perth, WA 6845, Australia
Marine
sponges are well established as a source of bioactive natural products,
many of which originate from the resident microorganisms rather than
from the sponge itself. Diverse communities of sponge microorganisms,
many of which appear to be symbionts can comprise up to 40% of the
sponge biomass. Metagenomic based approaches have been employed to study
the microbial ecology of both shallow water sponges such as Haliclona
simulans and Axinella dissimilis and deep sea sponges such as Inflatella
pellicula and Stelletta normanii sampled from depths ranging
from ~750 m to 2900 m to determine whether the sponge associated
microbiota contain bacterial genera that are likely producers of
bioactive compounds. Using culture based approaches and screening for
bioactivity against clinical pathogens such as Salmonella enterica serotype Typhimurium, methicillin-resistant Staphylococcus aureus (MRSA), and Clostridium difficile; three major groups of microorganisms with antibacterial activity, spore forming Bacillus, Streptomyces and Pseudovibrio
species have been identified. Our strategy has involved the sequencing
and annotation of the genomes of these bioactive strains, and the
subsequent analyses for gene clusters encoding potential novel bioactive
compounds using the web server AntiSMASH. Using this approach we have
identified Subtilomycin a novel lantibiotic produced by Bacillus strain MMA7, while genome mining of 12 Pseudovibrio
species has resulted in the identification of tropodithietic acid (TDA)
and other potentially novel small molecules. Genome sequencing of 15 Streptomyces
strains, a number of which are from deep sea sediments; indicate the
presence of multiple secondary metabolism gene clusters which appear to
be involved in the synthesis of polyketides, nonribosomal petides,
lantipeptides, terpenes and bacteriocins.
Marine Policy
Regulation of Access to Genetic Resources and Related Benefit-Sharing at the International and European Level—Learning the ABC of ABS
Thomas Greiber
Ocean Governance, Institute for Advanced Sustainability Studies e.V. (IASS), Potsdam, Germany
Innovation
based on genetic resources relies to some extent on having physical
access to genetic material. Such access, as well as related
benefit-sharing is regulated by a number of international, regional and
national legal instruments, some of which will be introduced to the
audience in this presentation. The presentation will provide a quick
introduction to the concept of access to genetic resources and fair and
equitable benefit-sharing (ABS) developed under the Convention on
Biological Diversity (CBD). In short, according to the ABS concept,
States hosting/providing genetic resources shall facilitate access to
these resources while users shall share in a fair and equitable manner
the benefits arising from the access to and use of the resources. The
presentation will then look in more detail at the Nagoya Protocol on
Access to Genetic Resources and the Fair and Equitable Sharing of
Benefits Arising from their Utilization. Being a supplementary agreement
to the CBD the objective of the Nagoya Protocol is to set an
international, legally binding framework to promote the implementation
of the ABS concept in the future. The Protocol aims at establishing
rules determining
- how researchers and companies who utilize genetic resources and/or traditional knowledge associated with genetic resources will obtain access to them (Access),
- how benefits arising from such utilization will be shared (Benefit-sharing), and
- which measures will need to be taken to ensure that users respect the ABS measures of the country providing the genetic resources and associated traditional knowledge (Compliance).
Furthermore,
the presentation will give an overview of the new EU ABS Regulation
implementing the Nagoya Protocol for the European Union. This Regulation
((EU) No 511/2014) applies from the date the Nagoya Protocol itself
entered into force for the Union, i.e., on 12 October 2014 (with some provisions of the Regulation only becoming applicable one year after that).
Access and Exploitation of Biological Biodiversity: New Challenges and Their Impact on Natural Products Research
Olga Genilloud
Fundación MEDINA, Health Sciences Technoogy Park, Granada, Spain
The
implementation of CBD in response to the need of a sustainable use of
biological diversity, with fair and equitable sharing of future
exploitation benefits, defined a new paradigm in the way industry
addressed international collaboration models with third countries. After
20 years of efforts to clearly manage the access and benefit sharing to
these genetic resources, and the transfer of relevant technology, we
have seen that wrong expectations about the potential economic returns,
emerging protective legislation in some countries, and the absence of
clear authorities to grant access, determined the introduction of
voluntary guidelines to assist the efficient management of exploitation
of ABS that were reinforced more recently with the Nagoya protocol.
Whereas this protocol aims at providing a legal framework for providers
and users of genetic resources, including a proposed infrastructure to
track materials and agreements, its conceptual and practical
implementation is becoming really challenging in many countries and has
set up the alarm for users both in the academic and industry
communities. The implications of this new policy and legislation on
natural product research will be discussed from the perspective of both
communities. These uncertainties derived from the poor developed models
for the country-to-country definitive implementation represent more than
ever clear barriers to the exploitation of these genetic resources.
These problems should be addressed jointly by the administration and all
stackeholders in the field to develop a long term sustainable model
fostering research in natural products.
Session 3. Abstracts Selected for Oral Presentations
Chemical Ecology
Secondary Metabolites of Phyllodesmium longicirrum and Their Role in Defense and Evolution
Alexander Bogdanov 1, Cora Hertzer 1,2, Stefan Kehraus 1, Samuel Nietzer 3, Sven Rohde 3, Peter J. Schupp 3, Heike Wägele 2 and Gabriele M. König 3
1 University of Bonn, Pharmaceutical Biology, Bonn, Germany
2 Zoologisches Forschungsmuseum Alexander König, ZFMK, Bonn, Germany
3 University of Oldenburg, ICBM Terramare, Wilhelmshaven, Germany
The
aim of the proposed project is to shed light on the evolution of the
defensive system of an opisthobranch group, the Cladobranchia. Within
Cladobranchia, the taxon Aeolidoidea is known to incorporate cnidocysts
from their prey for its own defense. This has been suggested as a major
driving force for speciation and an explanation for the success of the
Aeolidoidea. Another defensive strategy in shell-less opisthobranchs is
the incorporation of secondary metabolites from prey organisms. Our
project focuses on natural products in species belonging to the genus Phyllodesmium (Cladobranchia, Aeolidoidea) in correlation with their food. Members of the genus Phyllodesmium feed exclusively on different alcyonacean corals, which are a rich source of terpenoid secondary metabolites. P. longicirrum yielded several new secondary metabolites (1–5),
besides already known ones (e.g., sarcophytonin B, isosarcophytoxide
and polyhydroxylated steroids). The new compounds belong to the class of
cembrane diterpenes (1), secogorgosterols (2), more unusual tetracyclic and pentacyclic diterpenes (3–5).
The defensive role of the isolated compounds could be demonstrated by
feeding deterrence assays using the tropical pufferfish Canthigaster solandri as predator. VLC fractions of the crude extract were deterrent at levels below natural concentrations. Compound 3
exhibited significant deterrence at 0.5% of dry mass and thus is
suggested to play an important role in chemical defense against
predation. Our findings will finally be mapped onto a phylogenetic
reconstruction of the Cladobranchia to understand evolution of different
pathways of defensive strategies.
Acknowledgments
DFG Wa 618/10-1 and KO 902/8-1.
Natural Inducers for Larval Metamorphosis in Scleractinian Corals
Peter J. Schupp, Mareen Möller, Samuel Nietzer and Makoto Kitamura
Institute of Chemistry and Biology of the marine Environment, Oldenburg, Germany
Many
benthic marine invertebrates, including corals, disperse as plankton
before settlement and metamorphosis. Finding a suitable habitat is
crucial for sessile marine invertebrates. The ability of larvae to
detect habitat-specific cues has been recognized in a range of phyla,
but until recently, only a few studies identified the chemical structure
of compounds involved in larval settlement and metamorphosis. Biofilms
on Crustose Coralline Algae are a known inducer for settlement in
scleractinian corals but only little is known about specific bacteria
and chemical compounds involved in metamorphosis and settlement of coral
larvae. Here we present insights into the role of bacteria during the
coral larvae settlement, using larvae of the brooding coral Leptastrea purpurea
as a model. Experiments testing the involvement of CCA biofilms, as
well as biofilms from inert surfaces (e.g., tiles, bleached CCA),
revealed that biofilms on Hydrolithion and biofilms on certain inert surfaces more than three weeks old repeatedly induced settlement in L. purpurea and Acropora larvae. Continued studies lead also to the isolation of two settlement-inducing bacteria (both Pseudoalteromonas sp.).
Using bioguided fractionation of crude extracts from inducing bacteria
we could show that certain bacteria produce chemical cues, which induce
the settlement process. Two settlement and metamorphosis inducing
compounds have so far been isolated. Further assays also identified the
presence of a toxic compound in the bacterial extracts. Their structure
elucidation is currently under way.
Chemical Interactions between the Toxic Microalgae Ostreopsis cf. ovata and Mediterranean Benthic Diatoms
Eva Ternon 1, Sophie Marro 2, Rodolphe Lemée 2 and Olivier Thomas 1
1 Nice Institute of Chemistry—PCRE UMR7272 CNRS, University of Nice Sophia Antipolis, Nice, France
2
Sorbonne Universités, Université Pierre et Marie-Curie Paris 6, CNRS,
Laboratoire d’Océanographie de Villefranche, Villefranche-sur-mer,
France
Several species of
microalgae own a well-developed specialized metabolome yielding to the
production of toxic compounds. When highly concentrated and quickly
multiplying, these toxic microalgae are likely to induce negative
environmental or toxicological effects, by forming Harmful Algal Blooms
(HABs). During the past decade, a toxic benthic dinoflagellate belonging
to the genus Ostreopsis has bloomed repetitively along the
Mediterranean coastline, leading to toxic outbreaks on humans. Their
efflorescence has become a real concern in regard to public health and
several toxicological studies on the major toxins (ovatoxins) have been
carried out. On the other hand, their effects on the Mediterranean
benthic community have not been assessed yet. In this study we propose
to investigate the chemical mediation involved between Ostreopsis cf.
ovata and a Mediterranean benthic diatoms. Co-cultures without contact
were set up using dialyze bags, and the chemical content of both cells
and culture media was analyzed. Since the specialized metabolism of O. cf. ovata
remains scarcely known, an un-targeted metabolomics approach was used
to compare the chemical signals present in cultures thanks to a high
performance UHPLC-QTof. Chemical cues found to be involved in the
interactions were submitted to a MS-MS approach and compared to natural
products databases in order to propose a potential identity.
Irish Osmundea spp.: Food or Shelter for Aplysia sp.?
Sylvia Soldatou 1, Ryan Young 1,2, Candice Bromley 1, Svenja Heesch 3 and Bill Baker 1,2
1 National University of Ireland, Galway, Galway, Ireland
2 Department of Chemistry and Center for Drug Discovery and Innovation, University of South Florida, Tampa, FL, USA
3
Irish Seaweed Research Group, Ryan Institute for Environmental, Marine
and Energy Research, National University of Ireland, Galway, Galway,
Ireland
The Irish coastline is
approximately 7500 km long representing one of the most biodiverse and
rich-species habitats in Europe. With only few studies conducted in the
North East Atlantic region, Irish waters can be a great source of new
and unexplored chemical diversity. Four different Osmundea sp., red alagae commonly found in intertidal zone, have been described from Irish waters. Aplysia sp., is a sea hare which has been found to be associated with Osmundea algae. This project is focusing on the isolation and characterisation of secondary metabolites from Osmundea spp. and Aplysia
sp. samples collected from the shore of Western Ireland in county
Galway. The ultimate aim is to compare the chemistry produced by these
two marine organisms and determine whether the sea hares are
sequestering the compounds from the algae or they are using the Osmundea spp. as a shelter from predators and strong water currents. Thus half the Aplysia
sp. collected were allowed to fast prior to analysis affording an
opportunity to sample the chemistry contained within the sea hares
rather than that contained within the digestive tract. The algal and
animal samples were extracted separately in organic solvents followed by
purification and isolation of secondary metabolites by means of Medium
Pressure Liquid Chromatography (MPLC) and High Performance Liquid
Chromatography (HPLC). Comparisons between the algal and sea hare
extracts were carried out through metabolomics analysis using Liquid
Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography-Mass
Spectroscopy (GC-MS). Moreover, the structures of pure metabolites were
elucidated by means of 1D and 2D Nuclear Magnetic Resonance (NMR)
spectroscopy.
Dual Induction of Microbial Secondary Metabolites by Fungal/Bacterial Co-Cultivation
Mostafa Rateb 1,2, Jennifer Wakefield 1, Rainer Ebel 1 and Marcel Jaspars 1
1 Marine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Aberdeen, UK
2 Pharmacognosy Department, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt
Marine-derived
microorganisms are promising sources of new bioactive metabolites.
However, the frequent re-discovery of known compounds is a major
problem. Many biosynthetic genes are not expressed in vitro
thus limiting the chemical diversity of microbial compounds that can be
obtained through fermentation. On the other hand, the co-cultivation
(also called mixed fermentation) of two or more different microorganisms
helps to mimic complex microbial natural communities. The competition
during co-cultivation in most cases lead to an enhanced production of
constitutively present compounds, or to an induction of cryptic
compounds that are not detected in axenic cultures of the producing
strain. Herein, we report the induction of newly detected bacterial and
fungal metabolites by the mixed fermentation of the marine-derived
fungal isolate MR2012 and hyper-arid desert bacterial isolates.
Deep Sea and Polar Research
Isolation and Characterization of Bioactive Deep-Sea Marine Fungi
Stephen A Jackson 1, Erik Borchert 1, Jonathan Kennedy 1, Fergal O’Gara1,2 and Alan D.W. Dobson 1,2
1 School of Microbiology, University College Cork, Cork, Ireland
2 BIOMERIT Research Centre, University College Cork, Cork, Ireland
In
the search for novel marine natural products with antimicrobial
activities, the deep-sea remains an as yet largely under-sampled
environment. As terrestrial fungi are well known producers of
antimicrobial agents, marine fungi may be a promising source of novel
compounds with potential to combat the increasing threat of
antimicrobial resistances amongst human pathogenic bacteria. We have
isolated fungi from deep-sea sediment sampled from a depth of 1168 m and
from a marine sponge (Stelletta normani) sampled from a depth
of 751 m. Samples were obtained from the Atlantic Ocean off the west
coast of Ireland. Nineteen isolates (14 from the sponge and 5 from
sediment) were taxonomically characterized by PCR amplification
targeting 18S rRNA gene fragments using primers EukA and EukB [1]
followed by sequencing, BLAST analyses and phylogenetic tree-building.
All sediment isolates recruited to the genus Cladosporium and
are closely related to sequences derived from deep-sea or hypersaline
environments. Sponge derived isolates are closely related to the genera
Ascomycete, Cadophora and Geomyces. Phylogenetic analyses suggest these
isolates may be true marine fungi. All fungal isolates were tested for
antibacterial activities against clinically relevant Gram positive and
Gram negative test strains. Although little if any antibacterial
activity was observed from the sediment isolates, 7 sponge isolates
showed activity against one or more test strains. Three isolates in
particular (TS3, TS12 & TS13) inhibited all test strains in the agar
overlay assay. The secondary metabolite potential of these strains was
investigated by PCR through targeting of potential Polyketide synthase
(PKS) and Non Ribosomal peptide synthase (NRPS) genes in these fungal
genomes.
Reference
- Medlin, L.; Elwood, H.J.; Stickel, S.; Sogin, M.L. The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene 1988, 71, 491–499.
Investigation into the Bioactive Metabolites of Deep Sea Sponge Associated Fungi
Candice Bromley 1, Ryan Young 1,2, Stephen Jackson 3, Thomas Sutton 3, Alan Dobson 3 and Bill Baker 1,2
1 National University of Ireland Galway, Galway, Ireland
2 Center for Drug Discovery and Inovation, University of South Florida, Tampa, FL, USA
3
Marine Biotechnology Centre, School of Microbiology, University College
Cork, National University of Ireland Cork, Cork, Ireland
Marine
associated fungi, especially those isolated from extreme environments,
have been found to produce diverse bioactive secondary metabolites. The
ability to manipulate the fungal genome to amplify the production of
particular metabolites adds to their potential in the discovery of novel
bioactive compounds. In this study the ROV Holland I was used to
collect samples of the sponge Stelletta normani off the west
coast of Ireland from a depth of 751 m. Eleven deep sea sponge
associated fungi were isolated and cultured. Initial testing for
bioactivities was performed using a deferred antagonism assay against
gram negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, as well as gram positive bacteria such as Staphylococcus aureus and Bacillus subtilis.
In an attempt to increase the production of bioactive secondary
metabolites epigenetic modifiers, capable of activating silenced or
attenuated gene clusters in the fungi, were employed. Each of the fungal
strains were exposed to two such epigenetic modifiers, namely sodium
butyrate and 5-azacytidine, inhibiting histone deacetylase (HDAC) and
DNA methyltransferase (DNMT) respectively. To establish the effects of
epigenetic modifications on the propensity for the fungi to produce
bioactive secondary metabolites the activities of the organic extracts
were retested, this time covering a wider range of pathogens. In
addition, comparative investigations of the fungal metabolomic profiles
using LC-MS as well as NMR spectroscopy were conducted to establish any
potential bioactive natural products.
Antimicrobial Compounds from Antarctic Bacteria
Donatella de Pascale 1, Pietro Tedesco 1, Fortunato Palma Esposito 1, Antonio Mondini 1, Glen Brodie 2, Renato Fani 3 and Marcel Jaspers 2
1 Institute of Protein Biochemistry, CNR, Naples, Italy
2 University College of Aberdeen, The School of Natural and Computing Sciences, Aberdeen, UK
3 Department of Biology, University of Florence, Florence, Italy
The
increasing alarm of multidrug resistant bacteria in the last 20 years,
led scientific community to the discovery of novel source of
antimicrobials compounds. The bioprospecting from marine and extreme
environments has yielded a noteworthy number of novel molecules from a
wide range of organisms. Antarctica is the one of the most extraordinary
places on Earth and exhibits many distinctive features. It is Earth’s
southernmost continent and it is the coldest, driest, and windiest place
on the planet. Thus, Antarctica hides organisms, which have evolved
unique characteristics to face these harsh environmental conditions. In
particular, Antarctic microorganisms are known to produce novel
secondary metabolites that are valuable in a range of applications.
Herein, we report on the development of a six-step biodiscovery pipeline
starting with the collection of environmental samples and isolation of
novel bacteria, to the chemical identification of the bio-assay guided
purification of compounds with antimicrobial and antibiofilm activities.
Antarctic sub-sea sediments were used to isolate more 1000 bacteria.
The novel isolates were subjected to primary screening to determine
their bioactivity against a selected panel of human pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia, Burkholderia cenocepacia).
Isolates, positive to the first screening, were used to produce crude
extracts from microbial exhausted culture broths. A bioassay-driven
purification was performed using crude extracts of the most promising
isolates. LC-MS and NMR then structurally resolved the purified
bioactive compounds.
Marine Bacteria Isolated from Deep-Sea Hydrothermal Vents Are Valuable Sources of Glycosaminoglycan-Like Polysaccharides and Anti-Microbials
Christine
Delbarre-Ladrat, Laetitia Kolypczuk, Delphine Passerini, Jacqueline
Ratiskol, Corinne Sinquin, Agata Zykwinska, Sylvia Colliec-Jouault and
Françoise Leroi
Ifremer, Nantes, France
The
study of microbial life adapted to deep-sea hydrothermal vents
conditions is a promising way of discovering new biomolecules with
innovative properties and potential applications in human health. On one
hand, our collection of bacteria isolated from deep-sea hydrothermal
vents is screened for anti-microbial activities to fight the growing
threat of broad spectrum antibiotic resistant infections. A range of
Gram-positive and Gram-negative bacteria pathogens encountered in human
health field, aquaculture and food spoilage constitutes the selected
target bacteria. On the other hand, marine prokaryotes offer a source of
safe, biocompatible, biodegradable and valuable renewable products
especially carbohydrate polymers. Deep-sea marine bacteria have been
shown to produce exopolysaccharides (EPS) with unusual structure and
having glycosaminoglycan (GAG)-like biological activities; in
particular, some of these EPS are naturally sulfated; this is very rare
within the prokaryote domain. Data on three EPS-producing bacteria
isolated from deep-sea habitats will be presented. These would include
EPS production in bioreactors, molecular mechanisms of the
polysaccharide biosynthesis, chemical and enzymatic modifications,
carbohydrate active enzymes as well as some biological properties which
give them high value for the biomedical field. These studies provide a
better basic knowledge on the biosynthesis of bioactive polysaccharides
and would provide means to engineer the molecule for improving its
function.
Apoptosis Mediated Anticancer Activity of Streptomyces sp. MCCB 248 Isolated from an Arctic Fjord, Kongsfjorden, Svalbard, on NCI-H460 Human Lung Cancer Cell Line
Dhaneesha M. 1, Sajeevan T. P. 1, Krishnan K. P. 2 and Bright Singh I. S. 1
1 National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Kochi, Kerala, India
2 National Centre for Antarctic and Ocean Research, Headland Sada, Goa, India
A
total of 22 actinomycetes were isolates from the sediments collected
from the Kongsfjorden, Arctic fjord. On screening for the anticancer
activity of the metabolites of these isolates, one strain MCCB248 showed
promising activity on NCI-H 460 cell lines. 16S rRNA gene sequence of
the isolate revealed that the isolate MCCB 248 was a member of the genus
Streptomyces. The ethyl acetate extract of Streptomyces
sp. MCCB 248 was tested for the apoptotic induction. Apoptosis mediated
anticancer activity of the extract was evaluated and confirmed through
cell based assays. Hoechst 33342 staining assay revealed that treated
cells showed shrinkage of cell nucleus, fragmentation and chromatin
condensation. TUNEL assay also demonstrated that Streptomyces
sp. MCCB 248 triggered DNA damage as evident from the condensed TUNEL
positive chromatin with in cell nuclei. Confirmation of apoptosis by
staining with Annexin V-FITC/propidium iodide (PI) showed that the cells
treated with extract rapidly undergo apoptosis as obvious from the more
percentage of Annexin positive cells in the early hours of treatment.
Our result showed that Streptomyces sp. MCCB 248 isolated from
Arctic environment is a promising candidate for a potent anticancer
agent that is under current investigation.
Marine Toxins and Bioassays
Honaucin A, Mechanism of Action and Potential Role as a Cancer Prevention Agent
Lena
Gerwick, Samantha Mascuch, Gabrial Navarro, Paul Boudreau, Tristan Carland, Terry Gaasterland and William Gerwick
University of California San Diego, La Jolla, CA, USA
Three
related natural products, the honaucins A–C, were isolated from a
cyanobacterium overgrowing a coral reef in Hawaii. Subsequent biological
investigations revealed that these molecules inhibit both prokaryotic
quorum sensing and eukaryotic inflammation. The honaucins were
originally identified as molecules of interest in an in vitro
assay that quantified its ability to attenuate nitric oxide production
in LPS-stimulated macrophages. Continued experiments using honaucin A
displayed a transcriptional down-regulation of IL-6, TNFα, IL-1β, and
iNOS in these cells. Additionally, in vivo anti-inflammatory
activity in a murine model of ear edema was demonstrated. To uncover the
mechanism of action of honaucin, RNA deep sequencing was performed
using total RNA from honaucin A-treated macrophages. Analysis of
differentially regulated transcripts strongly suggested that honaucin A
is an activator of a pathway which results in the transcription of
cytoprotective genes. This signaling pathway has recently drawn interest
for its potential application to the treatment of neurodegenerative and
autoimmune diseases, as well as cancer. Experiments involving reporter
assays and protein pull down using a biotinylated probe to validate the
proposed target will be discussed.
Palytoxins from Marine Coastal Environments and Home Aquaria. What Role Do They Play in Human Inhalatory Poisonings?
Carmela Dell’Aversano 1, Luciana Tartaglione 1, Martino Forino 1, Patrizia Ciminiello 1, Andre Wieringa 2 and Aurelia Tubaro 3
1 Department of Pharmacy, University of Napoli Federico II, Napoli, Italy
2 Department of Clinical Pharmacy, Isala, Zwolle, The Netherlands
3 Department of Life Science, University of Trieste, Trieste, Italy
Since
the late 1990s’, hundreds of cases of respiratory illness and/or
dermatitis have been repeatedly recorded in people concomitantly with
massive proliferations of the benthic dinoflagellate Ostreopsis cf. ovata
in the Mediterranean area. Thanks to development of a liquid
chromatography-high resolution mass spectrometry (LC-HRMS) method, we
have characterized O. cf.
ovata as a producer of congeners of palytoxin, a highly potent
toxin whose inhalation hazard is however unknown. On the basis of the
concomitance of Ostreopsis blooms, respiratory illness in
humans, and detection of palytoxin congeners in algal samples, a cause
and effect relationship between the cases of malaise and the algal
toxins has been postulated but never substantiated. Further cases of
respiratory illness tentatively attributed to palytoxins have been
reported for aquarium hobbyists from incidental inhalation of steams
generated during cleaning operations of home aquaria containing soft
corals belonging to Palythoa genus. The only common feature between Palythoa spp. and Ostreopsis
spp. is that both, although phylogenetically distinct, may produce
palytoxins. The study reported herein serves the double purpose of
demonstrating the presence of palytoxins in home marine aquaria involved
in several inhalatory poisonings and of correlating the symptoms shown
by patients while handling Palythoa spp. in home aquaria with those reported for Ostreopsis-related
poisonings. From the chemical and symptomatological data it is
reasonable to hold palytoxins responsible for respiratory disorders
following inhalation. Although the exact mechanism through which
palytoxin congeners from Palythoa spp. and Ostreopsis
spp. exert toxicity by inhalation is still unknown, this represents a
step toward inhalatory risk assessment of palytoxin congeners in
domestic and open-air environments.
Discovery of Fish Killing Toxins from the Microalgae Prymnesium parvum
Thomas Ostenfeld Larsen 1, Silas Anselm Rasmussen 1, Kristian Fog Nielsen 1, Sebastian Meier 2, Jens Ølgaard Duus 2, Hannah Blossom 3, Nikolaj Gedsted Andersen 3 and Per Juel Hansen 3
1 Department of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark
2 Department of Chemistry, Technical University of Denmark, Kgs. Lyngby, Denmark
3 Marine Biology Section, University of Copenhagen, Helsingør, Denmark
Marine
fish farming has the potential to become an economically important
industry worldwide. However, blooms of ichthyotoxic (fishkilling)
harmful microalgae are a recurring phenomenon in coastal marine waters
with some times huge impacts on wild fish stocks as well as caged fish.
This has detrimental consequences for the implicated fish farmers, in
addition to recreational and commercial fishing. This paper will
describe results obtained in the collaborative Danish Strategic Research
project: “HABFISH—Harmful algae and fish kills”. Our initial efforts
have been directed towards analysis of the chemistry and bioactivities
related to Prymnesium parvum, an important microalgae in coastal waters. We found huge differences in ichtyotoxicity among 5 different P. parvum
strains. Chemical analysis based on LC-DAD-MS analysis showed that all
strains produced GAT toxins and oleamides [1]. However, we found no
evidence for ichthyotoxicity with ecological relevant concentrations,
thus excluding that these compounds can be the true ichtyotoxins as
recently reported. Furthermore, 13C feeding studies showed that oleamides are not true P. parvum
metabolites, but instead contaminants derived from plastics during
sample purification [1]. Excitingly, we found that different strains of P. parvum
produce at least to types of prymnesin-like molecules. One strain
produce prymnesin 1 & 2, whereas several Scandinavian strains
produce a novel type of prymnesins. Cultivation of >100 liters of
algal medium has allowed us to isolate enough material of a novel
prymnesin-like molecule, including a 13C enriched version.
This paper will report our efforts towards structural elucidation of
this large and complex polyketide derived polyether, based on various 2-
and 3-D NMR experiments.
Reference
- Blossom, H.E.; Rasmussen, S.A.; Andersen, N.G.; Larsen, T.O.; Nielsen, K.F.; Hansen, P.J. Prymnesium parvum revisited: Relationship between allelopathy, ichtyotoxicity, and chemical profiles in 5 strains. Aquat. Toxicol. 2014, 157, 159–166.
On the Mechanisms of Cancer Cell Death Induced by Marine Sponge Depsipeptides: A Comparison of Different in Vitro Methodologies
Marisa Rangel 1,2, Graziella A. Joanitti 3, Ricardo B. Azevedo 3, Wagner Fontes 4 and Mariana S. Castro 2,4
1 Lab. of Immunopathology, Butantan Institute, São Paulo/SP, Brazil
2 Lab. of Toxinology, Dept. of Physiological Sciences/IB, University of Brasilia Brasilia/DF, Brazil
3 Lab. of Nanobiotechnology, University of Brasilia, Brasilia/DF, Brazil
4 Lab. of Biochemistry and Protein Chemistry, Dept. of Cell Biology/IB, University of Brasilia, Brasilia/DF, Brazil
Geodiamolides
are depsipeptides from marine sponges that disrupt microfilaments of
sea urchin eggs and human breast cancer cell lines. Geodiamolide-H was
further tested in breast cancer cells cultures in three-dimentional
environment, showing stronger effect on the aggressive Hs578T cells,
inhibiting migration and invasion, and reversing its malignant phenotype
to polarized spheroid-like structures. The cell death pathways induced
by the geodiamolides in the cancer cells, however, have not yet been
elucidated. The goal of this work is to study the effects of the
geodiamolides-A and H in two different cancer cell lines (4T1, mouse
breast tumor; and A431, human nonmelanoma epithelial carcinoma) using
different methods, in order to elucidate the mechanisms of cell death.
First, using MTT, the geodiamolide-H did not reduce cell viability of
both lineages, even at 1 µM. Geodiamolide-A, however, reduced the
viability of A431 cells, with IC50 of 368 nM. On a second
assay, we used a kit to measure three different parameters. The cell
viability and cytotoxicity was measured by the activity of two
proteases, while apoptosis was measured by the caspase-3/7 activities
(Promega ApoTox-Glo™ Triplex Assay). In this assay, the
geodiamolides did not reduce the viability in the 4T1 cells, or
increased membrane permeability, but activated the caspase-3/7. In A431
cells, only the geodiamolide-A induced a decrease the viability,
confirming the MTT results. Additionally, geodiamolide-A activated the
caspase-3/7. These results indicate that the geodiamolides-A and H may
have different effects in these cell lineages, but activated the
apoptotic pathway in both. The human cancer cells lineage was more
susceptible to the treatment with geodiamolide-A than the murine cells,
what was also observed in previous studies, reinforcing the
pharmaceutical potential of these molecules. Further experiments will be
performed in order to unravel the mechanism of action triggered by
these promising antitumoral marine sponge depsipeptides.
Funding: CNPq
In Vitro Efficacy of Nannochloropsis Gaditana Extract on Primary Human Dermal Fibroblasts as Cosmeceutical Bioactive Ingredient
Sophia Letsiou 1, Konstantinos Gardikis 1, Lalia Lalia Mantecón 2, Carlos Unamunzaga 2 and Emmanouil Flemetakis 3
1 APIVITA S.A., Scientific Affairs, Industrial Park of Markopoulo Mesogaias, 19003 Markopoulo Attiki, Athens, Greece
2 Fitoplanton Marino, S.L. Dársena Comercial s/n (Muelle Pesquero), 11500 El Puerto de Santa María (Cádiz), Spain
3
Laboratory of Molecular Biology, Department of Biotechnology, School of
Food, Biotechnology and Development, Agricultural University of Athens,
Athens, Greece
Nowadays, there
is a huge interest on natural products obtained from marine organisms
that can promote the state of health and well-being for humans.
Microalgae represent a primary source of bioactive compounds and could
be used as functional ingredients in cosmetic formulations. The aim of
the present study is to evaluate in vitro effects of Nannochloropsis gaditana (NannoG) extract in cytoprotection against oxidative stress using H2O2
as stressor in primary normal human dermal fibroblasts (NHDF), so as to
investigate the potential applications of NannoG in cosmetics. In order
to gain an insight into the molecular mechanisms of NannoG bioactivity,
we employed a newly developed RT-qPCR platform for studying transcript
accumulation for an array of genes (more than 100) involved in many
skin-related processes, including anti-aging, hydration, oxidative
stress response etc. NHDF cells were purchased from Lonza Clonetics™. For the oxidative stress evaluation, H2O2 was used as stressor. Cells were incubated for 48 h with NannoG extract. After incubation and prior to H2O2 induction the cells were washed two times with PBS and then H2O2
was added to the cells for 3 h at varying concentrations. Cytotoxicity
was assessed by determining the ATP levels. Fibroblasts incubated with
NannoG extract and stressed with H2O2 showed a
significant increase in cell viability. Antioxidant, antiaging
protection of NannoG extract on NHDF was confirmed by the regulation of
several related transcripts (including GPX1, SIRT3) involved in the
relative pathways. In addition, an increase in the expression of genes
related to hydration process (acquaporins) was observed, under oxidative
stress, upon treatment with NannoG. These findings indicate that N. gaditana
extracts possess strong antioxidant properties and provide new insights
into the beneficial role of microalgae bioactive compounds in cosmetic
formulations protecting skin from oxidative stress and aging.
Dereplication Metabolomics, and Rational Approaches to Bioprospecting
Metabolomic Tools to Target and Accelerate the Isolation of Bioactive Compounds from Marine Microbial Symbionts
L. MacIntyre 1, M.L. Fazenda 1, C. Viegelmann 1, T. Zhang 1, C. Dowdells 1, L. Young 1, C. Clements 1, G. Abbott 1, K.R. Duncan 2, D. Green 2, L.M. Harvey 1, B. McNeil 1 and R. Edrada-Ebel 1
1 University of Strathclyde, Glasgow, Scotland, UK
2 Scottish Association for Marine Science, Oban, Scotland, UK
Marine
microbial symbionts produce a plethora of novel secondary metabolites
which may be structurally unique with interesting pharmacological
properties. Some of these compounds can be produced on a
biotechnological scale using fermentation processes and are therefore an
economically viable and sustainable source of commercial quantities of
metabolites of interest. Mass spectrometry (MS) and nuclear magnetic
resonance (NMR) spectroscopy-based metabolomics are readily applicable
to microbial drug discovery efforts, offering the ability to
comprehensively analyse the chemical composition of complex mixtures,
e.g., broth extracts, under a given set of conditions in a highly
efficient manner. Metabolomic methods were integrated with
chemoinformatic approaches and bioassay screening results to monitor
metabolite production by several strains of marine bacteria under
various culture conditions. Metabolomic tools were then used to
complement bioassay-guided fractionation by rapidly targeting novel
bioactive secondary metabolites, accelerating the isolation and
purification of new natural products. The application of this
methodology within the SeaBioTech* project shall be presented.
Acknowledgments
This
work was supported by the SeaBioTech project that is funded by the
European Commission within its FP7 Programme, under the thematic area
KBBE.2012.3.2-01 with Grant Number 311932.
An Integrated Approach for Bioprospecting Novel Bioactive Compounds from Marine Actinomycetes
Paula Jimenez 1,2, Larissa Guimarães 2, Danilo Rocha 2, Elthon Ferreira 2, Maria da Conceição Torres 2, Otília Pessoa 2, Eric Lau 3, Damian Mason 3, Eli Chapman 3, James La Clair 4 and Leticia Costa-Lotufo 5
1 Universidade Federal de São Paulo, Santos, SP, Brazil
2 Universidade Federal do Ceará, Fortaleza, CE, Brazil
3 University of Arizona, Tucson, AZ, USA
4 Xenobe Research Institute, San Diego, CA, USA
5 Universidade de São Paulo, São Paulo, SP, Brazil
Conventional
search strategies of new drugs from natural products comprise the
bioactivity-guided chemical fractionation of an extract leading to
isolation of the active principle. An innovative approach involves that
guided by a specific cellular protein or pathway as a biological target.
Herein, we describe the bioprospection of compounds from an extract
obtained from a strain of Actinomadura sp. recovered from
marine sediments applying both, bioactivity and target-directed
strategies. For bioactivity-guided fractionation, MTT assay was used to
evaluate the cytotoxicity of the crude extract, derived fractions and
pure molecules against the HCT-116 tumor cell line. For the
target-directed approach, a functional chromatography protocol, a
process of reverse affinity chromatography, was applied to collect
compounds that bound to survivin, an inhibitor of apoptosis protein.
Briefly, recombinantly expressed human survivin was covalently linked to
a resin, which, in turn, was incubated with the extract. Unbound
compounds were washed off and survivin was denatured with ethanol to
recover the retained material. The ethanolic extract was then analyzed
by LC-HRMS and µ-scale NMR to identify signals of molecules contained
therein. Theses signals were compared to molecular masses in the
AntiMarin database to search for matches. Bioactivity-guided
fractionation yielded nonilprodigiosin and cyclononilprodigiosin, two
members of the prodigiosins family, which are known for their cytotoxic
activity. These molecules were major components of this extract. For the
target-directed methodology, LC-MS analysis of the whole,
pre-chromatography extract returned masses for 36 compounds, while the
material eluted from resin contained 2 masses, 522.3038 and 536.3257,
which were unmatched in the database, suggesting they could be new
compounds. The next steps involve isolation of compounds and validation
of their modulatory effect using biological models. However, these
preliminary results highlight the efficiency of this approach in fishing
out molecules from a complex crude extract.
Comparative Metabolomics of Secondary Metabolites from Bacteria Using GNPS Molecular Networking
Katherine Duncan 1, Lynsey MacIntyre 2, David Green 1, RuAngelie Edrada-Ebel 2 and Michele Stanley 1
1 Scottish Association for Marine Science, Oban, UK
2 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK
Genome
sequencing has revealed many more microbial biosynthetic pathways then
would be predicted based on the numbers of secondary metabolites they
produce. These “silent” gene clusters may in fact be expressed but the
products missed using traditional natural product discovery tools. The
generation of molecular networks based on MS-MS data provides a rapid
and highly sensitive approach to visualize secondary metabolite profiles
in extracts of cultured bacteria. This technique facilitates strain
comparison and can be used to prioritize strains for more detailed
discovery efforts. Here we have generated molecular networks for several
biotechnologically valuable strains (including Micromonospora, Streptomyces, Vibrio splendidus and Rhodococcus sp.)
isolated from understudied marine environments (Antarctic, Scotland,
Mediterranean sponges) some of which have draft genome sequences
available. The networks were populated with standards that aid in
chemical identification. This has allowed identification of previously
isolated secondary metabolites (avoiding redundant chemical
purification) and the discovery of potentially novel secondary
metabolites. Select nodes have the potential to be linked to genome
sequencing data in an effort to identify the associated biosynthetic
pathways and facilitate structure predictions. Combining new
fermentation approaches with molecular networking provides opportunities
to improve the efficiency with which natural products are discovered.
Bioprospecting Portuguese Atlantic Coast Cyanobacteria for Bioactive Secondary Metabolites Reveals Untapped Chemodiversity
Ângela Brito 1,2, Joana Gaifem 1, Vitor Ramos 3, Evgenia Glukhov 4, Pieter C. Dorrestein 5,6, William H. Gerwick 4,6, Vitor M. Vasconcelos 2,3, Marta V. Mendes 1 and Paula Tamagnini 1,2
1 i3S—Instituto de Investigação e
Inovação em Saúde & IBMC—Instituto de Biologia Molecular e Celular,
Universidade do Porto, Porto, Portugal
2 Faculdade de Ciências, Departamento de Biologia, Universidade do Porto, Porto, Portugal
3 CIIMAR/CIMAR—Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Porto, Portugal
4
Center for Marine Biotechnology and Biomedicine, Scripps Institution of
Oceanography, University of California San Diego, La Jolla, USA
5 Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, USA
6 Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, USA
Cyanobacteria
produce a large array of bioactive compounds, some of which are toxic
to different organisms, including animals and humans, while others
possess promising therapeutic applications such as anticancer,
antibiotic and anti-inflammatory activities. Marine cyanobacteria, in
particular, are increasingly being recognized as important source of
structurally diverse secondary metabolites. In a previous study, several
cyanobacterial strains were isolated from the Portuguese coast and
characterized using a polyphasic approach [1]. To evaluate the potential
of our isolates to produce bioactive compounds, we performed a PCR
screening for the presence of genes encoding non-ribosomal peptide
synthetases (NRPSs) and polyketide synthases (PKSs), targeting the
adenylation (A) and ketosynthase (KS) domains, respectively. DNA
fragments were obtained for more than 80% of the strains tested and the
sequences obtained were used for an in silico prediction of the
PKS and NRPS systems. Moreover, in several selected strains RT-PCR
analyses revealed that these genes are transcribed under routine
laboratory conditions. Furthermore, LC-MS analysis coupled with
molecular networking, a mass spectrometric tool that clusters
metabolites with similar MS/MS fragmentation patterns [2], was used to
search for novel or otherwise interesting metabolites in crude extracts
of our isolates. Our results revealed an untapped chemodiversity in
marine cyanobacterial isolates from the temperate region of Portugal and
validate the LC-MS analyses coupled with molecular networking as a
powerful tool for their discovery [3].
References
- Brito, A.; Ramos, V.; Seabra, R.; Santos, A.; Santos, C.L.; Lopo, M.; Ferreira, S.; Martins, A.; Mota, R.; Frazão, B.; et al. Culture-dependent characterization of cyanobacterial diversity in the intertidal zones of the Portuguese coast: A polyphasic study. Syst. Appl. Microbiol. 2012, 35, 110–119.
- Winnikoff, J.R.; Glukhov, E.; Watrous, J.; Dorrestein, P.C.; Gerwick, W.H. Quantitative molecular networking to profile marine cyanobacterial metabolomes. J. Antibiot. 2014, 67, 105–112.
- Britoa, A.; Gaifema, J.; Ramosd, V.; Glukhove, E.; Dorresteinf, P.C.; Gerwicke, W.H.; Vasconcelosc, V.M.; Mendesa, M.V. Paula Tamagnini Bioprospecting Portuguese Atlantic coast cyanobacteria for bioactive secondary metabolites reveals untapped chemodiversity. Algal Res. 2015, 9, 218–226.
Natural Product Discovery Guided by Genome Sequence of Mangrove-12 ptDerived Streptomyces qinglanensis 172205
Kui Hong, Dongbo Xu, Yi Yu and Zixin Deng
Key Laboratory of Combinatorial Biosynthesis and
Drug Discovery, School of Pharmaceutical Sciences, Wuhan University,
Wuhan, Hubei, China
With the
rapid development of genome sequencing and the availability of genome
information in NCBI database, natural product discovery from
microorganisms is undergoing a transformation from traditional “forward”
approach based on bioactivity, color or chemical property tracking to
genome-driven “reverse” strategy including compounds structure and
properties prediction, OSMAC (one strain many compounds), promoter
strengthening, regulatory gene manipulation, heterologous expression, etc.
In this report, we present natural products discovery from a marine
actinomycete using the “reverse” strategy together with the “forward”
approach, which resulted at least 5 kinds of compounds. Streptomyces qinglanensis 172,205
is a novel species isolated from a mangrove soil sample in Hainan,
China. Its genome size is about 7.2 M. Twenty one gene clusters for
secondary metabolites biosynthesis were predicted by antiSMASH and
functional genes were annotated. Employing the “reverse” strategy,
guided by the gene cluster prediction, three compounds of enterocin,
ectoine and lysolipin were detected by LC-MS after OSMAC. Enterocin
which was the dominant compound of strain 172,205 was confirmed by
compound isolation and NMR characterization. Deletion of the gene
cluster responsible for biosynthesis of enterocin improved the detection
and isolation of the other low content compounds. With the “forward”
approach based on bioactivity screening and high-resolution MS database
searching, two other compounds PreQ0 base and coproporphyrin III were identified by LC-MS or NMR. Furthermore, the isolated compounds enterocin and PreQ0 were subjected to bioactivity screening such as antimicrobial, antitumor, α-glucosidase and β-amyloid protein (Aβ1–42) fibrillation inhibitory activities, and new bioactivities of these compounds were uncovered.
Advances in Isolation and Structure Elucidation
Isolation and Quantification of Biosynthetic Cyclic Peptides
Rosemary Adaba 1, Wael Houssen 1, Andrew McEwan 1, Jioji Tabudravu 1, Andrea Raab 1, Louis Thomas 1, Nathalie Pieiller 1, Andrew Bent 2, Marcel Jaspars 1 and Jim Naismith 2
1 University of Aberdeen, Aberdeen, UK,
2 University of St Andrews, St Andrews, UK
Cyclic
peptides hold a great promise as potential therapeutics for a range of
diseases [1]. One class of peptides, the cyanobactins, originating from
marine cyanobacteria are characterized by the incorporation of
heterocycles, d-stereocentres and possibly O-prenylated
residues [2–8]. One potential therapeutic application is the inhibition
of the drug efflux pump in cancer cells [9]. Generating analogues of
these compounds may improve their therapeutic utility. This range of
compounds cannot presently be made by chemical synthesis, but
biosynthetically through genetic engineering, milligram quantities of
these modified peptides have being successfully produced [10]. Optimal
purification and accurate quantification of these novel compounds is a
pre-requisite for biological evaluation. The use of analytical methods
such as UV detection, mass spectrometry or radioisotope labeling cannot
be use to accurately quantify such compound considering the individual
limitations of these methods. These peptides contain sulphur in form of
cysteines, methionines and other forms of sulphur containing amino acids
which can be used for species unspecific quantification by LC-ICP-MS.
We have subjected these cyclic peptides of varied amino acid sequences
to various purification steps followed by LC-ICP-MS quantification,
using ESMS for identification purposes only, since no pure compounds are
available for use as standards. The purpose was to confirm that the
choice of purification techniques for the synthesized mixtures are
adequate and accurate quantification of these pure compounds using the
amount of sulphur in each peptide sequence will become a critical part
of improving compound recovery.
References
- Giordanetto, F.; Kihlberg J. Macrocyclic drugs and clinical candidates: What can medicinal chemists learn from their properties? J. Med. Chem. 2014, 57, 278–295.
- Arnison, P.G.; Bibb, M.J.; Bierbaum, G.; Bower, A.A.; Bugni, T.S.; Bulaj G. Camarero, J.A.; Campopiano, D.J.; Challis, G.L.; Clardy, J.; et al. Ribosomally synthesized and post-translationally modified peptide natural products: Overview and recommendations for a universal nomenclature. Nat. Prod. Rep. 2013, 30, 108–160.
- Houssen, W.E.; Jaspars, M. Azole-based cyclic peptides from the sea squirt Lissoclinum patella: Old scaffolds, new avenues. ChemBioChem 2010, 11, 1803–1815.
- Schmidt, E.W.; Nelson, J.T.; Rasko, D.A.; Sudek, S.; Eisen, J.A.; Haygood, M.G.; Ravel J. Patellamide A and C biosynthesis by a microcin-like pathway in Prochloron didemni, the cyanobacterial symbiont of Lissoclinum patella. Proc. Natl. Acad. Sci. USA 2005, 102, 7315–7320.
- Long, P.F.; Dunlap, W.C.; Battershill, C.N.; Jaspars, M. Shotgun cloning and heterologous expression of the patellamide gene cluster as a strategy to achieving sustained metabolite production. ChemBioChem 2005, 6, 1760–1765.
- Donia, M.S.; Ravel, J.; Schmidt, E.W. A global assembly line to cyanobactins. Nat. Chem. Biol. 2008, 4, 341–343.
- Koehnke, J.; Bent, A.F.; Zollman, D.; Smith, K.; Houssen, W.E.; Zhu, X.; Mann, G.; Lebl, T.; Scharff, R.; Shirran, S.; et al. The cyanobactin heterocyclase enzyme: A processive adenylase that operates with a defined order of reaction. Angew. Chem. Int. Ed. 2013, 52, 13991–13996
- McIntosh, J.A.; Donia, M.S.; Schmidt, E.W. Insights into heterocyclization from two highly similar enzymes. J. Am. Chem. Soc. 2010, 132, 4089–4091.
- Aller, S.G.; Yu, J.; Ward, A.; Weng, Y.; Chittabonia, S.; Zhuo, R.; Harrell, P.M.; Trinh, Y.T.; Zhang, Q.; Urbatsch, I.L.; Chang, G. Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding. Science 2009, 323, 1718–1722.
- Houssen, W.E.; Bent, A.F.; McEwan, A.R.; Pieiller, N.; Tabudravu, J.; Koehnke, J.; Mann, G.; Adaba, R.I.; Thomas, L.; Hawas, U.W.; et al. An efficient method for the in vitro production of azol(in)e-based cyclic peptides. Angew Chem. Int. Ed. 2014, 53, 14171–14174.
Oxygenated Polyketides from a Chinese Plakortis Sponge. A Treasure Trove of Structurally Diversity and Biological Activity
Giuseppina Chianese 1, Fan Yang 2, Hue-Wen Lin 2, Shabana Khan 3 and Orazio Taglialatela-Scafati 1
1 Università degli Studi di Napoli Federico II, Naples, Italy
2 Shanghai Jiao Tong University, Shanghai, China
3 University of Mississippi, University, MS, USA
Chemical
analysis of the organic extract obtained from the sponge Plakortis
simplex collected in the South China Sea afforded a series of oxygenated
polyketides, belonging to different structural classes. Several of
these compounds were new and some of them proved to be characterized by
an unprecedented carbon skeleton. Their detailed stereostructural
characterization was based on extensive spectroscopic and computational
analyses, including GIAO 13C-NMR and ECD calculations. Among the
isolated compounds, endoperoxide derivatives exhibited in vitro
antimalarial activity against chloroquine-resistant Plasmodium
falciparum strains, while non-endoperoxide polyketides were tested on
PPAR-α and PPAR-γ, revealing an interesting profile of activity. The
isolation of this complex pattern of related compounds inspired an
investigation aimed at clarifying the origin of the many polyketides
found in sponges of this genus. Thus, the major polyketide endoperoxide
isolated from this organism, haterumadioxin A (1), was
allowed to react in reducing, acidic and basic media, and the extensive
rearrangements experienced by the natural product yielded to the
formation of several compounds, some of which previously described as
natural products. In this lecture, details on structure elucidation,
biological activities and possible biogenetic origin of the isolated
compounds will be discussed.
Residual Dipolar Couplings in the NMR-Based Configurational Analysis of NEW Cystochromanes from the Phaeophyta Cystoseira baccata
Julie Muñoz 1, Alexis Krupp 2, Stefan Immel 2, Michael Reggelin 2, Gerald Culioli 3 and Matthias Köck 1
1 Alfred-Wegener-Institut, Helmholtz-Zentrum für Polar- und Meeresforschung, Am Handelshafen 12, 27570 Bremerhaven, Germany
2
Clemens-Schöpf-Institut für Organische Chemie und Biochemie, Technische
Universität Darmstadt, Alarich-Weiss-Straße 4, 64287 Darmstadt, Germany
3 Université de Toulon, MAPIEM, EA 4323, 83957 La Garde cedex, France
The systematic investigation of the phaeophyta Cystoseira
baccata actually revealed seven new meroditerpenes, the
cystochromanes A–G. A detailed study of the relative and absolute
configurations of the new molecules as well as a model compound (the
structurally simplest member of the family) was carried out. For the
determination of the relative configuration, NOE-derived interproton
distances were used as input for floating chirality DG/DDD simulations.
Since the number of NOEs was not sufficient for an unambiguous
assignment of the relative configuration, residual dipolar couplings
(RDCs, measured in lyotropic liquid crystalline phases of chiral
polyarylacetylenes) were used to refine the structures. The absolute
configurations of the new compounds were assessed by comparison of their
circular dichroism (CD) spectra to the calculated ones. Cystoseira is one of the most studied genus of the Sargassaceae family and Cystoseira
spp. are known to produce a wide array of terpenes, such as linear
diterpenes or meroditerpenes. Even though these compounds have been
studied for more than 40 years, it was recently demonstrated that their
structures could still raise interesting issues, especially concerning
their stereochemistry. The isolation and structure elucidation of the
meroditerpenes from Cystoseira
baccata led to a strong indication for a revision of the bicyclo[4.3.0]nonane system characteristic for compounds of this family.
Chlorinated Peptide/Polyketide Hybrids from the Caribbean Sponge Smenospongia aurea: A Comprehensive Approach
Roberta Teta 1, Gerardo Della Sala 1, Germana Esposito 1, Alessia Caso 1, Roberta Miceli 2, Luca S. Ceccarelli 2, Rosa Camerlingo 2, Elena Irollo 2, Giuseppe Pirozzi 2, Valeria Costantino 1 and Alfonso Mangoni 1
1 The NeaNat Group, Dipartimento di Farmacia, Università degli Studi di Napoli Federico II, Napoli, Italy
2 Department of Experimental Oncology, Istituto Nazionale Tumori Fondazione “G. Pascale”, Napoli, Italy
The Caribbean sponge Smenospongia aurea was
recently shown to contain a series of chlorinated mixed-biogenesis
NRPS/PKS products with interesting cytotoxic activities, which make them
promising leads for antitumor drug design. In addition to the published
smenamide A (1) and B (2) [1] and smenothiazole A (3) and B (4) [2], S. aurea
contains several additional compounds belonging to this class. Overall,
the structures of these peculiar peptide/polyketide hybrids suggest
that they may be products of the cyanobacterial metabolism. The most
recent results of a comprehensive approach to the study of this peculiar
class of metabolites will be discussed, including structural
elucidation, synthesis, and biosynthetic origin.
References
- Teta, R.; Irollo, E.; della Sala, G.; Pirozzi, G.; Mangoni, A.; Costantino, V. Smenamides A and B, Chlorinated Peptide/Polyketide Hybrids Containing a Dolapyrrolidinone Unit from the Caribbean Sponge Smenospongia aurea. Evaluation of Their Role as Leads in Antitumor Drug Research. Mar. Drugs 2013, 11, 4451–4463.
- Esposito, G.; Teta1, R.; Miceli, R.; Ceccarelli, L.S.; della Sala, G.; Camerlingo, R.; Irollo, E.; Mangoni, A.; Pirozzi, G.; Costantino, V. Isolation and Assessment of the in Vitro Anti-Tumor Activity of Smenothiazole A and B, Chlorinated Thiazole-Containing Peptide/Polyketides from the Caribbean Sponge, Smenospongia aurea. Mar. Drugs 2015, 13, 444–445.
Industrial Biotechnology, Polymers and Biomolecules
Development of Synthetic Biology Tools to More Predictably Clone, Express and Select Biocatalytic Activities for Metabolic Pathway Optimization and High Yield Biomolecule Production
David McElroy, Ian Fotheringham and Alison Arnold
Ingenza, Ltd., Roslin, Edinburgh, UK
Ingenza’s
has developed a number of proprietary synthetic biology tools to enable
more predictably engineer biological systems for the production of
commercially relevant examples. These tools include protein engineering
to address poor response during the control of gene expression, the
development of synthetic landing pads to optimize the genomic operating
environment around delivered genes, the use of genome editing and RNA
trafficking systems to control gene expression, the application of
transciptomics and metabolomics to enhance cell system performance, the
development of synthetic gene expression regulatory elements to better
control gene expression and the deployment of our proprietary inABLE
combinatorial genetics platform for large scale gene/pathway assembly
and optimization. Together these tools have been used to rapidly clone,
express, select and optimize target activities for many separate
enzymatic reactions, from thousands of independent genes derived from
metagenomic and phylogenetic discovery approaches. Obvious synergy
exists between this approach and versatile, solid phase screening and
selection methods using growth-based, cross-feeding or colourimetric
methods to identify engineered cells of interest. This is illustrated
through the rapid identification of critical pathway enzymes, optimal
gene coding sequences and enzyme variants from inABLE®-derived
high quality variant libraries for industrial applications in bio-based
polymers, chemicals and personal care products with our commercial
customers. We will describe the success of modeling approaches to gene
design that enhance the predictability of heterologous gene expression
in diverse hosts. In developing this suite of technologies we aims to
bring increasing predictability and overcome persistent limitations
associated with today’s iterative and empirical processes for microbial
strain improvement resulting in faster routes to high yielding
biomolecule production. We will exemplify this approach with reference
to commercially relevant examples in the field of biopolymer and
therapeutic protein production.
Protein Superfamily Data Integration for Protein Function Elucidation and Optimisation
Tom van den Bergh 1,2 and Henk-Jan Joosten 1
1 Bio-Prodict, Nijmegen, The Netherlands
2 Wageningen University, Wageningen, The Netherlands
Nature
offers a wide variety of enzymes that can be utilized in many different
processes. Metagenomics studies allow us to find many new enzymes from
marine sources, however the exact function of new enzymes if not always
clear. Here we present 3DM, a protein superfamily analysis platform that
integrates many different data types (such as sequences, structures,
function data, literature, reaction data etc.) for complete
protein superfamilies. This platform can be used to assign function to
proteins and their amino acids. New enzymes often need to be optimized
by introducing mutations to meet the requirements of a
(biotechnological) process. In many cases multiple mutations are needed
to reach these goals, but finding the right combination of mutations
still is problematic. More and more protein-engineers use a strategy
referred to as “smart library design”. Smart mutant libraries contain
only a small number of mutants and are designed such that they contain a
high number of active clones with mutations at positions (called
hotspots) that are likely to show the desired effect. Using 3DM
different enzymes features, such as enantioselectivity, activity and
thermostability have been optimized. Comparisons with random designed
libraries show that using 3DM when designing a smart library results in
high quality libraries reducing not only the number of clones that need
to be screened but it also increases the chance of finding an enzyme
with the desired properties.
Bioprocessing of Marine Microbes for Industrial Exploitation
Christina Viegelmann, Mariana Fazenda, Lynsey MacIntyre, RuAngelie Edrada-Ebel, Brian McNeil and Linda Harvey
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK
Marine
microorganisms are a relatively untapped resource in the search for
biopolymers and bioactive metabolites. This was initially due to
limitations in sampling capabilities but now is primarily due to the
difficulties in the cultivation of organisms that have evolved to live
in unique environments. SeaBioTech, an EU-FP7 project, has isolated
microorganisms from extreme marine environments, such as geothermal
intertidal biotopes in Iceland, hydrothermal vents in the Eastern
Mediterranean Sea and areas of the Scottish coasts. These strains can
yield distinctive chemistries which produce metabolites of interest to
medicinal and industrial sectors as they have the potential to be used
in a wide range of applications. The challenge lies in providing the
optimum conditions for the production of these metabolites, as marine
microbes do not naturally experience the typical conditions found within
bioreactors used in industrial-scale fermentations. Physico-chemical
changes in the culture conditions during scale-up can result in
subsequent metabolomic variations. The target is therefore to improve
our knowledge of process physiology, thus optimising the potential for
the production of the metabolites of interest. Our aim is to develop
protocols for scaling up the organisms producing these target compounds
using laboratory and pilot scale systems. Thus, we will develop systems
which will allow exploitation on an industrial scale in the future. In
order to achieve this we will use multi-fermenter systems and
cross-scale process modelling, combined with metabolomic and chemometric
analysis, to deliver new products from marine sources with built-in
enhanced manufacturability.
Bio-Silica Glass Formation by Silicatein—Functional Dissection of a Sponge Enzyme with Multiple Applications
Heinz C. Schröder, Xiaohong Wang and Werner E.G. Müller
University Medical Center of the Johannes Gutenberg University, Mainz, Germany
Bio-silica
the material that forms the inorganic skeleton of the siliceous sponges
is characterized by exceptional mechanical and optical properties. We
have been the first to demonstrate that silicatein is a genuine enzyme
which synthesizes polymeric silica from ortho-silicate at concentrations
around its Michaelis constant of 100 μM, most likely via reactive
cyclic silicic acid species. Self-cleavage of the immature
pro-silicatein at the autocatalytic cleavage site glutamine [Q]/aspartic
acid [D] into the N-terminal pro-peptide and the mature
silicatein triggers the molecule not only to become enzymatically active
(structure-forming activity) but also to acquire structure-guiding
properties (providing the structural platform for the biosilica product
via self-assembly of the mature silicatein to dimers, tetramers,
pentamers, and finally long insoluble filaments). In order to dissect
the biocatalytic, structure-forming activity of silicatein from its
structure-guiding function, two differently mutated genes were
constructed from the silicatein-α gene of the demosponge Suberites domuncula.
(i) A gene encoding for a non-processed silicatein that was mutated, by
replacing Q/D by Q/Q, at the cleavage site of the primary translation
product; (ii) A gene encoding for a mature enzymatically-active
silicatein in which the serine [S] stretch (implicated in the binding to
silica) was replaced by a Q-stretch. The expressed recombinant proteins
were applied for micro-contact printing. The experiments revealed that
enzymatically active, structure-forming silicatein, coated around the
printed non-enzymatically acting structure-guiding silicatein has the
property to synthesize biosilica that can act as a light waveguide. Our
results show that the enzymes/proteins involved in bio-silica formation
can be applied for the development of novel bioinspired materials for
diverse applications in nano-optics and nano-biotechnology. (Supported
by EU FP7 grants “BlueGenics” no. 311848, “Bio-Scaffolds” no. 604036,
and WEGM: ERC Advanced Grant “BIOSILICA” no. 268476).
Evaluation of Microbial Production of Exopolysaccharide by Rhodothermus marinus Strains: Potential for Industrial Biotechnology
Roya R. R. Sardari, Evelina Kulcinskaja, Emanuel Ron and Eva Nordberg-Karlsson
Lund University, Lund, Sweden
The formation of extracellular polysaccharides (EPS) by Rhodothermus marinus DSM 4252 and Rhodothermus marinus
493, two wild type species of thermophilic bacteria has been screened
in different culture media. Marine broth containing either 1 or 10 g/L
of glucose, sucrose, lactose, and maltose, separately was used. The
results showed that these two strains have the ability to produce and
release the EPS. Marine broth containing 10 g/L lactose showed the
highest production of EPS in both strains. Besides, EPS production was
mainly during the stationary phase. The monosaccharide composition of
the produced exopolysaccharides was analyzed and quantified. The results
suggested a heteropolymer structure for produced EPS of both strains.
The most abundant monomers were xylose, arabinose, and mannose in all
media in both strains. Also, the presence of glucose, galactose was
varied depending on the type of media used for production of EPS. The
EPS of R. marinus DSM 4252 included high quantity of xylose, while in R. marinus
493 arabinose had the highest amount compared to the other produced
monosaccharides. The molecular mass of produced EPS by these two strains
was determined by size exclusion chromatography technique using
Sephacryl S-200 and Sephacryl S-500 columns. These results lead us for
further studies aimed at increasing the interest in the application of
produced EPS of Rhodothermus marinus in food, pharmaceutical, and
wastewater treatment industries by optimizing the condition for high
production of EPS and characterizing its physicochemical properties.
Organic Synthesis
Synthesis of Siderophores from Fish Pathogenic Bacteria
Carlos Jiménez 1, Yuri Segade 1, Katherine Valderrama 1, Rosa M. Nieto 1, Jaime Rodríguez 1, Miguel Balado 2 and Manuel L. Lemos 2
1 Universidade da Coruña, A Coruña, Spain
2 Universidad de Santiago de Compostela, Santiago de Compostela, Spain
The fish pathogenic bacteria Aeromonas salmonicida subsp. salmonicida (Ass) and Photobacterium damselae subsp. piscicida (Phdp)
are the aetiological agents of fish furunculosis and pasteurellosis,
respectively, diseases that cause large economic losses in marine
aquaculture worldwide [1,2]. We were able to isolate and identify some
of the siderophores involved in their iron uptake systems such as
acinetobactin from Ass and piscibactin from Phdp [3].
In order to perform several studies on the iron uptake mechanisms of
these bacteria, the synthesis of those siderophores and their analogues
were addressed. Acinetobactin (1) Piscibactin (2): In this communication we will focus on our results on the synthesis of compounds 1 and 2.
The synthesis of ent-acinetobactin and other simplified analogues were
already finished and the evaluation of their siderophore activity
allowed us to deduce several structure-activity relationships. On the
other hand, the advances on the total synthesis of piscibactin will be
also presented.
Acknowledgments
This work was supported by Grant AGL2012-39274-C02-02 (Ministerio de Economia y Competitividad, Spain).
References
- Osorio, C.R.; Juiz-Río, S.; Lemos, M.L. A siderophore biosynthesis gene cluster from the fish pathogen Photobacterium damselae subsp. piscicida is structurally and functionally related to the Yersinia high-pathogenicity island. Microbiology 2006, 152, 3327–3341.
- Najimi, M.; Lemos, M.L.; Osorio, C.R. Identification of siderophore biosynthesis genes essential for growth of Aeromonas salmonicida under iron limitation conditions. Appl. Environ. Microbiol. 2008, 74, 2341–2348.
- Souto, A.; Montaos, M.A.; Rivas, A.J.; Balado, M.; Osorio, C.R.; Rodríguez, J.; Lemos, M.L.; Jimenez, C. Structure and Biosynthetic Assembly of Piscibactin, a Siderophore from Photobacterium damselae subsp. piscicida, Predicted from Genome Analysis. Eur. J. Org. Chem. 2012, 2012, 5693–5700.
Synthesis and Antitumour Activity of PM050489 and PM060184 Analogues
Alberto Rodríguez, Isabel Digon, Cristina Mateo, María Garranzo, Carmen Murcia, Andrés Francesch, Simon Munt and Carmen Cuevas
Pharmamar, Madrid, Colmenar Viejo, Spain
PM050489 and PM060184 belong to a new family of compounds first isolated from the Madagascan sponge Lithoplocamia lithistoides
[1]. Both are known to be tubulin-binding agents showing antimitotic
properties in human tumour cell lines at subnanomolar concentrations and
displaying a distinct inhibition mechanism on microtubules [2,3]. A
synthetic route for PM060184 and PM050489 has already been developed [1]
and PM060184 is obtained on a multi-gram scale under GMP conditions for
use in phase I clinical trials. Herein, we describe the
structure-activity relationship studies carried out in our department
for this new family. Particularly, using variants of the previously
reported synthetic route to PM050489 and PM060184, we have prepared and
tested the in vitro cytotoxicity of many different analogues of this new family.
References
- Martín, M.J.; Coello, L.; Fernández, R.; Reyes, F.; Rodríguez, A.; Murcia, C.; Garranzo, M.; Mateo, C.; Sánchez-Sancho, F.; Bueno, S.; et al. Isolation and First Total Synthesis of PM050489 and PM060184, Two New Marine Anticancer Compounds. J. Am. Chem. Soc. 2013, 135, 10164–10171.
- Pera, B.; Barasoain, I.; Pantazopoulou, A.; Canales, A.; Matesanz, R.; Rodriguez-Salarichs, J.; García-Fernandez, L.F.; Moneo, V.; Jiménez-Barbero, J.; Galmarini, C.M.; et al. New Interfacial Microtubule Inhibitors of Marine Origen, PM050489/PM060184, with Potent Antitumor Activity and a distinct Mechanism. ACS Chem. Biol. 2013, 8, 2084–2094.
- PM060184, a new tubulin binding agent with potent antitumor activity including P-glycoprotein over-expressing tumors. Biochem. Pharmacol. 2014, 88, 291–302.
3D Natural Product Scaffolds: A Starting Point in Drug Discovery
Fatemeh Mazraati Tajabadi, Rebecca Pouwer, Marc Campitelli and Ronald J Quinn
Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Australia
The
vast majority of known synthetic scaffolds, and by extension screening
libraries, are planar (Fsp3 values < 0.45), whereas natural product
scaffolds tend to be less planar (Fsp3 values > 0.45) [1]. A number
of non-flat scaffolds embedded in NP have been identified through the
use of cheminformatics. A promising scaffold (cedrane) was subjected to a
series of molecular modeling studies. The overlaying of all natural
products that contain the cedrane scaffold demonstrates the ability of
the scaffold to direct the pendant groups in 3D space. Based on this
result, a six-step synthetic plan was utilized to make the scaffold with
three orthogonally reactive chemical handles.
Reference
- Tajabadi, F.M.; Campitelli, M.; Quinn, R.J. Scaffold Flatness: Reversing the Trend. Springer Sci. Rev. 2013, 1, 141–151.
Combining Dereplication, Metabolomics and Semi-Synthesis for the Discovery of Drug Lead Cryptic Analogs in Marine-Sourced Fungi
Olivier Grovel 1, Elodie Blanchet 2,3, Marieke Vansteelandt 4,5, Yann Guitton 6,7, Catherine Roullier 1, Ronan Le Bot 8 and Yves François Pouchus 1
1 Université de Nantes, Faculté de Pharmacie, MMS, F-44035 Nantes, France
2 LBBM, Station marine de Banyuls sur mer, USR3579, UPMC-CNRS, F-66650 Banyuls sur mer, France
3 MCAM, Museum National d’Histoire Naturelle, UMR7245, MNHN-CNRS, F-75006 Paris, France
4
Université de Toulouse, UPS, UMR 152 Pharma-DEV, Université Toulouse 3,
Faculté des Sciences Pharmaceutiques, F- 31062 Toulouse Cedex 09,
France
5 IRD, UMR 152 Pharma-DEV, F-31062 Toulouse Cedex 09, France
6 Université de Rennes 1, CNRS, IRISA UMR 6074, F-35000 Rennes, France
7 Université de Nantes, LINA UMR 6241, F-44000 Nantes, France
8 ATLANTHERA, F-44000 Nantes, France
The
rich biodiversity of marine-sourced microorganisms and the complexity
of their enzymatic equipment make them a promising source of
structurally diverse and biologically active compounds. Among them,
fungi of the genus Penicillium produce a wide range of
bioactive metabolites and it can be supposed that many more unknown
molecules are still to be discovered as new druggable compounds or as
new models for medicinal chemistry. Indeed, fungal biosynthetical routes
usually lead to many more derivatives than the major compounds which
have been isolated and described. However, targeting chemistry on these
“side-products” is a challenging task as they are often transient or
trace compounds, and their isolation is often bound to fail. To avoid
this pitfall, metabolomics can allow a rapid, accurate and highly
informative investigation of this cryptic chemical diversity by the
hyphenated use of UHPLC, mass spectrometers and bio-informatics tools.
The analysis of HRMS/MS fragmentations can also allow the automated and
fast detection of derivatives related to a chemical core inside a
metabolic cluster. Here, we will show how this strategy can be applied,
with the example of 4 strains belonging to a new marine-derived Penicillium
species selected for their antiproloferative activity against
osteosarcoma cell lines. Investigations of their metabolome led to the
isolation of several compounds among which a highly active and selective
new metabolite. The use of specially developed LC-HRMS metabolomics
tools allowed the detection in crude fungal extracts of some trace
analogs of this lead compound for which structural proposals were done
using HRMS/MS fragmentations modelisation. Their structures were then
confirmed by semisynthesis which allowed the establishment of their
structure-activity relationships against osteosarcoma. This exemple
illustrates that the combination of dereplication, metabolomics and
semi-synthesis is an interesting strategy for the elucidation of marine
chemodiversity and the discovery of new cryptic analogs of selected
compounds.
Variable Temperature NMR J-Based Configurational Analysis of Flexible Acyclic Systems
Jaime Rodríguez, Miriam Rega, Maria Isabel Nieto, Carlos Jiménez and Yuri Segade
Departamento de Quimica Fundamental, Facultad de
Ciencias, and Centro de Investigaciones Cientificas Avanzadas (CICA),
Universidad da Coruña, 15071-A Coruña, Spain
NMR
coupling constants and chemical shifts are the most used parameters to
deduce both skeleton frameworks and three-dimensional arrangements in a
molecule with an unknown relative stereochemistry. This difficult task
becomes a challenge, and sometimes this is the bottleneck in the full
characterization of a compound containing flexible moieties, such as
polysubstituted open chains. Several approaches have becoming very
popular in the last 15 years to study the relative stereochemistry in
these acyclic compounds since a key publication published in 1999 by
Murata and co-workers developed a new methodology for the organic
structure analysis. This robust and logical method, known as J-based
configurational analysis (JBCA), has been incorporated in the
“spectroscopic toolbox” of all research groups involved in the
organic-structure-elucidation field [1]. This methodology is based on
the general use of the J(HH) and 2,3J(CH) and it has been applied in different 1,2- and 1,3-dimethine systems.1
However, there are some situations where the analysis becomes very
complex because three different factors come in consideration: (a) The
three staggered rotamers are present in some extend in the equilibrium,
making all the coupling constants be averaged to medium values; (b) NOE
measurements cannot discriminate the antiperiplana or the synclinal
gauche-conformers equilibria in both erythro or threo configurations;
(c) The medium values of the coupling constants are on the edge of the
small/medium or medium/large Karplus-type curves, and therefore cannot
be classified.Completing our previous studies, in this communication we
want to present how all these cases can be solved using a methodology
where, the mentioned three factors are diminished [2].
References
- Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.; Tachibana. K. Stereochemical Determination of Acyclic Structures Based on Carbon−Proton Spin-Coupling Constants. A Method of Configuration Analysis for Natural Products J. Org. Chem. 1999, 64, 866–876.
- Ardá, A.; Nieto, M.I.; Blanco, M.; Jiménez, C.; Rodríguez, J. Low-Temperature NMR J-Based Configurational Analysis of Flexible Acyclic Systems. J. Org. Chem. 2010, 75, 7227–7232.
Biosynthesis of Marine Natural Products in Microbes
Bioprospecting Microbial Mats for Novel Bioactive Compounds
Silvia Cretoiu, Florianne Parel, Henk Bolhuis and Lucas Stal
Royal Netherlands Institute for Sea Research, Yerseke, The Netherlands
Microbial
mats are benthic communities of microorganisms, usually dominated by
cyanobacteria, which develop at the interface of water and sediment, on
rocks or on soil where they form microbial crusts. Microbial mats are
globally distributed and can be found on coastal, dessert and Polar
Regions, hypersaline environments and hot springs. Metagenomic
investigation of coastal microbial mats showed that these small-scale
ecosystems are repositories of a large number of genes from metabolic
pathways relevant for biotechnology. Highly abundant genes involved in
antibiotics and carbohydrates active enzymes synthesis indicate that
microbial mats may be suitable as habitat model for isolation of novel
organisms with biotechnological potential. A significant fraction of
these genes were taxonomically affiliated to Proteobacteria, Actinobacteria and Cyanobacteria—bacterial
groups well known for their usage in biotechnology. FP-7 EU project
MaCuMBA is investigating this great potential and proposing new
methodologies of assessing it for biotechnological purposes.
Natural Product Biosynthesis in Uncultivated Sponge Symbionts
Micheal C. Wilson and Jörn Piel
Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zürich, Zurich, Switzerland
Uncultivated filamentous bacteria from the candidate genus “Entotheonella” are common symbionts of geographically and taxonomically diverse marine sponges. Recently, we showed that members of the “Entotheonella” genus associated with the sponge Theonella swinhoei Y
from Japan are the source of nearly all major metabolites previously
isolated from the sponge. In our continued effort to study these
bacteria, we discovered that “Entotheonella” associated with
geographically and taxonomically distant sponges are also prolific
producers of many known and cryptic natural products. Here we report the
identification and characterization of natural product pathways from
the genomes of “Entotheonella” from diverse sponges.
Research on the Kenyan Marine Cyanobacterium Lyngbya Majuscula Opens New Frontiers in Marine Biodiscovery Efforts in Africa
Thomas Dzeha
University of Nizwa, Nizwa, Oman
Abstract:
Cancer still remains the undisputed number one challenge in societal
health care worldwide. A decade of research on the Kenyan marine
cyanobacterium Lyngbya majuscula has revealed the geno and
phenotypes for producing the anticancer modular cyclodepsipeptides
homodolastatin 16 and dolastatin 16; and antanapeptin A in the L. majuscula genome. I report here important milestones and contributions of the research on the Kenyan L. majuscula
for realizing a sustainable supply of these essentially important
anticancer compounds for drug discovery. These findings highlight the
urgent need for new frontiers in Marine Bio-discovery efforts in Africa.
Keywords: cancer; Lyngbya majuscula; modular cyclodepsipeptides; homodolastatin 16; dolastatin 16; antanapeptin A; genome
Exploring Ripp Biosynthetic Pathways in a Quest for a Novel Macrocyclase with Improved Catalytic Properties
Cristina-Nicoleta Alexandru-Crivac 1,2, Wael Houssen 1,2, Laurent Trembleau 1 and Marcel Jaspars 1
1 Marine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Aberdeen AB24 3UE, Aberdeen, UK,
2 Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, Aberdeen, UK
Cyclic
peptides are very important structures with high therapeutic potential,
as they proved capable of interfering with challenging targets such as
protein-protein interactions. Compared to their linear counterparts,
cyclic peptides are more stable, have increased target-binding affinity,
increased resistance to proteases and higher biological membrane
permeability. We are exploring biosynthetic clusters from different RiPP
classes to identify and engineer a novel macrocyclase with improved
catalytic properties and wider substrate tolerance. In our laboratories,
we currently use PatGmac macrocyclase from the patellamide pathway1, 2
to catalyse the N-C cyclization of linear peptides. However, PatGmac is a
slow enzyme with limited capability to process peptides of 6–11 amino
acids in length 3. The enzyme needs AYD signal at the C-terminal of the
linear substrate, which should be preceded by a proline or a heterocycle
to allow for cyclization. The latter will be incorporated in the final
structure of the peptide substrate. We have also overexpressed and
purified several homologues of PatGmac and tested their activity against
various substrates. The successfully active macrocyclases will be
combined with other enzymes from the cyanobactin biosynthetic pathways,
with the aim of generating highly diverse macrocyclic scaffolds
containing amino acids, enzymatically modified amino acids, non-natural
amino acids and non-amino acid building blocks. Furthermore,
understanding the structural differences between the various enzymes can
elucidate the required structural motifs for improved activity and
kinetics.
Biosynthetic Studies of Secondary Metabolites Produced by the Sponge-Derived Fungus Stachylidium sp.
Fayrouz El Maddah, Mamona Nazir, Stefan Kehraus and Gabriele M. König
Institute for Pharmaceutical Biology, University of Bonn, Bonn, Germany
Marine fungi are in the focus of research as a source of structurally novel compounds. The marine-derived fungus Stachylidium sp. was isolated from the sponge Callyspongia sp. cf. C. flammea.
Culture on a biomalt agar medium supplemented with sea salt yielded an
ethylacetate extract which was chemically investigated and led to the
isolation of a variety of metabolites including phthalides [1],
phthalimidines [2], tyrosine derived compounds [3], and N-methylated
cyclic peptides and diketopiperazines, with interesting bioactivities.
The present study aims at determining the biogenetic origin of the
isolated metabolites, employing classical isotope tracer experiments.
Interesting structural features, such as the rare and intriguing amino
acid N-methyl-3-(3-furyl)-alanine in the cyclic peptides, and
the phthalide and phthalmidine skeleton of the marilones and marilines,
respectively are targeted. Isotopic enrichment and 13C-13C coupling constants observed for N-methyl-3-(3-furyl)-alanine, after feeding of [U-13C] glycerol, [1-13C] glucose and [1-13C]phenylalanine,
points to a shikimate-related pathway for its biosynthesis, including
condensation of phosphoenolpyruvate and erythrose-4-phosphate to
3-deoxy-d-arabino-heptulosonic
acid-7-phosphate (DAHP) as an intermediate. However, these experiments
excluded the involvement of phenylalanine as a precursor. Feeding
studies with [1-13C] sodium acetate proved the tetraketide
nature of the phthalide and phthalimidine skeleton of the marilones and
marilines, respectively. Concerning the starter unit involved in their
biosyntheses, our results favour a methylated acetate starter unit over a
propionate unit.
References
- Almeida, C.; Kehraus, S.; Prudêncio, M.; König, G.M. Marilones A–C, phthalides from the sponge-derived fungus Stachylidium sp. Beilstein J. Org.Chem. 2011, 7, 1636–1642.
- Almeida, C.; Hemberger, Y.; Schmitt, S.M.; Bouhired, S.; Natesan, L.; Kehraus, S.; Dimas, K.; Gütschow, M.; Bringmann, G.; König, G.M. Marilines A–C: Novel Phthalimidines from the Sponge-Derived Fungus Stachylidium sp. Chem. Eur. J. 2012, 18, 8827–8834.
- Almeida, C.; Part, N.; Bouhired, S.; Kehraus, S.; König, G.M. Stachylines A–D from the sponge-derived fungus Stachylidium sp. J. Nat. Prod. 2011, 74, 21–25.
Marine Microbes
Novel Members of the Phylum Bacteroidetes—A Potent Source of Bioactive Compounds
Edda Olgudóttir 1, Sólveig K. Pétursdóttir 1, Sólveig K. Ólafsdóttir 1, Ólafur H. Friðjónsson 1, Beata Wawiernia 1, Elísabet E Guðmundsdóttir 1, Sigmar K. Stefánsson 1,2, Snædís H. Björnsdóttir 1,2 and Guðmundur Ó. Hreggviðsson 1,2
1 Matís ohf, Reykjavík, Iceland,
2 Institute of Life and Environmental Sciences, University of Iceland, Reykjavík, Iceland
Several
strains representing a novel taxonomic group were recently isolated
from intertidal geothermal areas on both the SW and NW coast of Iceland.
The strains derived from the vicinity of algal mats, from hot springs
that emerge during low tide but are submerged at high tide. This
environment is highly dynamic, where constant periodic fluctuations
occur during tidal cycles involving for example steep gradients of
temperature, salinity and mineral composition. Temperature gradients are
manifested most clearly in the contrast between the hot geothermal
water, emitted into the intertidal area from the hot springs, and the
cold seawater. The strains were red-colored, grew well in marine broth
and optimally at 60 °C. 16S rRNA gene analysis identified Rhodothermus marinus as
their closest cultured relative based on 90% sequence similarity. This
indicates that the strains represent a novel genus within the family Rhodothermaceae of the phylum Bacteroidetes. One
strain, MAT 4553 was subjected to whole-genome shotgun sequencing using
the 454 technology. The assembled sequence was of 4.6 Mb and a G + C
content of 67%. A total of 3740 genes were identified. Numerous genes
were found to encode carbohydrate-degrading enzymes. Several of these
are of a potential commercial values and the corresponding genes have
been cloned and expressed in E. coli. The genes encode enzymes
such as pectinases, alginate lyases, poly-galacturonidases,
rhamnosidases, galactosidases and a rhamnogalacturonan-esterase. This
indicates that the novel strains are able to use algal sugars. In
addition, operons were identified encoding genes that are potentially
involved in the synthesis of secondary metabolites such as polyketides,
bacteriocins and terpenes. The bioactive compounds of strain MAT 4553
are being investigated and the description of the strain as the type
strain of a novel genus is currently underway.
Actinomycete Metabolome Induction/Suppression with N-Acetylglucosamine
Yousef Dashti 1, Tanja Grkovic 1, Usama Ramadan Abdelmohsen 2, Ute Hentschel 2 and Ronald J Quinn 1
1 Eskitis Institute for Drug Discovery, Griffith University, Brisbane, QLD, Australia,
2 Department of Botany II, Julius-von-Sachs Institute for Biological Sciences, University of Würzburg, Würzburg, Germany
The metabolite profiles of three sponge-associated actinomycetes, namely Micromonospora sp. RV43, Rhodococcus sp. RV157 and Actinokineospora sp. EG49 were investigated after elicitation with N-acetylglucosamine. 1H
NMR fingerprint methodology was utilized to study the differences in
metabolic profiles of the bacterial extracts before and after
elicitation. Our study found that the addition of N-acetylglucosamine modified the secondary metabolite profiles of all three investigated actinomycete isolates. N-acetylglucosamine induced the production of 3-formylindole and guaymasol in Micromonospora sp. RV43, the siderophore bacillibactin and surfactin antibiotics in Rhodococcus sp. RV157, and increased the production of minor metabolites actinosporins E-H in Actinokineospora sp. EG49. These results highlight the use of N-acetylglucosamine
as an elicitor for the induction of silent biosynthetic pathways and
for increasing the chemical diversity of microbial natural products.
Diversity and Natural Products Repertoire of Marine Sponge-Associated Actinomycetes
Usama Ramadan Abdelmohsen 1, Cheng Cheng 1, Lynsey Macintyre 2, RuAngelie Edrada-Ebel 2 and Ute Hentschel 1
1 Julius-von-Sachs-Institute for Biological Sciences, University of Würzburg, Würzburg, Germany
2 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glascow, UK
Actinomycetes
are known for their unprecedented ability to produce novel lead
compounds of clinical and pharmaceutical importance. Our contribution
focuses on the diversity, abundance, and methodological approaches
targeting marine sponge-associated actinomycetes. Various approaches
encompassing co-cultivation, elicitation experiments, bioassay-guided
isolation, as well as -omics (genomics, metabolomics) were employed
towards this goal. We will report on the following findings: (i)
Chemical analysis resulted in the isolation of the novel cyclic
lipopeptides, cyclodysidins A–D, from Streptomyces sp. RV15 associated with the marine sponge Dysidea tupha.
From the same strain, one naphthoquinone derivative SF2446A2 was
isolated and showed new antichlamydial and antischistosomal activities;
(ii) Diazepinomicin, a dibenzodiazepine alkaloid, was isolated from
strain Micromonospora sp. RV115 derived from the marine sponge Aplysina aerophoba.
Using chemical as well as cell-based assays, a strong antioxidant
potential of diazepinomicin was demonstrated. Moreover, diazepinomicin
inhibited the proteases rhodesain and cathepsin L; (iii) A new O-glycosylated xanthone derivative, microluside A, was isolated from the broth culture of Micrococcus sp. EG45 cultivated from the Red Sea sponge Spheciospongia vagabunda. Microluside A exhibited antibacterial potential against Enterococcus faecalis JH212 and Staphylococcus aureus
NCTC 8325; (iv) Using metabolomics to dereplicate the marine
sponge-associated Actinokineospora sp. EG49 cultivated from the sponge Spheciospongia vagabunda,
20 compounds were identified, many of which are unknown.
Bioassay-guided isolation of the same strain led to the isolation of new
anti-trypanosomal and antioxidant angucyclines named actinosporins A–D.
Interestingly, co-cultivation of the two sponge-derived actinomycetes, Actinokineospora sp. EG49 and Nocardiopsis
sp. RV163, induced biosynthesis of three natural products that were not
detected in the single culture of either microorganism. These were N-(2-hydroxyphenyl)-acetamide, 1,6-dihydroxyphenazine and 5a,6,11a,12-tetrahydro-5a,11a-dimethyl-1,4-benzoxazino[3,2-b][1,4]benzoxazine. The phenazine derivative was active against Bacillus sp. P25, Trypanosoma brucei and interestingly, against Actinokineospora
sp. EG49. Our results highlight marine sponges as prolific resource for
taxonomically novel and rare actinomycetes with potential for drug
discovery.
Actinobacteria from the South Pacific: A Bioprospection for Natural Bioproducts
Beatriz Camara 1, Agustina Undabarrena 1, Fernanda Claverias 1, Valentina Gonzalez 1, Andres Cumsille 1, Myriam Gonzalez 1, Michael Seeger 1, Fabrizio Beltrametti 2 and Edward Moore 3
1 Universidad Tecnica Federico Santa Maria, Valparaiso, Chile
2 Actygea s.r.l., Gerenzano, Italy
3 Culture Collection University of Goteborg, Goteborg, Sweden
Marine
derived actinobacteria have demonstrated to be a source of a broad
variety of secondary metabolites with diverse biological activities,
including antibiotics, antifungal, antitumoral and extracellular
enzymes, among others. Most of these metabolites are synthesized by
complex metabolic pathways that involve polyketide synthases (PKS)
and/or non-ribosomal peptide synthetases (NRPS). Recently,
bioprospection in underexplored habitats has gained focus, since new
taxa of marine actinobacteria can be found, and thereof, possible new
metabolites. In this study, actinobacteria from marine sediments and
sponges of three locations along different latitudes of the supratidal
and subtidal coast of Chile (Chañaral de Aceituno Cove, III Region;
Valparaiso Bay, V Region and Cumau Fjord, X Region) were isolated and
its biotechnological potential was evaluated. Different culture
conditions and selective media that enrich the growth of this phylum
were used and approximately 300 bacterial strains were isolated.
Comparative analysis of the 16S rRNA gene sequences led to identifying
genetic affiliations of 27 known genera, belonging to 19 families. Also,
a putative new genus was found, belonging to the Nocardiopsaceae
family. The antimicrobial activity of representative isolates was
evaluated against laboratory test strains (Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica, Escherichia coli and Pseudomonas aeruginosa),
demonstrating the ability of many isolates to inhibit the growth of
gram-positive and/or gram-negative bacterial strains. Also, their
ability to produce extracellular lipases and proteases was evaluated,
and many isolates showed a significant activity. In addition, the
presence of PKS and NRPS biosynthetic genes was evaluated, and most of
the isolates were positive for at least one of the genes analyzed. This
study shows a remarkable biodiversity of culturable actinobacteria,
associated to marine environments along Chile. Our Chilean marine
actinobacterial culture collection represents an important resource for
the bioprospection of novel marine actinomycetes and its metabolites,
which evidences their potential as producers of natural bioproducts.
Metabolomics as a Tool to Search for New Potential Antibiotics from Mangrove Plant Avicennia Lanata and Its Endophytic Fungi
Noor Wini Mazlan 1,2, Shan Hui Sim 1, Rothwelle Tate 1, Carol Clements 1 and RuAngelie Edrada-Ebel 1
1 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK
2
Analytical Chemistry and Environment, School of Marine Science and
Environment, Universiti Malaysia Terengganu, Kuala Terengganu,
Terengganu, Malaysia
The
discovery of new secondary metabolites from plants and endophytes has
become more challenging in natural products chemistry. Bacteria and
fungi interact within the host plant and stimulate competition for
nutrients and spaces which is regarded as a major ecological factor that
induces the production of bioactive secondary metabolites. In this
study, the isolation of bioactive natural products was done on the crude
organic extract of the mangrove plant Avicenna lanata collected from
Terengganu, Malaysia using several high-throughput chromatographic
techniques which yielded three new derivatives of
2,3-dihydro-1,4-naphthoquinone along with three known congeners and
triterpenes. Meanwhile, three pure endophytic fungal strains were also
isolated from A. lanata: Aspergillus aculeatus (leaf); Lasiodiplodia theobromae (stem) and Fusarium sp.
(root). Prior to this study, metabolomics using high resolution mass
spectrometry and NMR spectroscopy was applied to identify and optimize
the production of bioactive secondary metabolites from the three strains
cultivated in both solid rice and liquid culture media at 7, 15 and 30
days. The spectral data was processed utilizing the quantitative
expression analysis software MZmine 2.10 coupled with the Antimarin
database for dereplication studies. SIMCA P+ 13.0 was used to prove that
the optimized models were statistically sound. The 15-day solid culture
of Fusarium sp. yielded four naphthazarin-related 1,4-naphthoquinones, ergosterol peroxide and β-sitosterol. The 30-day A. aculeatus rice culture afforded the new compound, 2-(3,4dihydroxyphenyl)-N,N-dimethylacetamide, four simple phenolics and secalonic acid B, while the 15-day L. theobromae
rice culture produced mellein along with its three derivatives.
Structure elucidation of the isolated compounds was established using 1D
and 2D-NMR and HRESI-MS. Aside from sterol compounds, all isolated
compounds from A. lanata and its fungal showed significant
anti-trypanosomal activity. This study proved that metabolomics is an
essential tool to identify biomarkers from mangrove plants and
endophytic fungi to target the isolation of potential anti-trypanosomal
effective drugs.
Poster Session 1. Chemical Ecology
Is the Specialized Metabolism Influenced by Environmental Conditions? The Case Study of the Mediterranean Sponge Crambe crambe
Eva Ternon 1, Erica Perino 2, Roberto Pronzato 2 and Olivier Thomas 1
1 Nice Institute of Chemistry—PCRE UMR 7272 CNRS, University of Nice Sophia Antipolis, Nice, France
2 Department for the Study of the Territory and its Resources of the University, Genoa, Italy
In
the age of marine biotechnology, research focusing on sponge secondary
metabolites production for industrial purposes has strongly increased.
Indeed, sponges are marine animals that produce a large array of
bioactive compounds. However, industrial production requires a large
amount of raw material, and sponges are characterized by low-growth, in
restricted areas. In order to supply large quantities of secondary
metabolites, sponges have been cultivated all around the world and the
most accurate environmental conditions for this purpose have been
investigated. In the Mediterranean Sea, the encrusting sponge Crambe crambe
has been showed to produce two bioactive families of metabolites, the
crambescins and the crambescidins. In the frame of the European project
BAMMBO, farming of C. crambe was conducted in two areas of the
Northwestern Mediterranean Sea that differ by their environmental
conditions. In the meantime, wild sponge specimens were collected close
to the farming area. Additionally, ex situ cultures of this
sponge species were set up in aquaria during 30 days, under a constant
flow and under various environmental conditions. The variation in the
specialized metabolome in relation with their living conditions was
assessed by both targeted and un-targeted metabolomics approaches, using
UHPLC-HRMS (QTof). Data showed that both seasons and sampling area are
factors affecting the specialized metabolome. On the opposite, no
significant correlation was found between the mode of growth (farming, ex situ cultures or wild) and any modification of the nature of the specialized metabolome.
Comparison Studies on Fatty Acid Profiles of Four Seaweeds Sargassum in China
Hongbing Liu and Zhen Chen
Institute of Marine Food and Drugs, School of Medicine and Pharmacy, Ocean University of China, Qingdao, Shandong, China
Sargassum is a genus of approximately 250 species in Sargassaceae and is geographically widespread in all tropical and temperate oceans. S. fusiforme, S. pallidum, S. honeri, and S. thunbergii
are mainly distributed in the coast of the North Pacific Ocean with an
abundant biomass in China. As important economic seaweeds, these species
have been used as nutritional foods and herbal medicine for treating
hyperlipidemia, hypertension, heart disease, inflammatory diseases, and
cancer in China for thousands of years. The chemical differences between
these medicinal species have not been systematically investigated. In
the present study, fatty acid profiles of S. fusiforme, S. pallidum, S. honeri, and S. thunbergii were established based on GC-MS, respectively (totally 87 batches). By means of principal component analysis (PCA), these Sargassum
species could be well differentiated from each other. It was alga
species that dominated the chemical diversity other than environmental
features such as growing region and water temperature in Sargassum. For S. fusiforme and S. pallidum,
which are both listed in the Chinese Pharmacopoeia, a further
comparison indicated that six potential key fatty acids (C16:0, C18:4 n-3, C20:2 n-6, C20:4 n-6, C20:5 n-3, and C22:1 n-9) could be employed to distinguish them. Meanwhile, S. fusiforme contained higher unsaturation index (UI) and lower n-6/n-3 ration than S. pallidum, which hinted that the former might be more beneficial for anti-cardiovascular disease.
Screening of Indonesian Macroalgae by Their Total Phenolic Contents and Antioxidant Activities: The Interest of Brown Seaweeds as a Source of Active Phlorotannins
Nur Azmi Ratna Setyawidati 1,2, Klervi Le Lann 1, Alizé Bagot 1, Ita Widowati 3 and Valérie Stiger-Pouvreau 1
1 LEMAR UMR 6539 UBO CNRS Ifremer
IRD, European Institute of Marine Studies (IUEM), Université de Bretagne
Occidentale (UBO), European University of Brittany (UEB), Technopôle
Brest-Iroise, Plouzané, France
2
Center for Marine and Fisheries Technology, Agency for Marine Affairs
and Fisheries Research and Development, Minister of Marine Affairs and
Fisheries, Jakarta, Indonesia
3 Faculty of Fisheries and Marine Sciences, Diponegoro University, Semarang, Indonesia
Biodiversity
of macroalgae are considered as an economic importance in Indonesia.
Within the framework of the international project called INDESO, we
assessed the potential and the added-value of natural populations of
abundant Indonesian seaweeds. We investigated the chemical composition
of 17 common red, green and brown seaweeds from two bays, Malasoro and
Ekas, sampled during dry and wet seasons. The conventional solid/liquid
extraction was conducted using two solvent mixtures: ethanol 75%
(EtOH-water 75:25) and 25% (EtOH-water 25:75). All of the crude extracts
were tested for their free radical scavenging activity; using the DPPH
(2,2-diphenil-1-picrylhydrazyl). From these procedures; the highest TPC
and strongest DPPH were showed in the ethanolic extraction (EtOH-water
75:25) of brown algae from Ekas Bay, Padina australis and Sargassum sp., and the aqueous (EtOH-water 25:75) of two brown algae from Malasoro bay, Sargassum sp. and Turbinaria conoïdes,
collected during the dry season. From the four interesting brown algae,
two different fractions were obtained by liquid/liquid purification
procedure: (i) an ethyl acetate fraction (AE) and (ii) an aqueous
fraction (AQ). We determined the phenolic content from the crude
extraction and the two purified fractions, AE and AQ, together with
their anti-scavenging activities: DPPH and ferric reducing antioxidant
power (FRAP) assay. The results of the screening and the activity of
purified fractions were discussed and could be indicated encouraging
perspectives for the use of Indonesian brown seaweeds. These seaweeds
would be a valuable source of antioxidant natural compounds for
Indonesia. This work was financed with the support of the INDESO project
(2013–2016), funded by the French Development Agency (AFD), being
sponsored and coordinated by the Indonesian Ministry of Marine Affairs
and Fisheries (MMAF).
Chemical Characterization, Anti-Microbial Inhibitory and Cytotoxic Activities of Crude Compounds from Mediterranean Ascidian Styela Plicata (Lesur, 1823)
Satheesh Kumar Palanisamy 1, Salvatore Giacobbe 1, Ronald J. Quinn 2, Donatella Del Bufalo 3, Daniela Trisciuoglio 3, Angelo Marino 1, Salvatore Cuzzocrea 1 and Antonello Mai 4
1 Department of Biological and Environmental Sciences, University of Messina, Messina 98166, Italy
2 Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Queensland 4111, Australia
3 Istituto Nationale Tumori Regina Elena, Rome 00144, Italy
4
Pasteur Institute, Cenci-Bolognetti Foundation, Department of Drug
Chemistry and Technologies, Sapienza University of Rome, Rome 00185,
Italy
Marine ascidians are sessile organisms with
a great ability to synthesize bioactive substances; they are well-known
reservoir for novel Marine Natural Products (MNPs). The primary
objective of the present study includes documentation of the taxonomy
and distribution of ascidian fauna in Mediterranean Sea-Messina, to
identify drug-like molecules from the ascidian Styela plicata and finds
their pharmacological properties. The Faro Lake, Messina at the
south-end of the Italy has a great number of ascidian fauna. In the
present study, we reported 15 ascidian species in 7 family and 11
genera, primarily from coastal lake, Ionian Sea, Messona. Maximum 6
species were reported from the family Styelidae, 2 species from the
family Aascidiadae, Ascidiiae and Didminidae and remaining others. The
chemical characterization of ascidian crude extract was determined by
FTIR, LC-MS and 1H-NMR. The crude extracts of S. plicata showed remarkable anti-microbial activity against B831—Pseudomonas sp. (16 mm) and B7—Burkholderia mallei
(12 mm). The crude compounds were showed modest cytotoxicity against
various tumour cell lines human epithelial kidney cells (HEK 293 Phoenix
cells), HeLa-cervical cancer and HT1080 human fibrosarcoma cells with
IC50 values of (93, 139 and >500 µm/mL) repectively. Pyrimidine
metabolites, dihydro-5-methylpyrimidine-2,4(1H,3H)-dione was isolated first time from S. plicata.
Their structure was determined by 2D-NMR techniques. Nevertheless,
further studies are recommended to verify which fraction is responsible
for the biological activity of the whole extract.
Keywords: ascidians; cell; crude extract; cytotoxic; FTIR; LC-MS; NMR
Marine Toxins and Bioassays
Discovery of Peptide Toxins in the Bootlace Worm, the World’s Longest Animal
H.S Andersson 1, E. Jacobsson 2, C. Eriksson 2, K.J. Rosengren 3, P. Andrén 4, M. Strand 5,6 and U. Göransson 2
1 Linnaeus University Centre for
Biomaterials Chemistry, Department of Chemistry and Biomedical Sciences,
Linnaeus University, Kalmar, Sweden
2 Division of Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, Uppsala, Sweden
3 School of Biomedical Sciences, The University of Queensland, Brisbane, Australia
4 Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden
5 Department of Biological and Environmental Sciences, University of Gothenburg, Gothenberg, Sweden
6 Swedish Species Information Centre, Swedish University of Agricultural Sciences, Uppsala, Sweden
Nemerteans
(ribbon worms) are marine predators, which capture their prey using a
proboscis containing a mixture of toxins which brings on rapid paralysis
[1]. In addition, the epidermis of nemerteans contains a thick mucus of
similar toxic constitution. Although the presence of low-molecular
weight toxins such as anabasine and tetrodotoxin have been described
previously, the presence of peptide based toxins in nemerteans, with one
notable exception [2] have not been reported. We have now found such
toxins in the mucus of the bootlace worm (Lineus longissimus), a
spectacular species that can reach up to 50 meters in length. We have
been able to confirm the expression of peptide toxins from this worm via
LC-MS and MALDI imaging. Two peptides have been isolated and the
peptide sequences have been elucidated; these peptides contain three
disulfides and represent a novel family of nemertean toxins. Purified
extract of one of these peptides was shown to produce rapid cramping
followed by death in shore crabs. In addition, a third peptide, with
strong resemblance to the only peptide toxin previously characterized
from nemerteans, Neurotoxin B-IV, a 55 aa peptide, has been discovered.
In addition, we have sequenced the transcriptome of the bootlace worm
and obtained the transcriptomes of a series of other nemerteans,
presently under investigation. In this presentation, we intend to
discuss these findings and their implications further.
References
- Kem, W.R. Structure and activity of Nemertine Toxins. Integr. Comp. Biol. 1985, 25, 99–111.
- Kem, W.R. Purification and characterization of a new family of polypeptide neurotoxins from the heteronemertine Cerebratulus lacteus (Leidy). J. Biol. Chem. 1976, 251, 4184–4192.
Anti-Tumor and Anti-Angiogenic Activity of the Cyanobacterial Metabolite Coibamide A
Jeffrey Serrill, Xuemei Wan, Adam Alani, Kerry McPhail and Jane Ishmael
Oregon State University, Corvallis, OR, USA
Coibamide A is an N-methyl-stabilized
depsipeptide that was isolated from a marine cyanobacterium collected
using SCUBA from the Coiba National Park as part of the International
Cooperative Biodiversity Groups (ICBG) program based in Panama. Previous
testing of coibamide A in the National Cancer Institute in vitro
60 cancer cell line panel revealed an unmatched selectivity profile
indicative of a unique mechanism of action. Coibamide A displayed
sub-nanomolar potency as a growth inhibitory agent and good histological
selectivity for several solid tumor cell types including CNS, breast,
colon and ovarian cancer. Although the cellular target of this structure
is unknown, we have determined that coibamide A induces cell-cycle
arrest at the G1 stage and is a potent and effective cytotoxin with the
ability to trigger apoptosis or alternate cell death signaling in human
cancer cells. Coibamide A also induces a rapid, mTOR-independent
autophagy response that is not required for cell death but occurs as a
cell stress response to treatment. We report that coibamide A shows
promising in vivo efficacy in a subcutaneous mouse model of
human glioblastoma; treated animals showed significant decreases in
tumor growth and increased survival relative to control animals.
Coibamide A also displays potent anti-angiogenic activity in vitro
and effectively inhibits the ability of human umbilical vein
endothelial cells (HUVECs) to proliferate, migrate, and form
three-dimensional networks. Coibamide A selectively decreases the
expression of several candidate membrane and secreted proteins that are
particularly important for angiogenesis, such as VEGF-A, VEGF receptor 2
and VCAM-1. Coibamide A remains a lead structure in natural product
drug discovery by virtue of its ability to modulate the growth and
development of tumors in a multi-faceted manner through its effects on
both cancer and endothelial cell types.
Seabiotech: From Sea-Bed to Test-Bed: Harvesting the Potential of Marine Biodiversity for Industrial Biotechnology: An Overview of the Advances in Year 2
Lynsey MacIntyre, Mariana Fazenda, Tong Zhang, Linda Harvey,
Carol Clements, Louise Young, Grainne Abbott, Christina Viegelmann,
Catherine Dowdells, RuAngelie Edrada-Ebel and Brian McNeil
University of Strathclyde, Glasgow, Strathclyde, UK
Discover
the progress that an interdisciplinary consortium of European
Scientists has made in their explorations of marine microbial diversity
to discover new products with the potential for development as
antibiotics anti-infectives and anti-cancer treatments; and the
developments for sustainable manufacture of these products on an
industrial scale. The advances made in the second year of the project
will be presented: illustrating the biodiversity and number of samples
collected; the preliminary results obtained from a comprehensive
screening campaign; the use of metabolomics as a tool to monitor
secondary metabolite production under different culture conditions; and
the scale-up to bio-fermenter level. SeaBioTech is an SME-driven EU-FP7
project designed to create innovative marine biodiscovery pipelines as a
means to convert the potential of marine biotechnology into novel
industrial products for the pharmaceutical, cosmetic, food and
industrial chemistry sectors. The program brings together experts in
biology, genomics, natural product chemistry, bioactivity testing,
industrial bioprocessing, legal aspects, market analysis and
knowledge-exchange. SeaBioTech targets novel marine endosymbiotic
bacteria from unique and untapped habitats, including geothermal
intertidal biotopes in Iceland, hydrothermal vent fields and deep sea
oligotrophic basins of the Easter Mediterranean Sea and Iceland as well
as areas of the Scottish coasts that are likely highly productive
sources of new bioactive compounds. The sampling process utilises
metagenomics to ensure the quality of marine resources for further
industrial development. SeaBioTech combines metabolomics with systems
biology and functional bioassays to increase the ability to uncover
positive hits with a cheaper faster approach: an affordable innovative
and efficient method to separate, elucidate the structure and identify
bioactive metabolites; and through well controlled metabolic engineering
increase their yield at lab and industrial scales. UNCBD Nagoya
protocol compliant. The SeaBioTech project is funded by the European
Commission within its FP7 Programme, under the thematic area
KBBE.2012.3.2-01 Grant Number 311932.
An Integrated Lead Discovery Programme within Seabiotech to Address Major Parasitic Infections in Aquaculture
Christer Wiik-Nielsen 1, Louise Young 2, Laura Stucchi 3, Lynsey MacIntyre 2, Grainne Abbott 2, Elin Aksnes 1, Ruangelie Edrada-Ebel 2, Daniele Carettoni 3, Michele Stanley 4, Cheng Cheng 5, Ute Hentschel Humeida 5 and John Day 4
1 Pharmaq AS, Oslo, Norway
2 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK
3 Axxam SpA, Bresso (Milan), Italy
4 The Scottish Association for Marine Science, Scottish Marine Institute, Oban, UK
5 Department of Botany II, Julius-von-Sachs Institute for Biological Sciences, University of Würzburg, Würzburg, Germany
A
limiting factor for the identification of novel antiparasitics in
aquaculture is the lack of high-throughput assays. Indeed,
low-throughput phenotypic assays on living parasites are mainly
available, making them incompatible to assist the screening of large
sample collections. To enable the drug discovery efforts aimed at
counteracting the infesting parasite sea lice, a joint collaborative
program within SeaBioTech was implemented between Strathclyde
University, the Scottish Association for Marine Science (SAMS),
University of Würzburg, Axxam and Pharmaq. The strategy relies on the
identification of high-throughput assays as pre-selection tools for
phenotypic screening. The pre-selection assays were identified according
to two complementary principles. First, functional assays developed on
an orthologous sodium channel and on eel acetyl-cholinesterase were made
available by Axxam and Strathclyde University, respectively. These
assays measures the activity of validated targets, whose function is
impaired by antiparasitics used in industrial settings. In the second
strategy, an array of cell-based assays was validated by Axxam for their
sensitivity to reference compounds used as antiparasitic agents in
aquaculture. As a result, four preselection assays were identified in
total. To date, over 650 extracts of marine origin assembled along the
SeaBioTech program were screened at Axxam and Strathclyde University on
the four preselection assays. In total, 135 extracts were identified as
candidates for screening on sea lice at Pharmaq. Two extracts obtained
by SAMS and by the University of Würzburg and identified by Axxam for
their antagonistic activity on two preselection assays were confirmed to
display high parasiticidal activity against sea lice. The two extracts
displayed a selective activity profile on 26 functional assays used
within SeaBioTech, and preliminary dereplication data generated by
Strathclyde University indicated a potentially interesting chemistry
profile. The SeaBioTech project is funded by the European Commission
within its FP7 Programme, under the thematic area KBBE.2012.3.2-01,
Grant Number 311932.
Seabiotech: From Sea-Bed to Test-Bed: Harvesting the Potential of Marine Biodiversity for Industrial Biotechnology
RuAngelie Edrada-Ebel1 1, Lynsey MacIntyre 1, Mariana Fazenda 1, Tong Zhang 1, Carol Clements 2, Louise Young 2, Grainne Abbott 2, Linda Harvey 1, Alan Harvey 2 and Brian McNeil 1
1 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow, UK
2 Strathclyde Innovations in Drug Research (S.I.D.R), University of Strathclyde, 161 Cathedral Street, Glasgow, UK
SeaBioTech
is an EU-FP7 project designed and driven by SMEs to create innovative
marine biodiscovery pipelines as a means to convert the potential of
marine biotechnology into novel industrial products for the
pharmaceutical (human and aquaculture), cosmetic, functional food and
industrial chemistry sectors. To achieve its goals, SeaBioTech brings
together leading experts in biology, genomics, natural product
chemistry, bioactivity testing, industrial bioprocessing, legal aspects,
market analysis and knowledge-exchange. SeaBioTech targets novel marine
endosymbiotic bacteria from unique and previously untapped habitats,
including geothermal intertidal biotopes in Iceland, hydrothermal vent
fields and deep sea oligotrophic basins of the Eastern Mediterranean Sea
and unsampled areas of Scottish coasts that are likely to be highly
productive sources of new bioactive compounds. This poster describes the
first year’s activity of the SeaBioTech project which resulted in a
robust, validated workflow suitable for evaluating unexplored activity
of marine samples. UNCBD Nagoya protocol compliant. The SeaBioTech
project is funded by the European Commission within its FP7 Programme,
under the thematic area KBBE.2012.3.2-01 with Grant Number 311932.
Microalgal Natural Products with Bioactivities Relevant for Human Health
Chiara Lauritano 1, Jeanette H. Andersen 2, Espen Hansen 2, Marte Albrigtsen 2, Laura Escalera 1, Francesco Esposito 1, Kirsti Helland 2, Kine Ø. Hanssen 2, Giovanna Romano 1 and Adrianna Ianora 1
1 Stazione Zoologica Anton Dohrn, Napoli, Italy
2 Marbio, University of Tromsø, Tromsø, Norway
The
EU project PharmaSea is a large consortium of 24 partners whose aim is
to discover new bioactive compounds from marine microorganisms for the
treatment of various human diseases. Marine microalgae have been shown
to be valuable sources of novel biologically active molecules for
applications in the food industry (additives, pigments, vitamins and
colorants) as well as in the pharmaceutical and cosmetic sectors. For
the PharmaSea project we tested crude extracts of different microalgal
classes for possible antioxidant, anti-inflammatory, anticancer,
anti-diabetes, antibacterial and anti-biofilm activities. Previous
studies have shown that many microalgae produce defense metabolites with
potentially interesting biotechnological applications when they are
grown in stressful conditions. Hence, selected microalgae were grown
under different culture conditions in order to identify those that
produced higher amounts of the compounds of interest. Our results
indicate that various species belonging to the Bacillariophyceae and
Coscinodiscophyceae displayed anti-inflammatory, anticancer (blocking
human melanoma cell proliferation) and anti-biofilm (against the
bacteria Staphylococcus epidermidis) activities. We also
observed that different clones and species grown in nutrient starvation
conditions had different effects. An additional dereplication step has
identified 10-Hydroxyphaeophorbide A in various Bacillariophyceae as
possibly responsible for the observed anti-inflammatory activity, while
Phaeophorbide A and Phaeophytin A have been identified in
Coscinodiscophyceae as the possible agents for anticancer and
anti-biofilm activities, respectively. The tested extracts will be
further studied for possible isolation and identification of new
compounds with these bioactivities. In addition, RNA-seq of the
bioactive species is currently underway in order to identify the
enzymatic pathways involved in the production of the metabolites of
interest.
Inhibitory Activity against the Chikungunya Virus of Extracts from the Soft Coral Lobophytum microlobulatum
Yik Sin Chan, Nam Weng Sit and Kong Soo Khoo
Faculty of Science, Universiti Tunku Abdul Rahman, Kampar, Perak, Malaysia
Chikungunya fever is an arboviral disease transmitted by Aedes
mosquitoes. It has resulted in epidemics of the disease in tropical
countries in the Indian Ocean and South East Asian regions. This study
aims to investigate the anti-chikungunya virus activity of extracts from
a soft coral, Lobophytum microlobulatum, collected from
Malaysian waters. Following lyophilization and grinding to powder form,
the sample was subjected to sequential solvent extraction using hexane,
chloroform, ethyl acetate, ethanol, methanol and distilled water in
order to extract bioactive compounds. The antiviral activity was
evaluated in three different modes (pre-, concurrent and post-treatment)
using monkey kidney epithelial (Vero) cells infected with the virus
(multiplicity of infection = 1). The cell viability was determined by
Neutral Red uptake assay. Strong antiviral activity (cell viability 70%)
was observed only in the concurrent mode whereby the hexane and ethanol
extracts resulted in cell viabilities of 73.3% ± 2.6% and 79.2% ± 0.9%
respectively (mean ± SD; n = 3). The corresponding mean 50% effective concentrations (EC50)
for the extracts were 14.2 ± 0.2 and 115.3 ± 1.2 µg/mL respectively.
The chloroform extract showed moderate antiviral activity (cell
viability 31%–69%) in the concurrent mode (cell viability = 57.9% ±
0.8%) as well as in the pre-treatment mode (cell viability = 51.2% ±
2.4%). Neither strong nor moderate antiviral activity was shown in the
post-treatment mode. The two extracts with strong antiviral activity
were selected for analysis of viral load in the assay using quantitative
Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR). The viral
loads in the assay treated with the hexane (20 mg/mL) and ethanol (320
mg/mL) extracts were 5.62 × 106 ± 0.11 × 106 and 4.97 × 107 ± 0.67 × 107
copies/mL respectively, indicating a reduction of 99.6% and 96.3%
respectively compared to the virus control. The ethanol extract appears
to hold the most promise for further characterization of active
compounds as it possessed greater selectivity index (SI > 5.6)
compared to the hexane extract (SI = 2.1).
Neuroprotective Effect of Seaweeds Extracts against Dopamine-Induce Cell Death in Human Neuroblastoma Sh-Sy5y Cells
Joana Silva, Celso Alves, Susete Pinteus, André Horta, Susana Mendes and Rui Pedrosa
MARE—Marine and Environmental Sciences Centre, Polytechnic Institute of Leiria, Peniche, Leiria, Portugal
The
Parkinson Disease (PD) is a neurodegenerative disease of the central
nervous system that results in the depletion of dopamine production
cells in a specified region of the brain, affecting about 1% of the
world population. Although the causes of PD pathogenesis remains
incomplete, some evidences from human and animal studies has suggested
that oxidative stress is an important mediator in its pathogenesis. The
aim of this study was to evaluate the protective effects of seaweeds
extracts on dopamine (DA)-induced neurotoxicity in the human
neuroblastoma cell line SH-SY5Y, as well the associated intracellular
signaling pathways. Cell viability studies were assessed by
(3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) MTT assay
and the intracellular signaling pathways associated to oxidative stress
induced by DA: H2O2 generation, mitochondrial
membrane potential and caspase-3 activity. Exposure of SH-SY5Y cells to
30–3000 µM of DA caused significant effects on the reduction of cell
viability, in a concentration (24 h) and time-dependent manner (6 h, 12
h, 24 h, 48 h). The data suggest that the cell death induced by DA was
mediated by the increase of H2O2 production,
depolarization of mitochondrial membrane potential and the increase of
caspase-3 activity. The treatment with seaweeds extracts (1 mg/mL; 24 h)
evidenced a neuroprotection effect on toxicity induced by DA (1000 µM;
24 h), namely by recovering cell viability between 10% and 25%. The
protective effect obtained by seaweeds extracts against DA-induced
toxicity could be mediated by an anti-apoptotic effect (mitochondrial
protection and decrease of caspase-3 activity). These results suggest
that seaweeds can be a promising source of new compounds with
neuroprotective potential.
Gambierone, a Ladder-Shaped Polyether from the Dinoflagellate Gambierdiscus belizeanus
Kevin Calabro 1, Inés Rodrìguez 2, Grégory Genta-jouve 3, Carmen Alfonso 4, Eva Alonso 2, Jon Sànchez 2, Amparo Alfonso 2, Luis Botana 2 and Olivier Thomas 1
1 Institut de Chimie de Nice, Université Nice Sophia Antipolis, UMR 7272 CNRS, Faculté des Sciences, Nice 06108, France
2 Departamento de Farmacología, Universidad de Santiago de Compostela, Facultad de Veterinaria, Lugo 27002, Spain
3
Laboratoire de Pharmacognosie et de Chimie des Substances Naturelles,
Université Paris Descartes, COMETE UMR 8638 CNRS, Paris 75006, France
4 Laboratario CIFGA S.A., Plaza de Santo Domingo, n° 20, 5ª Planta, Lugo 27001, Spain
Ciguatoxin
(CTX) intoxications are a serious and global public health issue and
the identification of compounds associated to this toxin group is
critical but usually highly challenging due to structural complexity and
low amount available. We report herein the isolation, structure
elucidation and biological activity of gambierone (1), a new natural product from the dinoflagellate Gambierdiscus belizeanus.
This compound exhibits unprecedented polyether skeleton and right-hand
side-chain. Its relative configuration was fully determined by
interpretation of NOESY experiment and comparison between experimental
and theoretical NMR data. Although the succession of cycles has no
chemical similarity with CTXs, 1 has molecular formula and biological activity similar to CTX-3C, although lower in intensity.
Antibacterial Compounds from Indonesian Rhodophyta against Aeromonas hydrophilla and Vibrio sp.
Adhika P. A. Wijnana, Mamluatul Hikmah, Amirul Mu'minin, Noer Kasanah, Triyanto and Alim Isnansetya
Fakulty of Agriculture, Gadjah Mada University, Yogyakarta, Indonesia
Rhodophyta
have potential as high source of bioactivity secondary metabolites. The
study was conducted to screen the activity of secondary metabolites
from three Gracilaria edulis, Gracilaria arcuata, and Gelidium spinosum against two fish pathogenic bacteria (Aeromonas hydrophila and Vibrio
sp.). The Rhodophytas were collected from Drini beach, Yogyakarta,
Indonesia. For each species, 100 g dried samples were extracted using
ethyl acetate. Bioassay guided fractionation was conducted on column
chromatography using silica gel and step gradient eluent. Bioactivity
compounds were identified using GC-MS and LC-MS in conjunction with
database. The results showed that three fractions from G. arcuata, five fractions from G. edulis, and two fractions from G. spinosum have bioactive compounds against Aeromonas hydrophila, while only two fractions from G. arcuata and G. spinosum have bioactive compounds against Vibrio sp.
The MIC concentrations were around 0.625 μg/μL to 2.5 μg/μL. Several
chemical componens that had been identified were terpenes, phenolic,
steroid, and fatty acid compounds.
Analysis of Iodine in Fucus serratus Seaweed Bathwater and Its Potential for Uptake by the Body
Tarha Westby, Aodhmar Cadogan and Geraldine Duignan
Institute of Technology Sligo, Sligo, Ireland
Iodine,
found in seaweed, is a vital micronutrient for normal growth,
development and function. The levels of iodine in seaweed vary depending
on species, locality and season and can account for up to 1.2% of the
dry weight of some seaweed. The tradition of seaweed baths has been
revived recently in Ireland and it is suggested that there is a
considerable uptake of minerals by the body during such treatments.
Traditional seaweed baths are prepared using fresh water and seawater
along with the seaweed Fucus serratus. Simulated seaweed
bathwater samples were prepared and stored weekly over 12 months. These
were analysed using the classical spectrophotometric Sandell-Kolthoff
(SK) assay in order to establish the seasonal variation in iodine
levels. Results show that the concentration of total iodine ranges from
0.8 to 14 ppm. Results of pH analysis of the bathwater indicate an
acidic environment (pH 5.8). The acidity of the bathwater affects the
species of iodine present where iodine gas (I2) and iodate (IO3−) predominate over iodide (I−).
As approximately 90% of iodine present in the body is excreted in the
urine, analysis of the urinary iodine concentration (UIC) of bathers’
samples collected immediately prior to bathing and following bathing
over 32 h was carried out to establish if there is an increase in UIC as
a result of taking a seaweed bath. Almost all samples, from bathers and
controls, analysed by the SK assay demonstrate an increase in UIC
following a seaweed bath. The control subjects showed a spike in UIC
despite not immersing in the bathwater but sitting beside it inhaling
iodine gas only. This suggests that iodine gas contributes greatly to
the increase in UIC for all subjects as there was no possibility of
dermal absorption for the control.
Oxidative Stress Condition Induced by H2O2 Is Blunted by Fucus spiralis Fractions
Joana Silva 1, Susete Pinteus 1, Celso Alves 1, André Horta 1, Olivier Thomas 2 and Rui Pedrosa 1
1 MARE—Marine and Environmental Sciences Centre, Polytechnic Institute of Leiria, Peniche, Leiria, Portugal
2 Institut de Chimie de Nice, University of NiceSophia Antipolis, Nice, France
Oxidative
stress is one of the main causes for the development of diseases with
huge impact in world societies such as cardiovascular diseases,
neurodegenerative diseases, metabolic syndrome, cancer, etc. In line
with these, becomes of extremely importance to find new molecules with
capacity to inhibit, delay or prevent oxidative stress situations.
During the last decades marine organisms have demonstrated the ability
to produce molecules with high antioxidant properties, of which brown
algae have shown high potential. Fucus spiralis (spiral wrack)
is a brown algae (Phaeophyceae), living on the Atlantic coasts of Europe
and North America. Previous studies of our research group found high
antioxidant potential in methanolic extracts of Fucus spiralis,
collected on the Peniche coast. In order to go further to the
purification of the antioxidant compounds it was performed a Vacuum
Liquid Chromatography (VLC) with the following solvent system: 100% H2O (F1); 1:1 H2O:CH3OH (F2); 100% CH3OH (F3); 3:1 CH3OH: CH2Cl2 (F4) and 100% CH2Cl2 (F5) in a C18 column. All fractions (1 mg·mL−1;
24 h) were tested for anti-oxidative stress potential in Human breast
adenocarcinoma model (MCF-7 cells). The cytotoxicity was evaluated by
the MTT method in a presence or in absence of an induced oxidative
stress situation promoted by H2O2 (0.2 mM; 24 h).
Finally, in order to understand possible mechanisms of action of the
active compounds, the mitochondrial membrane potential (MMP) and the
caspase 9 activity were evaluated. The VLC fraction did not revealed
cytotoxicity. All fractions revealed capacity to revert an oxidative
stress condition by the following potency order F4 > F2 > F3 >
F5 > F1. Almost all the fractions reduced the cell’s membrane
depolarization and suggested anti-apoptotic mechanisms attesting the
protective effect against hydrogen peroxide toxicity. It is concluded
that Fucus spiralis fraction has high antioxidant activity able to
inhibit the MCF-7 oxidative stress condition induced by H2O2.
Bioprospecting Marine Microalgae, New Sources of Bioactive Compounds
Caterina Rodríguez de Vera 1,2, Guillermo Díaz Crespín 1,2, Antonio Hernández Daranas 1,3, José Javier Fernández 1,2, María Luisa Souto 1,2, Päivi Tammela 4, Heiko Rischer 5, Christian D. Muller 6,7 and Manuel Norte 1,2
1 University Institute of Bio-Organic
Chemistry “Antonio González”, Center for Biomedical Research of the
Canary Islands (CIBICAN), University of La Laguna, La Laguna, 38206
Tenerife, Spain
2 Departament of Organic Chemistry, Univesity of La Laguna, La Laguna, 38206 Tenerife, Spain
3 Departament of Chemical Engineering and Pharmaceutical Technology, University of La Laguna, La Laguna, 38206 Tenerife, Spain
4
Centre for Drug Research, Division of Pharmaceutical Biosciences,
Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland
5 VTT Technical Research Centre of Finland, Fi-02044 Espoo, Finland
6 Laboratoire d’Innovation Thérapeutique, Faculté de Pharmacie, Université de Strasbourg, 67401 Illkrich, France
7
Plateforme eBioCyt, Faculté de Pharmacie & Féderation
Translationelle de Médecine, Université de Strasbourg, Illkrich, France
Bioprospecting
consists on the exploration of biodiversity for new resources of social
and comercial value [1]. In this context, the oceans host an unparallel
biological and chemical diversity [2]. In particular, microalgae and
other phytoplanktonic species like dinoflagellates have contributed a
large number of unprecedent structures, some of which could become
therapeutically useful agents. Okadaic acid is an example of how
compounds from marine organisms may provide opportunities to obtain
valuable metabolites. This lipophilic polyether produced by Dynophysis and Prorocentrum genera,
is an inhibitor of protein phospatase 2A (PP2A) and its tumor promoting
and cytotoxic activities have been of a great interest in
pharmacological research at the molecular level [3]. Furthermore, it is a
novel tool for Alzheimer’s disease [4]. Herein, the results of a
bioprospecting process comprising 31 strains of marine microalgae
(including dinoflagellates, haptophytas, heterokontophytas and one
chlorophyta) are showed. The assays were targeted to provide information
on antimicrobial, antiproliferative, apoptotic and anti-inflammatory
potential of the studied extracts.
Acknowledgments
To
MINECO SAF2011-28883-C03-01, FP7-KBBE-2009-3-245137 (MAREX EU) and
FP7-REGPOT-2012-CT2012-316137-IMBRAIM projects. C.R. de Vera to MINECO
FPU program. To Santiago Fraga, IEO Vigo, for donating the strain. The
research group acknowledge the financing granted to ULL by Agencia
Canaria de Investigación, Innovación y Sociedad de la Información, being
85% cofinancied by the European Social Fund.
References
- Abida, H.; Ruchaud, S.; Rios, L.; Humeau, A.; Probert, I.; de Vargas, C.; Bach, S.; Bowler, C. Bioprospecting Marine Plankton. Mar. Drugs 2013, 11, 4594–4611.
- Costa, M.; García, M.; Costa-Rodrigues, J.; Costa, M.S.; Ribeiro, M.J.; Fernandes, M.H.; Barros, P.; Barreiro, A.; Vasconcelos, V.; Martins, R. Exploring Bioactive Properties of Marine Cyanobacteria Isolated from the Portuguese Coast: High Potential as a Source of Anticancer Compounds. Mar. Drugs 2014, 12, 98–114.
- Fernández, J.J.; Candenas, M.L.; Souto, M.L.; Trujillo, M.M.; Norte, M. Okadaic acid, a useful tool for studying cellular processes. Curr. Med. Chem. 2002, 9, 229–262.
- Kamat, P.K.; Rai, S.; Swarnkar, S.; Shukla, R.; Nath, C. Molecular and cellular mechanism of okadaic acid (OKA)-induced neurotoxicity: A novel tool for Alzheimer's disease therapeutic application. Mol. Neurobiol. 2014, 50, 852–865.
Anti-Diabetic Properties of Seaweed Extract Using the Adipocyte Cell Model 3T3-L1
Margrét Eva Ásgeirsdottir 1,2, Eva Kuttner 1, Hörður G. Kristinsson 1,2 and Guðmundur Óli Hreggviðsson 1
2 Matis ohf, Vinlandsleid 12, 113, Reykjavik, Iceland
2 Faculty of Food Science and Nutrition, University of Iceland, Eiríksgata 29, 101, Reykjavik, Iceland
Obesity,
where the body accumulates an excess amount of fatty tissue is one of
the major risk factors for type 2 diabetes. Adipocytes constitute a
major part of the fatty tissue and perform key endocrine functions, one
of them insulin sensitivity. Regulating adipogenesis through dietary
supplementation could be one way of alleviating complications of type 2
diabetes. Polyphenols have been shown to have anti-diabetic properties.
Extracts from seaweed, having a high polyphenol content, have revealed
antidiabetic and antioxidant properties. The aim of this study was to
further investigate the effects of different seaweed extracts on
differentiation (adipogenesis), influence on protein expression levels,
proliferation and apoptosis of adipocytes. Undifferentiated murine
adipocytes (3T3-L1 cell line) were exposed in different concentrations
to the seaweed extract and the several key protein isolated and used for
expression studies.
Paralytic Shellfish Poisoning Toxins from Mediterranean Alexandrium minutum and A. catenella: Toxin Profile and SXT Gene Content
Luciana Tartaglione 1, Carmela Dell’Aversano 1, Patrizia Ciminiello 1, Federico Perini 2, Samuela Cappellacci 2, Valentina Sparvoli 2, Silvia Casabianca 2, Michele Scardi 3, Mariagrazia Giacobbe 4 and Antonella Penna 2
1 Dipartimento di Farmacia, Università degli Studi di Napoli Federico II, Napoli, Italy
2 Dipartimento di Scienze Biomolecolari, Università di Urbino, Urbino, Italy
3 Dipartimento di Biologia, Università di Tor Vergata, Roma, Italy
4 Istituto per l’Ambiente Marino Costiero, CNR, Messina, Italy
Paralytic
shellfish poisoning (PSP) toxins are potent water-soluble neurotoxins
including the parent compound saxitoxin (STX) and a number of its
congeners. They are tetrahydropurine derivatives that can be subdivided
into three main groups according to substitution of the side chain:
carbamoyl-, N-sulfocarbamoyl-, and decarbamoyl-toxins. The
carbamoyl derivatives (STX, NEO and GTX1-4) are reported to be the most
potent. Due to their accumulation in filter feeding shellfish, PSP
toxins can move through the food chain inducing a toxic syndrome in
seafood consumers. Symptoms are neurological with rapid onset (30–60 min
from ingestion) and include paraesthesia, vertigo, numbness, tingling
of the face, tongue, and lip, ataxia, blocking of respiration and even
death. Due to the high risk posed to human health by PSP toxins, a
multidisciplinary integrated approach based on liquid chromatography
high resolution mass spectrometry (LC-HRMS and MS2) and qPCR-based assay has been used to depict the PSP toxin scenario in the Mediterranean Sea. As the sxtA and the sxtG genes are known as the starting genes of PSP toxin synthesis in dinoflagellates, different populations of the Mediterranean A. minutum
from NW Adriatic, Ionian, Tyrrhenian and Catalan Seas were grown in
culture and analyzed by qPCR in order to obtain the quantification of
these genes. In parallel, LC-HRMS2 analyses were performed on the A. minutum
cultured strains and revealed for all of them a toxin profile
consisting of only GTX1 and GTX4. Toxin production was in the fg/cell
range. Concomitantly with a massive natural bloom of A. minutum and A. catenella
that occurred in Spring 2014 along the Syracuse coasts (Sicily, Italy),
four seawater samples were collected and analyzed by LC-HRMS and MS2. The analyzed extracts were found to contain a variety of PSP toxins, namely STX, NEO, the gonyautoxins GTX1-4, the N-sulfocarbamoyl derivatives C1/C2, B1 and B2 and the decarbamoyl STX.
Towards the Search of Novel Bioactive Molecules from North Atlantic Ocean
Nipun Mahajan 1, Daria Firsova 1, Sylvia Soldatu 1, Candice Bromley 1, Ryan Young 1 and Bill Baker 1,2
1 National University of Ireland, Galway, Ireland
2 Center for Drug Discovery and Innovation, University of South Florida, FL, USA
The
marine microbial community has proven records of providing bioactive
molecules useful for the treatment of human diseases. Macro-organisms
and sediments from the coastal and deep sea region of North Atlantic
Ocean were collected to grow their associated microbes. Using a variety
of specialised media enabled the growth of hundreds of bacteria,
actinomycetes and fungi. The isolated pure colonies of bacteria were
cultured using broth media whilst fungal strains were cultured on
sterilised rice with and without stress factors such as genetic
modifiers. These cultured bacteria and fungi were subsequently extracted
with lipophilic solvent. The resultant extracts were submitted for high
throughput screening against ESKAPE pathogen related microbial
diseases, cancer and osteoarthritis, to identify potentially
biologically useful microbes. Upon identification of the hit
microorganism(s), large scale culturing of the microbes of interest will
be undertaken to get sufficient biomass for activity guided isolation
of hit molecule(s).
Dereplication Metabolomics, and Rational Approaches to Bioprospecting Oceancharcot: A Program Dedicated to the Screening of Marine Chemodiversity
Benoît Serive 1,2, Ronald J. Quinn 1 and Stéphane Bach 2
1 Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Australia
2 Kinase Inhibitor Specialized Screening Facility, USR3151 CNRS/UPMC, Station Biologique de Roscoff, Roscoff, France
Oceans
dominate the surface of our planet and they are the chest of a huge
biodiversity, resulting from an adaptive evolution to many ecological
niches. Amongst the 33 known phyla on earth, 21 are exclusively marine.
The aim of the OCEANCHArCoT project is to explore the chemodiversity
from marine organisms to find new chemical scaffolds able to inhibit
cellular targets. Amongst the latter, protein kinases are involved in
various human pathologies such as cancer and neurodegenerative diseases,
or can be targeted to alter the life cycle of various parasites. Some
protein kinases are also implicated in cellular events leading to
necroptosis. Necroptosis is of central pathological relevance in
neurodegenerative diseases, inflammatory diseases, viral and bacteria
infections, allograft rejection, ischemia-reperfusion injury, brain
injuries, atherosclerosis, Gaucher’s disease or in age-related macular
degeneration. We decided to focus on some key protein kinases of the
necrosome to guarantee a large potential of pharmacological
applications. The workflow was designed to make efficient the way
leading to the identification of new inhibitors by combining several
advantages:
- (i)Exploitation of a huge potential of chemodiversity: Nature Bank library (Eskitis Institute for Drug Discovery, Brisbane, Australia) and planktonic extracts from cultivatable strains (OCEANOMICs project, Station Biologique de Roscoff—CNRS, Roscoff, France).
- (ii)Using advanced technologies such as a high-throughput screening robot (BioCel 900, Agilent Technologies) and bioaffinity mass spectrometry (ESI-FTICR-MS, Bruker).
This program of Excellence is supported by a Marie Curie International Outgoing Fellowship grant from European Commission.
Accurate Dereplication of Bioactive Secondary Metabolites from Marine-Derived Fungi by UHPLC-DAD-QTOFMS and a MS/HRMS Library
Sara Kildgaard, Maria Mansson, Ina Dosen, Andreas Klitgaard, Jens C. Frisvad, Thomas O. Larsen and Kristian F. Nielsen
Department of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark
Microorganisms
from the marine environment are a promising source of new bioactive
compounds based on new chemical scaffolds. Although the majority of
known compounds originate from bacterial species, marine-derived fungal
strains have yielded a plethora of biologically active compounds [1,2].
The most common sources are isolates of Penicillium and Aspergillus.
Due to the cosmopolitan occurrence of many bioactive compounds, most
natural product extracts contain compounds that have previously been
characterized. In drug discovery, reliable and fast dereplication of
known compounds is therefore essential for identification of novel
bioactive compounds. This study presents an integrated approach using
ultra-high performance liquid chromatography-diode array
detection-quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOFMS)
with tandem high resolution MS (MS/HRMS) analysis to screen extracts
from bioactive marine-derived Aspergillus, Penicillium, and Emericellopsis
strains [1]. The chosen strains were selected from a screening
conducted as part of the PharmaSea project [3]. Dereplication of known
compounds, including small polyketides, non-ribosomal peptides, terpenes
and meroterpenoids in the bioactive extracts were done by searching the
MS/HRMS data against a newly conducted in-house MS/HRMS library of
~1300 compounds (10, 20, and 40 eV spectra) using the Agilent software
[3].
Acknowledgments
We
acknowledge support from EU for the FP7 PharmaSea project and from
Agilent for Thought Leader Award of new MS-instrumentation.
References
- Kildgaard, S.; Mansson, M.; Dosen, I.; Klitgaard, A.; Frisvad, J.C.; Larsen, T.O.; Nielsen, K.F. Accurate Dereplication of Bioactive Secondary Metabolites from Marine-Derived Fungi by UHPLC-DAD-QTOFMS and a MS/HRMS Library. Mar. Drugs 2014, 12, 3681–3705.
- Debbab, A.; Aly, A.H.; Lin, W.H.; Proksch, P. Bioactive Compounds from Marine Bacteria and Fungi. Microb. Biotechnol. 2010, 3, 544–563.
The Role of Marinlit as a Dereplication Tool: Biogeography
Serin Dabb 1, Helen Potter 1, Jeff White 1, John Blunt 2 and Murray Munro 2
1 Royal Society of Chemistry, Cambridge, UK
2 University of Canterbury, Christchurch, New Zealand
To
actively pursue bioactive compounds from natural sources data covering
five disparate sources are required—biogeography, taxonomy, access to
biodata as well as dereplication and characterisation tools. Fortunately
for those working in marine natural products one database, MarinLit
covers all these areas. There are three aspects of MarinLit that are not
found in any other database:
- biogeography;
- functional group recognition; and
- access to biodata.
These data are vital in the selection of collecting sites, dereplication and appropriate assays for compounds of interest. Biogeography
is described in this poster and the functional group recognition
aspects are described in an adjacent poster. Biogeography, the
description and understanding of the spatial patterns of biodiversity,
is an ecological tool seldom associated with natural products research.
But, information on biodiversity hotspots, species distribution and
latitudinal/longitudinal gradients can be vital to understand and
predict the distribution of marine natural products, lead to a better
understanding of spatial and temporal patterns in chemotaxonomic studies
of taxa of interest, and ultimately increase the success of natural
product discovery. The paucity of biogeographical information for marine
species has been addressed by MarinLit by the incorporation of
geographical/depth data in a searchable web-based format and is the only
natural product database able to provide and search biogeographical
data, or combine those data with other typical database features such as
substructure searching. Examples will be provided to demonstrate the
potential of biogeography as a tool to provide new insights into future
bioprospecting efforts.
The Role of Marinlit as a Dereplication Tool: Functional Group Recognition
Serin Dabb 1, Helen Potter 1, Jeff White 1, John Blunt 2 and Murray Munro 2
1 Royal Society of Chemistry, Cambridge, UK
2 University of Canterbury, Christchuch, New Zealand
To
actively pursue bioactive compounds from natural sources data covering
five disparate sources are required—biogeography, taxonomy, access to
biodata as well as dereplication and characterisation tools. Fortunately
for those working in marine natural products one database, MarinLit
covers all these areas. There are three aspects of MarinLit that are not
found in any other database:
- biogeography;
- functional group recognition; and
- access to biodata.
These data are vital in the selection of collecting sites, dereplication and appropriate assays for compounds of interest. Functional
group recognition is described in this poster and the biogeography
aspects are described in an adjacent poster. Functional group
recognition. This approach is very simple, but extraordinarily discriminating. 1H-NMR
spectra are information-rich and allow the ready recognition of a wide
variety of functional groups—methyl groups, acetal protons, a-protons in
peptides, carbinol, and olefinic protons and aromatic substitution
patterns. MarinLit uses a specifically developed algorithm that
recognizes a wide range of functional groups within a structure leading
to each entry in MarinLit being seeded with the numbers of these
functional groups observable in a 1H-NMR spectrum in
searchable fields. By simply counting the number and types of features
(methyl groups, aromatic substitution patterns, etc.) in a 1H-NMR
spectrum and searching MarinLit all compounds that match are
recognised. If there is no match the compound is new to marine natural
products. Recent advances in the detection algorithm allow for the
recognition of a wider range of aromatic and heteroaromatic systems.
MarinLit is the only database with functional group recognition and it
is possible to combine those data with other typical database features
such as substructure searching. Examples will be provided that
demonstrate the potential of functional group recognition as a
dereplication tool.
An Easy, Reproducible and Semi-Automated Protocol for Bioassay-Guided Fractionation of Marine Extracts
Genoveffa Nuzzo 1, Adele Cutignano 1, Elvira Luongo 1, Carmela Gallo 1, Giovanna Romano 2, Adrianna Ianora 2 and Angelo Fontana 1
1 CNR-ICB, Pozzuoli, Napoli, Italy
2 SZN, Napoli, Italy
Marine
biodiversity is a rich source of a great variety of novel chemical
entities some of which are endowed with pharmaceutical properties [1,2].
The discovery of these products relies on different approaches but the
most common entails bioassay-guided fractionation of marine extracts.
Several academic and industrial groups are familiar with high-throughput
screening (HTS) to assay a large number of potential chemical agents
against a chosen set of defined biological targets. The success of this
method requires the overcoming of several crucial points, as removing of
salts that led to overlooking bioactive components and emphasizing the
presence of minor compounds often masked in the raw material. Here we
present an easy, versatile and efficient fractionation procedure to
screen marine organism extracts based on Solid Phase Extraction (SPE).
The protocol relies on a polystyrene based adsorbent as solid support
and a stepwise organic solvent elution. The method has been validated on
three selected marine organisms, i.e., Dendrilla membranosa, Reniera sarai and Amphidinium carterae,
whose secondary metabolism has been previously investigated. The
results indicated that the main classes of primary and secondary
metabolites exhibited a predictable and reproducible distribution within
five fractions. By using an automated chromatographic platform, the
method has been miniaturized thus allowing to quickly handle several
samples at laboratory scale with significant saving of time and solvent.
Combined with routinely NMR and MS analyses, the platform produces a
rich data set over a relatively short space of time and supports the
development of a home-made library of natural products. In support of
this, we briefly report the application of our protocol to two discovery
programs for immuno-modulatory and anti-infective agents from sponges
and cultivable marine organisms.
References
- Blunt, J.W.; Copp. B.R.; Keyzers, R.A.; Munro, M.H.; Prinsep, M.R. Marine natural products. Nat. Prod. Rep. 2015, 32, 116–211.
- Newman D.J.; Giddings L.A. Natural products as leads to antitumor drugs. Phytochem. Rev. 2014, 13, 123–137.
A Time-Scale Metabolomics Study Reveals Biosynthetic Pathways in Marine-Sourced Penicillium Cultures
Catherine Roullier, Mathilde Peigné, Yves François Pouchus and Olivier Grovel
Faculté de Pharmacie, Université de Nantes, MMS, Nantes, France
The
field of research combining natural products chemistry and metabolomics
studies is currently growing. When trying to unravel cryptic
biosynthetic pathways from microorganisms, different methods are usually
performed such as targeted mutagenesis, changes in culture media, use
of different types of elicitors, as well as co-cultivation of organisms.
Most research in this field statistically compares the metabolic
profiles (either LC-MS or NMR or both) generated after a selected time
of cultivation under the different treatments applied to the
microorganisms in order to identify interesting compounds. Even though
it is well assumed that metabolism is a function of the development
stage of an organism and consequently changes over time, so far, few
studies have compared metabolic profiles through time. Then, it can be
assumed that such studies can reveal new metabolic intermediates in
biosynthetic pathways. In the work presented here, a time-scale
metabolomic study has been performed on different marine-derived Penicillium
strains, for which crude culture extracts were analyzed by
HPLC-DAD-HRMS each day for an 18-day period. Differences in terms of
final metabolites, intermediates and kinetics of production have been
highlighted for strains belonging to different Penicillium species.
The results of this study will be given with a particular focus on a
common biosynthetic pathway in all strains leading to griseofulvin, a
drug which is still on the market and regains interest in the field of
cancer.
13C NMR as a Metabolomic Tool for Identification of Individual Components of Algal Extracts: An Application to the Chemiodiversity of Laurencia microcladia from Corsica (Ajaccio Bay)
Sylvain Sutour, Ange Bighelli, Vincent Castola and Mathieu Paoli
University of Corsica, Equipe Chimie et Biomasse, Ajaccio, France
The genus Laurencia
is one of the most prolific algal sources of highly diverse secondary
metabolites exhibiting tremendous biological activity. In order to
realize a metabolomic study of extracts from Laurencia microcladia growing wild on the Corsican Coast, we applied the analytical method based on 13C-NMR developed in our laboratory. The recorded 13C
NMR spectra of pentane and ethyl acetate extracts followed by a
computer-aided analysis, allowed the identification 9 compounds
belonging to terpene, acetogenin and sterol families. 3(E)-laurenyne is the main compound, followed by 3(Z)-laurenyne,
cholesterol, obtusallene IA and a dibromochamigrene derivative. Among
the non polar compounds (pentane extract) we can identify four known
compounds bearing a laurane (laurene and dihydrolaurene) and a cuparane
(A-bromocuparene and A-isobromocuparene) skeleton. All the previously
identified compounds are described for the first time in extracts from Laurencia microcladia. Moreover, no compounds previously described from Laurencia microcladia and Laurencia obtusa
from Greece and Italy were identified from the Corsican extracts. As
suggested by F. Pietra, there is a close relationship between a species
and a chemical family more than individual compound. Thus, Laurencia microcladia from Corsica is different from Laurencia microcladia
from Greece which produces mainly sesquiterpenes bearing cuparane and
laurane skeleton. Its metabolomic profile is closer to those described
in Italy by the occurrence of C15 acetogenin represented by 3E-laurenyne as major component. However, a difference is still remaining between C15
acetogenins from extracts from Italy and from Corsica. Indeed, oxepanes
characterize extracts from Italy whereas seven members ring acetylenic
ethers characterize extracts from Corsica. 13C NMR as proved
to be an efficient metabolomic tool for identification of individual
components of algal extracts. The unambiguously identification of
stereoisomers exhibiting close or net enough differentiated mass spectra
like: 3E vs. 3Z-laurenyne and A-bromocuparene vs. A-isobromocuparene appear to be a strong advantage of this method.
Biodiversity, Anti-Trypanosomal Activity Screening, and Metabolomics Profiling of Actinomycetes Isolated from Mediterranean Sponges
Cheng Cheng 1, Lynsey MacIntyre 2, Usama Ramadan Abdelmohsen 1,4, Hannes Horn 1, Paraskevi N. Polymenakou 3, RuAngelie Edrada-Ebel 2 and Ute Hentschel 1
1 Department of Botany II, Julius-von-Sachs Institute for Biological Sciences, University of Würzburg, Würzburg, Germany
2 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK
3 Hellenic Centre for Marine Research, Institute of Marine Biology, Biotechnology and Aquaculture, Crete, Greece
4 Department of Pharmacognosy, Faculty of Pharmacy, Minia, Egypt
Marine
sponge–associated actinomycetes are considered as promising sources for
the discovery of novel biologically active compounds. In the present
study, a total of 64 actinomycetes were isolated from 12 different
marine sponge species that had been collected from offshore the islands
of Milos and Crete, Greece, eastern Mediterranean. The isolates were
affiliated to 23 genera representing 8 different suborders based on
nearly full length 16S rRNA gene sequencing. Four putatively novel
species belonging to genera Geodermatophilus, Microlunatus, Rhodococcus and Actinomycetospora
were identified based on a sequence similarity <98.5% to validly
described 16S rRNA gene sequences. Eight actinomycete isolates showed
bioactivities against Trypanosma brucei brucei TC221 with half maximal inhibitory concentration (IC50)
values <20 µg/mL. Thirty four isolates from the Milos collection and
12 isolates from the Crete collection were subjected to metabolomics
analysis using high resolution LC-MS and NMR for dereplication purposes.
Two isolates belong to the genera Streptomyces (SBT348) and Micromonospora
(SBT687) were prioritized based on their distinct chemistry profiles as
well as their anti-trypanosomal activities. These findings demonstrated
the feasibility and efficacy of utilizing metabolomics tools to
prioritize chemically unique strains from microorganism collections and
further highlight sponges as rich source for novel and bioactive
actinomycetes.
Affinity of Spongionella Isolated Compounds by Cyclophilins
Amparo Alfonso 1, Jon A Sánchez 1, Eva Alonso 1, Mostafa E. Rateb 2,3, Wael E. Houssen 2,4, Rainer Ebel 2, Marcel Jaspars 2 and Luis M Botana 1
1 Universidad de Santiago de Compostela, Lugo, Spain
2 Marine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Aberdeen, Scotland, UK
3 Beni-Suef University, Beni-Suef, Egypt
4 Institute of Medical Sciences, University of Aberdeen, Aberdeen, Scotland, UK
Gracilins H, A, L and Tetrahydroaplysulphurin-1 are bioactive diterpene compounds isolated from the marine sponge Spongionella
able to protect mitochondria from oxidative stress. Cyclophilins (Cyp)
are a group of proteins belong to the immunophilins family with
different localization, Cyp D in the mitochondrial matrix and Cyp A in
the cytosol. The activation of these molecules has important
implications in diseases such us alzheimer, cancer and inflammatory
alterations. Cyclosporine A (CsA) is a cyclic peptide useful as
immunosuppressant agent with high affinity by different immunophilins.
Since the effects of Spongionella diterpenes over mitochondria and
calcium fluxes were similar to the effects of CsA, the aim of this work
was to study the association between all these compounds and
cyclophilins by using a biosensor. This instrument measures biomolecular
interactions in real time and allows the calculation of kinetic
constants. Cyp A and D were immobilized onto the sensor surface at pH
4.5 or 6. Over these immobilized ligands the association with CsA and
Spongionella compounds was studied. When CsA was used typical
association curves were obtained at any condition studied, showing
higher affinity when the immobilization was done at pH 6. In the
presence of Spongionella compounds different results were obtained. When
cyclophilins were immobilized at pH 6 no interactions with Spongionella
derivatives were observed. With Cyp D immobilized at pH 4.5, typical
association curves were obtained when Gracilin L or
Tetrahydroaplysulphurin-1 were added, while no interactions were
observed with Gracilin A and H. When Cyp A was immobilized at pH 4.5,
good association profiles were obtained in the presence of Gracilin L
and H, although lower than CsA. However Gracilin A and
Tetrahydroaplysulphurin-1 showed higher affinity by Cyp A than CsA.
These results suggest that Spongionella diterpenes are novel
cyclophilins inhibitors with affinity comparable to that of
immunosuppressive CsA.
From Chemical Profile to Biological Profile, Brominated Metabolites from the Bryozoan Amathia tortuosa Produce Phenotypic Effects on Parkinson’s Disease Cells
Yousef Dashti, Marie-Laure Vial, Stephen A. Wood, George D. Mellick, Catherine Roullier and Ronald J Quinn
Eskitis Institute for Drug Discovery, Griffith University, Brisbane, QLD, Australia
NMR metabolite fingerprints of fractions from an extract of the bryozoan Amathia tortuosa
allowed identification of three known and evidence of a new
tribrominated metabolite. The new compound, kororamide B, was purified
through NMR-guided isolation and the structure assigned by mass and
detailed NMR analysis. The absolute configuration of the new compound
was established by comparison of the experimental and calculated
electronic circular dichroism spectra. The compounds displayed effects
on early endosomes when profiled on human olfactory neurosphere-derived
cells (hONS) derived from a Parkinson’s disease patient using a
multidimensional phenotypic assay.
Scottish Seaweed Species as a Source of Bioactive Natural Products
Kenneth Boyd and Robbie Mutton
University of the HIghlands and Islands, Thurso, UK
In
the 1970’s marine natural products research was largely focused on
cataloguing novel metabolites from red and brown algae. However interest
in macroalgae was quickly overtaken by studies on other phyla,
especially sponges, tunicates and latterly microorganisms. The movement
away from studying macroalgae coincided with the development of
screening programmes aimed at identifying compounds with promising
pharmacological properties. As a result the pharmacological potential of
macroalgal metabolites has been largely unexplored, leading to them
being described as one of the forgotten phyla in marine natural products
chemistry. Macroalgae are ubiquitous in the majority of near shore
habitats and therefore represent an easily accessible and potentially
valuable resource to the natural products chemist. Here we examine
marine algae from Caithness (Scotland) as a source of bioactive natural
metabolites. A survey of 18 sites revealed a wide diversity of seaweed
species in the area, with 22 green, 43 brown and 69 red algal species
identified. From these, metabolites have previously been reported from
only 20 (15%) with no reports of natural products from the remaining 114
(85%). Polar and non-polar extracts of 13 species of algae were
prepared and subjected to a battery of assays including antioxidant,
antimicrobial, anti-inflammatory and cyctotoxicity. From the results
57.6% of the polar extracts showed activity in at least one of the
bioassays with 84.6% of the non-polar extracts showing activity. A
series of meroterpenes from H. siliquosa exhibited potent antioxidant
activity. This demonstrates the potential of regional collection of
macroalgae as a source of bioactive metabolites.
Biosynthesis of Marine Natural Products in Microbes
Rational Design of Patgmac, the Macrocyclase from Patellamide Pathway, to Accelerate the Production of Cyclic Peptides
Fabio Tamaki 1,2, Wael Houssen 1,2 and Marcel Jaspars 1
1 Marine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Meston Walk, Aberdeen AB24 3UE, UK
2 Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK
The
biosynthesis of the ribosomally-synthesised and post-translationally
modified patellamides involves enzymatic processing of a precursor
linear peptide that contains a leader sequence for recognition by the
heterocyclase PatD, a core sequence to be processed and cleavage signals
for the protease PatA and the macrocyclase PatGmac. PatGmac
is a very slow enzyme with a turnover rate of one molecule/day. It can
process core sequences containing 6–11 amino acids of which the last
residue is either a proline or a heterocyclised amino acid. PatGmac
helix-loop-helix insertion plays a crucial role for macrocyclisation
since its deletion resulted in enzymes presenting only proteolytic
acticity but no macrocyclisation activity [1]. We used a combination of
bioinformatics and structural data to identify positions that can be
varied to improve PatGmac kinetics and to eliminate the requirement for proline or heterocycle in the core sequence. Co-variation analyses using PatGmac
Protein Family Statistic [2] yielded, amongst others, five co-variant
positions near the active site residues as targets for introducing
variability aiming speed up macrocyclase enzymes. Moreover, three
additional positions near the subsite that houses the substrate
heterocyclised residue (PDB:4AKT) [1], will be further targeted for
variability introduction aiming new enzymes with no need for
heterocyclised amino acids.
References
- Koehnke, J.; Bent, A.; Houssen, W.E.; Zollman, D.; Morawitz, F.; Shirran, S.; Vendome, J.; Nneoyiegbe, A.F.; Trembleau, L.; Botting, C.H.; et al. The mechanism of patellamide macrocyclization revealed by the characterization of the PatG macrocyclase domain. Nat. Struct. Mol. Biol. 2012, 19, 767–772, doi:10.1038/nsmb.2340.
- Bleicher, L.; Lemke, N.; Garratt, R.C. Using Amino Acid Correlation and Community Detection Algorithms to Identify Functional Determinants in Protein Families. PLoS ONE 2011, 6, e27786, doi:10.1371/journal.pone.0027786.
The in Vitro Use of Cyanobactin Prenylases in the Preparation of Bioactive Modified Cyclic Peptides
Luca Dalponte 1,2, Wael Houssen 1,2, David Fewer 3, Christian Umeobika 1, Laurent Trembleau 1 and Marcel Jaspars 1
1 Marine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Aberdeen, UK
2 Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK
3
Microbiology and Biotechnology Division, Department of Food and
Environmental Sciences, University of Helsinki, Helsinki, Finland
In
the last decade, the interest in macrocycles and constrained cyclic
peptides has grown exponentially due to their high resistance to
enzymatic digestion and high receptor binding activity when compared to
their linear counterparts. However, a main challenge to develop these
compounds as therapeutics is the limited ability for many compounds to
cross membrane barriers. Prenylation, a post-translational modification
that involves addition of a prenyl group to amino acid residues, could
increase the lipophilicity of the cyclic product and thus its ability to
cross cellular membranes. Cyanobactins include many examples of
prenylated cyclic peptides such as trunkamides from Prochloron spp. and aestuaramides from Lyngbya aestuarii.
Our group has identified the structure of PatF prenylase from
Prochloron. However PatF is a non-functional prenylase which is in
accordance with the fact that none of the patellamides is prenylated.
More recently, Schmidt group studied LynF prenylase that is capable of
processing oxygen containing amino acids such as Ser, Thr and Tyr. In
tyrosine, the process is followed by spontaneous nonenzymatic Claisen
rearrangement generating the C-prenylated derivative. The aim of this
project is to study homologues of LynF, in order to identify enzymes
that can process other amino acids, e.g., tryptophan and identifying the
key structural features responsible for substrate specificity.
Tamd: An α-Oxoamine Synthase Enzyme Involved in the Biosynthesis of the Marine Natural Product Tambjamine YP1
Piera Marchetti 1, Daynea Wallock-Richards 1, Van Kelly 1, Stephen McMahon 2, James H. Naismith 2 and Dominic Campopiano 1
1 University of Edinburgh, Edinburgh, UK
2 University of St. Andrews, St. Andrews, Fife, UK
Tambjamine YP1 is a natural product produced by the marine microorganism Pseudoalteromonas tunicata, which colonises the surface of the sea lettuce Ulva australis
and prevents biofouling. The tambjamines are a family of bipyrrolic
natural products with cytotoxic and antimicrobial activity that also act
as anion transporters. The gene cluster encoded in the P. tunicata genome for tambjamine YP1 production was identified by Kjelleberg et al. and contains 19 genes in one operon. The TamD gene is predicted to encode an unusual two domain enzyme that is composed of an N-terminal acyl-carrier protein (ACP) fused to a C-terminal SerT domain which is a member of the α-oxoamine synthase (AOS) family. We hypothesise that TamD condenses l-serine
with an ACP-bound substrate using a pyridoxal 5′-phosphate (PLP)
dependent Claisen-like decarboxylation reaction. The TamD product would
subsequently cyclise to form the bipyrrole moiety of tambjamine YP1. Not
only are these enzymes interesting for the production of tambjamines
for clinical studies but pyrrole structures are also important motifs in
many other drugs [3]. The full length TamD enzyme has been
heterologously expressed in Escherichia coli as well as the
separate ACP and SerT domains. Loading of the ACP and binding of the
substrates to the AOS domain are being investigated by mass spectrometry
and UV/Visible spectroscopy. Efforts to obtain a crystal structure of
the enzyme are underway and will hopefully shed light on the substrate
binding and mechanism of action of the enzyme. In the future the enzyme
kinetics will be probed using synthetic substrates and other recombinant
enzymes from the tambjamine YP1 biosynthetic pathway.
Phenalenones: Insight into the Biosynthesis of Polyketides from the Marine Alga-Derived Fungus Coniothyrium cereale
Mamona Nazir 1, Fayrouz El Maddah 1, Stefan Kehraus 1, Ekaterina Egereva 1, Jörn Piel 2, Alexander O. Brachmann 2 and Gabriele M. König 1
1 University of Bonn, Institute for Pharmaceutical Biology Nussallee 6, 53115 Bonn, Germany
2 Institute for Microbiology, ETH Zürich, Wolfgang Pauli Strasse 10, 8093 Zürich, Switzerland
The
marine-derived fungus Coniothyrium cereale was isolated from the Baltic
sea alga Enteromorpha sp., is a profile producer of phenalenones. Upon
cultivation on solid marine medium, it produced an impressive range of
polyketides with unique structures and remarkable biological activities
[1–3]. To establish the biosynthetic precursors of the polyketides
(−)-cereolactam and (−)-cereoaldomine, which inhibited Human Leukocyte
Elastase (HLE) with an IC50 value of 9.28 and 3.01 µM respectively; C. cereale
was cultivated with 13C-labeled precursors. We successfully elucidated
the biosynthetic origins of the carbon skeleton of (−)-cereolactam and
trypethelone by intensive 13C NMR techniques. Fractions containing the
(−)-cereoaldomine were analysed by UPLC-MS. MS spectra showed signals
indicating the presence of seven acetate units, five for the polyketide
part and two for the terpene unit (M + 1 to M + 7). According to our
putative biosynthetic pathway, respective metabolites originate from
heptaketide precursors of which some carbons are lost by oxidative
cleavage. In the same way we established the biosynthetic building
blocks of the antimicrobial polyketides coniosclerodin and cereolactone.
References
- Elsebai, M.F.; Nazir, M.; Kehraus, S.; Egereva, E.; Ioset, K.N.; Marcourt, L.; Jeannerat, D.; Gütschow, M.; Wolfender, J.L.; König, G.M. Polyketide Skeletons from the Marine Alga-Derived Fungus Coniothyrium cereale. Eur. J. Org. Chem. 2012, 2012, 6197–6203.
- Elsebai, M.F.; Kehraus, S.; Lindequist, U.; Sasse, F.; Shaaban, S.; Gütschow, M.; Josten, M.; Sahl, H.G.; König, G.M. Antimicrobial phenalenone derivatives from the marine-derived fungus Coniothyrium cereale. Org. Biomol. Chem. 2011, 9, 802–808.
- Elsebai, M.F.; Kehraus, S.; Mohamed, I.E.; Schnakenburg, G.; Sasse, F.; Shaaban, S.; Gütschow, M.; König, G.M. HLE-Inhibitory Alkaloids with a Polyketide Skeleton from the Marine-Derived Fungus Coniothyrium cereal. J. Nat. Prod. 2011, 74, 2282–2285.
New Amphidinols from the Dinoflagellate Amphidinium carterae Collected at Fusaro Lake (Naples): Structural Characterization and Biosynthesis
Adele Cutignano, Genoveffa Nuzzo, Angela Sardo and Angelo Fontana
CNR-ICB, Pozzuoli, Napoli, Italy
Amphidinols are a family of antifungal and hemolytic polyhydroxy-polyene polyketides typical of dinoflagellates of the genus Amphidinium [1]. The first member of the series was isolated from Amphidinium klebsii in
1991 [2] and a few congeners have been reported so far [3,4]. In the
frame of our ongoing research on bioactive natural products from marine
protists, we started assembling a collection of marine dinoflagellates
from the gulf of Naples. An epiphytic strain was isolated from the brown
macroalga Dictyota dichotoma collected in the brackish waters of Fusaro Lake (Naples) and taxonomically identified as Amphidinium carterae.
From the methanolic extract of the microalgal pellet we characterized
two new amphidinol analogs, named amphidinol-20 (AM20) and its 7-sulfate
derivative (amphidinol-21, AM21). Along with the identification of
these molecules and the evaluation of the antifungal activity of AM20,
here we report the elucidation of their biosynthesis by NMR studies on
the 13C-labelled derivatives after experiments with 1-13C acetate, 2-13C
acetate, 1,2-13C acetate and 1-13C glycolate.
References
- Kobayashi, J.; Kubota, T. Comprehensive Natural Products II Chemistry and Biology; Mander, L., Lui, H.-W., Eds.; Elsevier: Oxford, UK, 2010; Volume 2, pp. 263–325.
- Satake M.; Murata, M.; Yasumoto, T.; Fujita, T.; Naoki, H.J. Amphidinol, a polyhydroxypolyene antifungal agent with an unprecedented structure, from a marine dinoflagellate, Amphidinium klebsii. J. Am. Chem. Soc. 1991, 113, 9859–9861.
- Espiritu, R.A.; Matsumori, N.; Tsuda, M.; Murata, M. Direct and Stereospecific Interaction of Amphidinol 3 with Sterol in Lipid Bilayers. Biochemistry 2014, 53, 3287–3293.
- Nuzzo, G.; Cutignano, A.; Sardo, A.; Fontana, A. Antifungal amphidinol 18 and its 7-sulfate derivative from the marine dinoflagellate Amphidinium carterae. J. Nat. Prod. 2014, 77, 1524–1527.
Citrinin and Other Polyketides from Penicillium citrinum, an Endophyte from the Marine Red Alga Dichotomaria marginata
Teresinha Andrade 1,2, Luciano Puzer 3, Angela Araujo 2, Marcel Jaspars 4 and Dulce Silva 2
1 IFPI, Teresina, Brazil
2 Inst Chem. UNESP, Araraquara, Brazil
3 UFABC, Sao Paulo, Brazil
4 University of Aberdeen, Aberdeen, UK
Marine
organisms may host a variety of endophytic fungi, which induce or
enhance production of secondary metabolites associated to important
features in adaptation, defense against predators, and represent an
important source for bioprospection. Dichotomaria marginata is a marine red alga collected in the Brazilian southeast coast which afforded a fungal strain identified as Penicillium citrinum.
Its crude extract afforded citrinin and three derivatives: dicitrinin
A, decarboxydihydrocitrinone, and esclerotinin A, along with one
xanthone, one anthraquinone and other polyketides, by chromatographic
fractionation and purification by HPLC. The secondary metabolites
identification was carried out mainly by ESIMS, NMR analyses and search
in Antimarin2011®, MarinLit® and ChemSpider®
databanks. In addition, their inhibitory activities towards kallikreins
were evaluated. Kallikreins are serine proteases comprising a group of
15 peptidases, which may be expressed in several organs, are involved in
cell signaling and constitute important tumor biomarkers. KLK3 and KLK2
are prostate cancer markers, whereas KLK1 is expressed in kidneys,
intestine and salivary glands, KLK7 is expressed in esophagus, kidneys
and liver; and KLK5 is expressed in breast, CNS, prostate and trachea.
Best results were observed for citrinin, which inhibited KLK1, KLK3,
KLK5 and KLK7 with IC50 values of 1.27 mM, 9.57 mM, 6.60 mM and 2.51 mM,
respectively. Citrinin inhibitory activity was also evaluated towards
trypsin and papain with IC50 values of 0.16 mM and 0.38 mM,
respectively. Such results corroborate the huge chemodiversity of
endophytic marine fungi secondary metabolites and may represent an
important contribution for the sustainable exploration of marine
biodiversity in the search for bioactive compounds.
Antifouling Thielavins from the Marine-Derived Fungus Thielavia sp.
Zhuang Han 1,3, Ying Xu 2,3 and Pei-Yuan Qian 3
1 Sanya Institute of Deep-sea Science and Engneering, Chinese Academy of Sciences, Sanya, China
2 Division of Life Science, College of Life Science, Shenzhen University, Shenzhen, China
3 The Hong Kong University of Science and Technology, Hong Kong, China
Marine
natural products are regarded as a potential source of non-toxic
antifoulants that can effectively prevent larval settlement of fouling
organisms on man-made marine surfaces. In this study, we used
bioassay-guided isolation procedures to purify and characterize 14 new
depsides, thielavin U–Z (1–6) and thielavin Z1–Z8 (8–14), together with four known compounds, thielavin A, H, J, and K (15–18), from the EtOAc extract of the marine-derived fungal strain Thielavia
sp. UST030930-004. All of the compounds were evaluated with respect to
their antifouling potential against cyprids of the barnacle Balanus amphitrite, and the results showed that compounds 4–6 and 9–16 were active (EC50
values ranging from 2.95 ± 0.59 to 69.19 ± 9.51 μM, respectively). The
inhibitive effects of some active compounds were reversible. This is the
first report to describe antifouling activities of thielavins against
barnacle cyprids.
Bioactive Compounds from Five Marine-Derived Fungal Strains of the South China Sea
Shu-Hua Qi, Xiao-Yong Zhang, Xin-Ya Xu, Xu-Hua Nong and Jie Wang
South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China
It
is now well known that diverse fungal community is abundant in marine
environments, and marine fungi have received considerable attentions as
important sources for new drug discovery. In order to obtain new
bioactive compounds from marine microorganisms, we isolated culturable
fungi from different deep-sea sediment cores and coral samples from the
South China Sea. Combined with chemical analysis by HPLC and LC-MS, and
various bioassay tests, such as antibacterial, antifungal, antifouling
and cytotoxic activity, some bioactive fungal strains were chosen for
further study of their bioactive secondary metabolites. Recently, we
have obtained more than 30 new compounds with obvious antifouling,
cytotoxic, antioxidant or enzyme inhibiting activity from five
marine-derived fungal strains including Penicillium sp., Aspergillus sp., Xylariaceae sp., Engyodontium sp., Trichobotrys
sp. For several antifouling compounds, their antifouling activities
were further evaluated in field test. Two new chromones showed
significant cytotoxicity against several carcinoma cell lines, their
structure-activity relationships were discussed, and their mechanisms of
antitumor were probed. Three compounds with strong inhibiting activity
towards acetylcholinesterase with IC50 < 0.1 μM. And several
compounds showed stronger antioxidant activity than Vitamin C.
Searching of Non Ribosomal Peptides Anti Vibrio from Collection of Indonesan Marine Actinobateria
Noer Kasanah, Triyanto Triyanto, Drajad Seto, Bagash Kurniadi, Muhammad Arrahman and Wisnu Adisusila
Universitas Gadjah Mada, Yogyakarta, Indonesia
Vibrio spp. are a Gram-negative bacteria that dominate the aquatic habitat. Infection by Vibrio spp. can occur in humans through contaminated seafood, open wound and can cause cholera and vibriosis. V. parahaemolyticus causes a diarrhea and V. vulnificus infection can be fatal in immunocompromised patients. Infection by Vibrio
spp. also one of major problem in fish and shrimp aquaculture. The
choice of antibiotic for therapy in humans are limited as well as
antibiotic use in aquaculture is restricted. Therefore we need a new
anti-infective compounds to control of Vibrio spp. infections
in human and aquaculture. The objective of this research was to discover
new bioactive compounds as Antivibrio based on selection of potential
marine Actinobacteria. Collection of marine Actinobacteria collected
from Tulamben, Indonesia were subjected for screening based on the
presence of nrps gene and bioactivity against Vibrio sp.
Detection of nrps gene was done by PCR and bioactivity was examined by
96 well format and bioautography. We were able to select potential
marine Actinobacteria with desired activity and target gene for further
structure determination.
Keywords: Indonesia; actinobacteria; non ribosomal peptide; Vibrio
Anti-Cancer and Apoptosis-Inducing Activities of Streptomyces A16-1 an Isolate from Coastal Soil in the East Gulf of Thailand
Rattanaporn Srivibool 1 and Chantarawan Saengkhae 2
1 Institute of Marine Science, Burapha University, Chonburi, Thailand
2 Faculty of Allied Health Sciences, Burapha University, Chonburi, Thailand
Marine
microorganisms have been investigated for decades for novel natural
anti-cancer products that are hard to formulate by traditional
synthesis. Streptomyces are a prolific source of secondary
metabolites used in clinical cancer therapy such as daunomycin,
doxorubicin, bleomycin. This study Streptomyces A16-1 was
isolated from coastal soil in Chonburi, Thailand. Cytotoxic activity and
apoptotic mechanisms were investigated with partial purification of
crude red pigment of the strain. The fractionates were applied to human
nasopharynx cell lines (KB cells) and peripheral blood mononuclear cells
(PBMCs). Percent cell viability was assessed by MTT assay. The
apoptotic effects were evaluated by DAPI nuclear staining, agarose gel
electrophoresis, mitochondria staining and Caspase-3 activity. Of 21
fractions, fr7-9, fr10-12, and fr13-16 displayed strong inhibitory
effects against KB cells in a dose-dependent manner but less effective
against PBMCs. Fraction fr7-9 showed the greatest effect with IC50
values of 0.04 ± 0.005, whereas fr10-12, fr13-16 and doxorubicin were,
0.20 ± 0.02, 0.55 ± 0.05 and 1.35 ± 0.23 μg/mL, respectively.
Morphological observations showed cell shrinkage, irregular in shape
with cytoplasmic granules. Molecular mechanisms of cell death were
associated with mitochondrial transmembrane depolarization, chromatin
condensation and DNA fragmentation as well as sub G1 fraction of cell
cycles. Furthermore, the induction of apoptosis in KB cells was mediated
by activated caspase-3 which was significantly diminished in a
caspase-3 inhibitor. By 16S rRNA gene sequence analysis and BLAST
matching from GenBank database revealed Streptomyces A16-1 was 99.101% similarity to Streptomyces indiaensis.
On chromatograms there were at least eight components in fr7-9, the
strong cytotoxic activity and cell apoptosis might cause by a
synergistic effect of some or all components while little cytotoxic
effect on PBMCs was found which was a good sign. Streptomyces A16-1
might have many promising active compounds for cancer chemotherapy.
Further efforts to identify the structure and to explore the therapeutic
strategy are necessary.
Biological and Chemical Analysis of Bacteria Associated with Common Marine Sponges from Singapore
Deepak Kumar Gupta, Rownak Nazia and Lik Tong Tan
National Institute of Education, Singapore, Singapore
Marine
invertebrates, such as sponges and corals, are hosts to a myriad of
microbes and they represent potential sources of bioactive natural
products. In this study, we present biological and chemical data of 24
marine microbial strains isolated from three common marine sponges, Gelliodes fibulata, Clathria reinwardti, and Xestospongia testudinaria.
Using culture-dependent method, these marine microbes were isolated and
cultured using different marine media, namely M1, M2, and M3. The
organic extracts of the isolated marine microbes were screened for
cytotoxicity property (MOLT-4) and antibacterial activity based on Escherichia coli and Bacillus cereus.
The organic extracts from 12 marine microbial strains were found to
display significant anticancer (MOLT cell line) property as well as
antibacterial activity against Methicillin-Resistant Staphylococcus aureus (MRSA) BAA40, Escherichia coli EC598, Escherichia faecalis V583, Pseudomonas aeruginosa, Burkholderia cenocepacia WT-Hill, and Acinetobacter baumannii.
The phylogenetic data based on 16S rRNA genes of selective marine
bacterial strains that showed significant biological activities is also
presented. In addition, preliminary chemical data based on LCMS and
1D-NMR spectroscopy of bioactive bacterial-derived extracts/fractions
indicated the presence of unique secondary metabolites. This study
revealed the biotechnological applications of local marine
invertebrate-associated microbes in drug discovery and development
efforts.
Sphaerococcus coronopifolius Associated Bacteria: A New Source of Antimicrobial and Antitumor Compounds
André Horta, Nádia Fino, Celso Alves, Susete Pinteus, Joana Silva, João Francisco, Américo Rodrigues and Rui Pedrosa
MARE—Marine and Environmental Sciences Centre, Polytechnic Institute of Leiria, Peniche, Leiria, Portugal
The
increase of resistant tumor cells and emergence of antibiotic-resistant
bacteria and the need for novel antimicrobial and antitumor compounds
stress the search for new bioactive substances. The aim of this study
was the isolation and identification of associated bacteria from Sphaerococcus coronopifolius and
the evaluation of the antitumor and antimicrobial activities of the
extracts obtained from the isolated strains. The identification of
associated bacteria was determined by 16S rRNA gene sequencing. Bacteria
extracts were obtained with methanol and dichloromethane (1:1)
extraction. The antimicrobial activity of bacteria extracts was
evaluated against seven microorganisms: Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Bacillus subtilis (ATCC 6633), Salmonella enteritidis (ATCC 13076), Staphylococcus aureus (ATCC 25923), Saccharomyces cerevisiae (ATCC 9763) and Candida albicans
(ATCC 10231). The cell viability and the cell proliferation studies
were performed on human breast adenocarcinoma cell line (MCF-7 cells)
and a human hepatocellular cancer (HepG-2) according to MTT method. A
total of 28 Sphaerococcus coronopifolius associated bacteria were isolated and 9 were identified as Vibrio sp. (32.14%), Pseudoalteromonas sp. (17.86%), Halomonas sp. (10.71%), Shewanella sp. (17.86%) and Bacillus sp. (7.14%). Four (14.29%) of the 28 Sphaerococcus coronopifolius associated
bacteria presented less than a 90% Basic Local Alignment Search Tool
(BLAST) match, and could be new. In antimicrobial assays, four different
strains (SP2, SP4, SP7 and SP36) inhibited the Bacillus subtilis and Staphylococcus aureus growth. The highest antimicrobial activity against Bacillus subtilis was exhibited by SP7 with an IC50 12.99 µg·mL−1. The SP36 bacteria extract had a highest antibacterial activity against Staphylococcus aureus with an IC50 126.00 µg·mL−1. In antitumor tests, SP16 bacteria extract inhibited more than 50% on MCF-7 cell proliferation. In conclusion, the Sphaerococcus coronopifolius associated bacteria can be used as source of new marine natural compounds with high antibacterial and antitumoral activity.
Characterization and Photoprotector Activity of Fungal Pigment Isolated from Indonesian Coastal Plant Sarang Semut (Hydnophytum formicarum)
Kustiariyah Tarman 1,2, Mada Triandala Sibero1 1 and Novriyandi Hanif 3
1 Department of Aquatic Products
Technology, Faculty of Fisheries and Marine Sciences, Bogor Agricultural
University, Jl. Agathis 1, Kampus IPB Darmaga, Bogor, Indonesia
2
Center for Coastal and Marine Resources Studies, Bogor Agricultural
University, Jl. Raya Pajajaran 1, Kampus IPB Baranangsiang, Bogor,
Indonesia
3 Department of Chemistry,
Faculty of Mathematic and Natural Sciences, Bogor Agricultural
University, Jl. Lingkar Akademik, Kampus IPB Darmaga, Bogor, Indonesia
Endophytic fungus RS3 isolated from epiphytic plant sarang semut (Hydnophytum formicarum)
produced extracellular black pigment as a secondary metabolite. The
research aimed to extract melanin pigment from RS3, characterize and
analyze SPF activity of the pigment. This research consisted of several
steps including determination of precipitation solvent, extraction,
qualitative analysis and melanin characterization, SPF analysis, and
toxicity analysis using Brine Shrimp Lethality Test (BSLT) method. The
result showed that the pigment could be extracted in acid solvent with
pH 2.5 and showed positive in melanin qualitative analysis. According to
FTIR and UV-Vis analyses, pigment from RS3 was proposed to be eumelanin
possessing UV-Vis spectrum at UV-A (367.8 nm and 350.0 nm), UV-B (317.2
nm) and UV-C (271.6 nm; 266.8 nm; 264.0 nm; 260.8 nm; 223.6 nm). It has
also several functional groups such as hydroxy, aromatic, phenol, and
amine. The level of toxicity was 557.95 µg/mL. The fotoprotector value
using SPF method was 11.33.
Keywords: ant plant; endophytic fungi; eumelanin; SPF
New Hexacyclic Polyketides from a Marine Streptomyces
José Ma Sánchez López, Francisco Romero Millán, Marta Martínez Insua and Antonio Fernández Medarde
Instituto Biomar, S. A, Leon, Spain
The
search for new compounds with potential biological activity remains the
main objective of natural products development. During the last 40
years, marine organisms have proven to be a rich source of metabolites
and have been able to provide novel lead compounds for the development
of new pharmaceutical agents [1]. The protein kinases include a large
number of family members, which play a central role in regulating a wide
variety of cellular function. In the course of a screening program for
new kinase inhibitors, five hexacyclic poliketides were identified, from
the mycelium of a Streptomyces sp., isolated from an
unidentified alga collected at the Spanish’s Bay of Biscay Coast
(Cantabrian Sea). Three of the compounds are new natural products, the
other two are previously reported compounds [2,3]. Details of the
producer microorganism, isolation and spectroscopic data leading to the
structure elucidation of the compounds, as well as its biological
activities will be presented.
References
- Blunt, J.W.; Copp, B.R.; Keyzers, R.A.; Munro, M.H.G.; Prinsep, M.R. Marine natural products. Nat. Prod. Rep. 2013, 30, 237–323.
- Herath, K.B.; Jayasuriya, H.; Guan, Z.; Schulman, M.; Ruby, C.; Sharma, N.; MacNaul, K.; Menke, J.G.; Kodali, S.; Galgoci, A.; et al. Anthrabenzoxocinones from Streptomyces sp. as Liver X receptor ligands and antibacterial agents. J. Nat. Prod. 2005, 68, 1437–1440.
- Kojiri, K.; Nakajima, S.; Fuse, A.; Suzuki, H.; Suda, H. BE-24566B, a new antibiotic produced by Streptomyces violaceusniger. J. Antibiot. 1995, 48, 1506–1508.
Deep Sea and Polar Research
Identification of Bioactivities and Bioactive Compound Structure Determination from Deep-Sea Sponge Associated Microbes
Stephen A Jackson 1, Jonathan Kennedy 1, Jioji Tabudravu 2, Marcel Jaspers 2, Fergal O’Gara 2,3 and Alan D. W Dobson 1
1 School of Microbiology, University College Cork, Cork, Ireland
2 Marine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Aberdeen, UK
3 Biomerit Research Centre, University College Cork, Cork, Ireland
Marine
sponges (Porifera) host extensive and diverse populations of symbiotic
microbes with sponges and their symbionts being the source of the
largest fraction of all new marine natural products described annually.
However, sponges from the deep-sea remain relatively unexplored. We
sampled sponges from Irish waters in the Atlantic Ocean from depths
ranging from ~750 m to 2900 m. Bacteria were isolated from sponge
tissues on a range of isolation media. Actinobacterial isolates were
subsequently fermented in four growth media designed to elicit
production of bioactive compounds. Subsequently, culture extracts of
aqueous and organic soluble compounds were prepared through
chromatographic techniques and the extracts were tested for
bioactivities. Significant bioactivities (anti-bacterial and
anti-fungal) have been identified from 42 individual extracts. Very
strong activity was observed from a further 15 individual extracts
including broad-range antimicrobial activities against clinically
relevant strains of bacteria and/or fungi. Scale-up fermentations of 2
bacterial strains allowed for the production of compounds of interest in
sufficient quantities to allow for compound purification and structure
elucidation. The known bioactive compounds, Amicoumacins B and C [1,2];
hybrid PKS-NRPS gene products were identified. Genomic analyses has now
clearly established that “silent” or “cryptic” secondary metabolism
biosynthesis pathways are common in Actinobacteria. We are currently
using methods including genome “mining” and co-cultivation of our 15
marine Actinomycete species to uncover novel metabolites. Analysis of
genome sequence data from the deep sea Actinobacteria forms part of the
PharmaSea project, where we will seek to both identify gene clusters for
isolated metabolites and target novel gene clusters for activation.
References
- Itoh, J.; Omotoh, S.; Nishizawa, N.; Kodama, Y.; Inouye, S. Chemical structures of Amicoumacins produced by Bacillus pumilus. Agric. Biol. Chem. 1982, 46, 2659–2665.
- Pinchuk, I.V.; Bressollier, P.; Sorokulova, I.B.; Verneuil, B.; Urdaci, M.C. Amicoumacin antibiotic production and genetic diversity of Bacillus subtilis strains isolated from different habitats. Res. Microbiol. 2002, 153, 269–276.
Chemical Ecology of the Antarctic Nudibranch Charcotia granulosa
Juan Moles 1,2, Adele Cutignano 2, Angelo Fontana 2 and Conxita Avila 1
1 University of Barcelona, Barcelona, Spain
2 Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Pozzuoli (Napoli), Italy
Sea
slugs are characterized by the presence of a wide array of natural
products, often used as defense against sympatric predators. In
Antarctica, even if only few species have been chemically studied,
evidence has been progressively increasing that many natural products
act as protective agents against predation by sea stars and other
putative predators. The Antarctic nudibranch Charcotia granulosa
Vayssière, 1906 (Mollusca: Gastropoda) was collected in Deception
Island during our last cruise and analyzed for the presence of new
natural products. Several adults together with some egg masses and its
bryozoan prey, Beania erecta, were chemically analyzed by chromatographic and spectroscopic techniques. A new homosesterterpene, named granuloside (1), was characterized from the notum of the sea slug by spectroscopic studies on natural compound and its methyl derivatives (1a and 1b).
Despite the apparently simplicity, granuloside exhibits an
unprecedented linear C26 skeleton that arises a few questions about its
biogenesis and origin in the Antarctic nudibranch. Here we report the
description of the novel natural product and discuss the key
characteristics of its putative biosynthesis and function. Our results
suggest that C. granulosa synthesizes de novo the
compound in early stages of its ontogeny, this being the first evidence
of biosynthesis within the family Charcotiidae. In fact, granuloside is
the first example of terpenes reported in the family Charcotiidae and,
in analogy with similar compounds from other marine and terrestrial
organisms, its occurrence may be beneficial for the nudibranh and be one
of the biochemical factors that have supported its radiation upon a
wide geographic area around the South Pole.
Poster Session 2: Isolation and Structure Elucidation
Salarin C, A Potent Inducer of Apoptosis
Yoel Kashman 1, Lee Zur 1, Ashgan Bishara 1, Maurice Aknin 2, Drorit Neumann 3 and Nathalie Ben-Califa 3
1 School of chemistry, Tel Aviv University, Ramat Aviv 69978, Israel
2
Laboratorie de Chimîe des Substances Naturelles et des Aliments,
Faculté des Sciences et Techniques, Université de Réunion, 15 Avenue
Rene Cassin, B.P. 7151, 97715 Saint Denis, Cedex 9, France
3 Department of Cell and Developmental Biology, Tel Aviv University, Ramat Aviv 69978, Israel
Four groups of novel nitrogen atom containing compounds have been isolated from the Madagascan sponge Fascaplysinopsis
sp. The chemical content of the various samples collected in Salary bay
(100 km north to Tulear) changed significantly from one site to the
other suggesting the metabolites to origin from guest micro organisms. A
notion supported by resemblance of several of the compounds to earlier
reported micro organism metabolites. The sponge extract was tested for
its effect on proliferation of the K562 leukemia cell line. Salarin C
was the most effective, among the salarins tested, in inhibiting cell
proliferation, as measured by the MTT assay, exhibiting inhibitory
activity down to 0.005 µg/mL. Once purified, salarin C becomes very
unstable and changes under light in the air to salarin A. Possessing
eight functional groups complicates the chemistry of the salarins. Next
to transformation of the oxazole ring to a triacetylamine, the second
sensitive moiety is the vinylepoxide. The latter becomes a good site for
chemical transformations and inter alia may explain the
biogenesis of tausalarin C. Namely, opening of the vinylepoxide of
salarin A by nucleophilic attack of pre-taumycin A.
New Polycyclic Xanthones Isolated from Marine Actinomadura sp.
Librada M Cañedo, Carmen Schleissner, Ana M Peñalver and Fernando de la Calle
Carmen Cuevas Pharmamar, Madrid 28770, Spain
Historically,
terrestrial microorganisms have been a plentiful source of structurally
diverse bioactive substances, and have provided important contributions
to the discovery of pharmaceutically useful compounds, many of them
isolated from actinomycetes [1]. Marine derived bacteria constitute a
new and promising source of unique metabolites with considerable
pharmaceutical and therapeutic potential [2,3], in particular, marine
actinobacteria are an attractive resource for new bioactive compounds
screening [4]. In the context of our interest in discovering new
cytotoxic compounds from marine sources, three new polycyclic xanthones
PM140035, PM140036 and PM140108, with general structure 1, have been isolated from marine bacteria belonging to the genus Actinomadura.
Polycyclic xanthones are a family of polyketides which are
characterized by their highly oxygenated angular hexacyclic frameworks
[5]. A literature search for related compounds led to the closely
related core structure of IB-00208 which was also isolated from a marine
derived species of Actinomadura [6]. Citreamycins,
cervinomycins and simaomicins also contain a 1,4-dioxygenated xanthone
subunit, and are structurally related to 1 but belong
to the family of xanthone derivatives with a cyclic amide, which is rare
amongst aromatic polyketides [7]. More details about the producer
microorganism, isolation and spectroscopic data leading to the structure
determination of these new polycyclic xanthones and their biological
activities will be reported.
References
- Bauer, A.; Brönstrup, M. Industrial natural product chemistry for drug discovery and development. Nat. Prod. Rep. 2014, 31, 35–60.
- Blunt, J.W.; Copp, B.R.; Keyzers, R.A.; Munro, M.H.; Prinsep, M.R. Marine natural products. Nat. Prod. Rep. 2015, 32, 116–211.
- Molinski, T.F.; Dalisay, D.S.; Lievens, S.L.; Saludes, J.P. Drug development from marine natural products. Nat. Rev. Drug Discov. 2009, 8, 69–85.
- Subramani, R.; Aalbersberg, W. Marine actinomycetes: An ongoing source of novel bioactive metabolites. Microbiol. Res. 2012, 167, 571–580.
- Winter, D.K.; Sloman, D.L.; Porco, J.A., Jr. Polycyclic xanthone natural products: Structure, biological activity and chemical synthesis. Nat. Prod. Rep. 2013, 30, 382–391.
- Rodríguez, J.C.; Puentes, J.L.F.; Baz, J.P.; Cañedo, L.M. IB-00208, a New Cytotoxic Polycyclic Xanthone Produced by a Marine-derived Actinomadura II. Isolation, Physico-chemical Properties and Structure Determination. J. Antibiot. 2003, 56, 318–321.
- Hopp, D.C.; Milanowski, D.J.; Rhea, J.; Jacobsen, D.; Rabenstein, J.; Smith, C.; Romari, K.; Clarke, M.; Francis, L.; Irigoyen, M.; et al. Citreamicins with Potent Gram-Positive Activity. J. Nat. Prod. 2008, 71, 2032–2035.
Pretrichodermamide C and N-methylpretrichodermamide B, Two New Cytotoxic Epidithiodiketopiperazines from Hyper Saline Lake Derived Penicillium sp.
Raha Orfali 1,2, Amal Ali 1, Weaam Ebrahim 1,3, Mohamed Abdel-Aziz 4, Werner Muller 5, WenHan Lin 6, Georgios Daletos 1 and Peter Proksch 1
1 Heinrich-Heine-University, Dusseldorf, Germany
2 King Saud University, Riyadh, Saudi Arabia
3 Mansoura University, Mansoura, Egypt
4 National Research Center, Cairo, Egypt
5 Medical Center of Johannes Gutenberg University, Mainz, Germany
6 Peking University, Beijing, China
The
aim of this study is to isolate and structural elucidate bioactive
fungal secondary metabolites from a hypersaline ecosystem.
Epidithiodiketopiperazines (ETPs) are a diverse group of natural
products characterized by a disulfide linkage across the dioxopiperazine
ring. These metabolites possess a broad spectrum of biological
activities, including antibacterial, antiviral and antifungal
activities. Ascomycetes are known to accumulate numerous new and
bioactive secondary metabolites including ETPs. The genus Penicillium comprises more than 300 known species and contains a highly diversified array of active compounds. Thousands of Penicillium
isolates have probably already been screened in bioprospecting
programmes since the discovery of penicillin, and still new biologically
active secondary metabolites continue to be discovered from these fungi
indicating their importance as a source for novel bioactive molecules
with interest to the pharmaceutical industry. In this study, a fungal
strain WN-11-1-3-1-2, identified as Penicillium sp., was
isolated from the sediment of Wadi El-Natrun Lake (a hyper saline lake),
Egypt, 80 km northwest of Cairo. The crude ethyl acetate extract of the
fungus was subjected to different chromatographic techniques to yield
two new epidithiodiketopiperazines (ETPs) derivatives,
pretrichodermamide C (1) and N-methylpretrichodermamide B (2). The structures of (1) and (2)
were unambiguously determined on the basis of one- and two-dimensional
NMR spectroscopy and by high-resolution mass spectrometry, as well as by
comparison with the literature. Compound (2) showed pronounced cytotoxicity against the murine lymphoma L5178Y cell line with an IC50
value of 2 mM. The results presented here suggest that halotolerant
fungi from hypersaline environments are a rich source of bioactive
secondary metabolites which could have implications for drug discovery
in the future.
Antimalarial Endoperoxide Polyketides from the Chinese Marine sponge Plakortis cfr. simplex
Giuseppina Chianese 1, Marco Persico 1, Fan Yang 2, Hou-Wen Lin 2, Nicoletta Basilico 3, Silvia Parapini 4, Donatella Taramelli 4, Orazio Taglialatela-Scafati 1 and Caterina Fattorusso 1
1 Department of Pharmacy, University of Naples Federico II, Naples, Italy
2
Key Laboratory for Marine Drugs, Department of Pharmacy, Renji
Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai,
China
3 Dipartimento di Scienze Biomediche, Chirurgiche e Odontoiatriche, Università di Milano, Milan, Italy
4 Dipartimento di Scienze Farmacologiche e Biomolecolari Università di Milano, Milan, Italy
Marine sponges of the genus Plakortis
(Demospongiae, Plakinidae) have been intensively investigated for their
secondary metabolites over the last decades. The most prominent and
peculiar class of Plakortis metabolites is given by propionate-
and butyrate-based polyketides, exemplified by the simple 1,2-dioxane
plakortin [1,2], often endowed with a promising antimalarial activity
[3]. An intense research activity on Plakortis metabolites led
to postulate a likely mechanism of action [4], basing on the information
coming from natural and synthetically prepared analogues. Chemical
investigation of the organic extract obtained from the sponge Plakortis simplex collected
in the South China Sea afforded five new polyketide endoperoxides four
of which containing a 1,2-dioxene ring, never evaluated before for
antimalarial activity. The stereostructures of these metabolites have
been deduced on the basis of spectroscopic analysis and chemical
conversion. The isolated endoperoxide derivatives have been tested for
their in vitro antimalarial activity against Plasmodium falciparum strains, showing IC50
values in the low micromolar range. Other apolar fractions of the same
organic extract afforded novel polyketide-based metabolites, testifying
the incredible chemical diversity produced by this organism. Some
examples will be provided in this presentation.
References
- Higgs, M.D.; Faulkner, D. John. Plakortin, an antibiotic from Plakortis halichondrioides. J. Org. Chem. 1978, 43, 3454–3457.
- Fattorusso, E.; Taglialatela-Scafati, O; Di Rosa, M; Ianaro, An. Metabolites from the Sponge Plakortis simplex. Part 3: Isolation and Stereostructure of Novel Bioactive Cycloperoxides and Diol Analogues. Tetrahedron 2000, 56, 7959–7967.
- Fattorusso, E.; Parapini S.; Campagnuolo, C.; Basilico, N.; Taglialatela-Scafati, O; Taramelli, D. Activity against Plasmodium falciparum of cycloperoxide compounds obtained from the sponge Plakortis simplex. J. Antimicrob. Chemother. 2002, 50, 883–888.
- Taglialatela-Scafati, O.; Fattorusso, E.; Romano, A,; Scala, F.; Barone, V.; Cimino, P.; Stendardo, E.; Catalanotti,B.; Persicoa, M.; Fattorusso, C. Insight into the mechanism of action of plakortins, simple 1,2-dioxane antimalarials. Org. Biomol. Chem. 2010, 8, 846–856.
New 36-Membered Antifungal Macrolides from Streptomyces caniferus
Rodney Lacret, Daniel Oves-Costales, Caridad Díaz, Ignacio
Pérez-Victoria, Jesús Martín, Mercedes de la Cruz, Nuria de Pedro,
Francisca Vicente and Olga Genilloud
Fundación Medina, Armilla, Granada, Spain
Macrolides
constitute one of the most interesting groups of natural products,
mainly produced by actinomycetes and fungi. They exhibit various
biological activities, including antitumor, antifungal, antiparasitic,
citotoxic and immunosuppressant activities. Bioactive macrolides
containing a 32 or 36-membered macrocyclic lactones have been described
in the past and include, among others, brasilinolide, liposidolide, and
novonestmycins A and B. However, macrolides linked to sugar derivatives
and a 1,4-naphtoquinone are fairly uncommon. At present, they are
restricted to the axenomycins and langkolide, isolated from culture
broths of Streptomyces lisandri and Streptomyces sp.
Acta 3062. As part of the PharmaSea project, over 400 actinomycetes were
grown on carefully selected fermentation media, and their fermentation
extracts were assayed against clinically relevant pathogenic microbial
strains. In this work, we detected that acetone extracts from culture
broths of Streptomyces caniferus (CA-271066) possesed antifungal activity against Aspergillus fumigatus and Candida albicans.
Herein we report the bioassay guided isolation, structural elucidation,
antifungal and citotoxic properties of a family of new macrolides
(structurally related to axenomycins) isolated from culture broths of
this actinomycete. The producing strain was isolated from an Ascidia
collected off-shore in Sao Tome and Principe and it was grown in
APM9-modified medium. The extraction of culture broths with acetone
afforded an aqueous crude extract (ACE), which displayed antifungal
activity. Liquid-liquid extraction of ACE with ethyl acetate followed by
reversed-phase semipreparative HPLC yielded several new 36-membered
antifungal macrolides as the compounds responsible for the observed
bioactivity. Structural elucidation of the family of compounds was based
on 1D and 2D NMR and High Resolution Mass Spectrometry (ESI-TOF). Their
relative stereochemistry was determined by J-based configuration analysis combined with the presence of key NOESY correlations and data reported for similar compounds.
Novel Adociaquinone Derivatives from the Indonesian Marine Sponge Xestospongia sp.
Fei He 1, Linh H. Mai 1, Arlette Longeon 1, Brent R. Copp 2, Nadège Loaëc 3, Amandine Bescond 3, Laurent Meijer 3 and Marie-Lise Bourguet-Kondracki 1
1 Laboratoire Molécules de
Communication et Adaptation des Micro-organismes, UMR 7245 CNRS/MNHN,
Muséum National d’Histoire Naturelle, 57 rue Cuvier (C.P. 54), 75005
Paris, France
2 School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
3 ManRos Therapeutics, Perharidy Research Center, 29680 Roscoff, France
Marine sponges of the genus Xestospongia
have proved to be an extremely rich source of secondary metabolites
with unprecedented molecular structures and various bioactivities.
Adocia-, halena- and xesto-quinone are the three main quinone-type
skeletons identified from sponges of the genus Xestospongia. Among the most significant compounds, adociaquinones A and B, first isolated from the sponge Adocia sp. and then from the Philippine sponge Xestospongia
sp. revealed inhibition of topoisomerase II in catalytic DNA unwinding
and decatenation assays as well as inhibition of enzyme in the potassium
sodium dodecyl sulfate assay. Previous investigations on the South
Pacific Xestospongia sp. by our group led to the isolation of a
series of halenaquinone-type compounds, including xestosaprol C
methylacetal, 3-ketoadociaquinones A and B, tetrahydrohalenaquinones A
and B, halenaquinol sulfate, halenaquinone and orhalquinone.
Orhalquinone demonstrated significant inhibitory activities against both
human and yeast farnesyltransferase enzymes, with IC50
values of 0.4 μM [1]. In the frame of the European Bluegenics program,
we have chemically investigated the Indonesian sponge of Xestospongia
sp. collected off North Sulawesi because its methanol crude extract had
showed kinase inhibition as well as antimicrobial and antioxidant
activities. Bio-guided fractionation of the extract led to the isolation
of seven new adociaquinone derivatives 1a–4c,
together with seven known compounds, adociaquinone A and B,
secoadociaquinones A and B, 15-chloro-14-hydroxyxestoquinone,
14-chloro-15-hydroxyxestoquinone and xestoquinol sulfate. The known
compounds were identified by comparison of their spectroscopic data with
those of the literature. The isolation and structural elucidation of
the new compounds as well as their biological activities will be
presented and discussed [2].
References
- Longeon, A.; Copp, B.R.; Roué, M.; Dubois, J.; Valentin, A.; Petek, S.; Debitus, C.; Bourguet-Kondracki, M.-L. New bioactive halenaquinone derivatives from South Pacific marine sponges of the genus Xestospongia. Bioorg. Med. Chem. 2010, 18, 6006–6011.
- He, F.; Mai, L.H.; Longeon, A.; Copp, B.R.; Loaëc, N.; Bescond, A.; Meijer, L.; Bourguet-Kondracki, M.-L. Novel Adociaquinone Derivatives from the Indonesian Sponge Xestospongia sp. Mar. Drugs 2015, 13, 2617–2628.
Erythrins, New Toxic Metabolites from the Marine Ciliate Pseudokeronopsis erythrina Used as Chemical Defense against Predators
Andrea Anesi 1, Federico Buonanno 2, Graziano DiGiuseppe 3, Claudio Ortenzi 2 and Graziano Guella 1
1 University of Trento, Trento, Italy
2 University of Macerata, Macerata, Italy
3 University of Pisa, Pisa, Italy
Marine
protozoa are known for their ability to produce a vast and chemically
diverse array of secondary metabolites that are involved in different
ecological functions. Morphospecies belonging to genus Euplotes have
been extensively studied for their ability to produce chemically diverse
secondary metabolites and, interestingly, it was found that strains
belonging to same genetic clades were characterized by a different
profile of bioactive compounds [1]. From the genus Pseudokeronopsis only two classes of pigments have been so far isolated, keronopsins as defensive molecules of Pseudokeronopsis rubra [2] and, more recently, keronopsamides from cell culture of the marine ciliate Pseudokeronopsis riccii [3]. We report here on the characterization of new secondary metabolites, erythrins, produced by cell cultures of Pseudokeronopsis erythrina
(Ciliophora, Hypotricha). Their structure have been elucidated by
extensive NMR and high resolution MS measurements and are characterized
by a central 4-hydroxy-unsaturated delta-lactone ring bearing an alkyl
saturated chain at C(2) and a butyl -benzenoid group at C(5). The
simultaneous presence of the corresponding 4-sulphate analogues has also
been ascertained and a reasonable proposal of their biosynthesis will
be reported. Cold-shock treatment has been performed to induce the
discharge of these metabolites from cell pigment granules. The analysis
of cytotoxic activity on a panel of free-living ciliates and
micro-invertebrates, together with some observation on the defensive
behavior by P. erythrina, indicated that erythrins are very effective for its chemical defence.
References
- Guella, G.; Skropeta, D.; di Giuseppe, G.; Dini, F. Structures, biological activities and phylogenetic relationships of terpenoids from marine ciliates of the genus Euplotes. Mar. Drugs 2010, 8, 2080–2116.
- Höfle, G.; Pohlan, S.; Uhlig, G.; Kabbe, K.; Schumacher, D. Keronopsins A and B, Chemical Defence Substances of the Marine Ciliate Pseudokeronopsis rubra (Protozoa): Identification by Ex Vivo HPLC. Angew. Chem. Int. Ed. Engl. 1994, 33, 1495–1497.
- Guella, G.; Frassanito, R.; Mancini, I.; Sandron, T.; Modeo, L.; Verni, F.; Dini, F.; Petroni, G. Keronopsamides, a new class of pigments from marine ciliates. Eur. J. Org. Chem. 2010, 2010, 427–434.
Cyclic Peptides from the Indonesian Marine Sponge Callyspongia aerizusa with Potent and Selective Antitubercular Activity
Georgios Daletos 1, Rainer Kalscheuer 2, Rudolf Hartmann 3 and Peter Proksch 1
1 Institute for Pharmaceutical Biology and Biotechnology, Heinrich-Heine-University, Duesseldorf, Germany
2 Institute for Medical Microbiology and Hospital Hygiene, Heinrich-Heine-University, Duesseldorf, Germany
3 Institute of Complex Systems: Strukturbiochemie, Forschungszentrum Juelich, Juelich, Germany
Chemical investigation of the Indonesian sponge Callyspongia aerizusa
afforded 13 cyclic peptide derivatives, namely callyaerins. The planar
structures of the isolated compounds were unambiguously elucidated on
the basis of 1D and 2D NMR spectroscopic data and MS interpretation. The
absolute configurations of their constituent amino acid residues were
determined using Marfey’s method. The basic structural unit of the
callyaerins comprises a cyclic peptide with a linear peptide side chain,
both of variable size, linked through a non-proteinogenic (Z)-2,3-diaminoacrylic
acid (DAA) functional group. The peptides are unusual in containing a
considerable number of proline residues, of which one proline is always
positioned at the beginning of the side chain, while all others are
found in the ring system. All compounds were investigated in vitro against Mycobacterium tuberculosis,
as well as against THP-1 (human acute monocytic leukemia), and MRC-5
(human fetal lung fibroblast) cell lines in order to assess their
general cytotoxicity. Callyaerins were found to inhibit M. tuberculosis at low micromolar concentrations making these compounds interesting candidates for further studies.
Epipyrones from the Marine-Derived Fungus Epicoccum Nigrum Link Inhibit the Proteases Cathepsin K and S
Peter Hufendiek 1, Stefan Kehraus 1, Michael Gütschow 2 and Gabriele M. König 1
1 Institute of Pharmaceutical Biology, University of Bonn, Bonn, Germany
2 Institute of Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Bonn, Germany
Fungi
belonging to the Ascomycota are an excellent source of bioactive
compounds, among others, antibiotics, immunosuppressants and cholesterol
lowering agents. This project aims to identify new fungal secondary
metabolites useful as lead structures in pharmacology, especially
compounds which show enzyme inhibitory activity. Since the marine
environment and fungi occurring there are not yet well researched
concerning the presence of pharmacologically active compounds, our
project targets fungi isolated from marine algae. Here we focus on a
marine-derived strain of the fungus Epicoccum nigrum, isolated from the
surface of a green alga. Cultivation and fractionation of the crude
extract led to the isolation of the epipyrones, which are isomeric
polyketides with an unusual C-glycosyl-moiety. Biological testing revealed selective activity against two different cysteine proteases, i.e., cathepsin K and S. The IC50-values
are 11.4 and 6.6 μM, respectively. Cathepsin K inhibitors may be used
to combat osteoporosis, whereas cathepsin S plays a role in tumor
proliferation. Interestingly, no activity was seen against cathepsin B
and L. For the serine proteases human leukocyte elastase and trypsin,
lower or no activity was measured, underlining the selectivity of the
compounds. Since the epipyrones tend to isomerize quickly, we aimed to
block isomerization by acetylation of the glycosyl-unit. Indeed,
acetylation led to one single isomer, which will also be tested against
the aforementioned targets, to compare the inhibitory activity. In
conclusion, the epipyrones were shown to exhibit selective activity
against cathepsin K and S. The role of these enzymes in osteoporosis and
tumor growth renders the epipyrones interesting lead compounds for
further studies.
Acknowledgments
This research is funded by the NRW International Graduate Research School BIOTECH-PHARMA.
Isocoumarins and Cyclic Hexapeptide from the Sponge-Associated Fungus Aspergillus similanensis sp. nov. Kufa 0013
Chadaporn Prompanya 1, Tida Dethoup 2, Madalena Pinto 3 and Anake Kijjoa 1
1 Instituto de Ciências Biomédicas Abel Salazar and CIIMAR, Universidade do Porto, Porto, Portugal
2 Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand
3
Laboratório de Química Orgânica e Farmacêutica, Departamento de
Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Porto,
Portugal
In our ongoing search for new
natural products with antibacterial and anticancer activities produced
by the marine-derived fungi of the genera Neosartorya and Aspergillus, we have investigated the secondary metabolites of a Thai collection of a new species of Aspergillus, which we have named Aspergillus similanensis (KUFA0013), isolated from the marine sponge Rhabdermia sp.,
collected from the Similan Islands in Southern Thailand. The ethyl
acetate extract of the culture of this fungus furnished several
isocoumarine derivatives including new isocoumarins similanpyrones A (1), B (2) and C (3), a new chevalone analog, chevalone E (4), a new pyripyropene analog, pyripyropene T (5), and a new cyclic peptide, named similanamide (6)
[1,2]. Some of the isolated compounds were evaluated for their
antimicrobial activity against Gram-positive and Gram-negative bacteria
and multidrug-resistant isolates from the environment.
Acknowledgments
This work was partially supported by the Project MARBIOTECH (reference NORTE-07-0124-FEDER-000047).
References
- Prompanya, C.; Dethoup, T.; Bessa, L.J.; Pinto, M.M.M.; Gales, L.; Costa, P.M.; Silva, A.M.S.; Kijjoa, A. New Isocoumarin Derivatives and Meroterpenoids from the Marine Sponge-Associated Fungus Aspergillus similanensis sp. nov. KUFA 0013 Mar. Drugs 2014, 12, 5160–5173.
- Prompanya, C.; Fernandes, C.; Cravo, S.; Pinto, M.M.M.; Dethoup, T.; Silva, A.M.S.; Kijjoa, A. A New Cyclic Hexapeptide and a New Isocoumarin Derivative from the Marine Sponge-Associated Fungus Aspergillus similanensis KUFA 0013. Mar. Drugs 2015, 13, 1432–1450.
Indole Alkaloids and Dihydroisocoumarin from the Alga-Associated Fungus Neosartorya takakii KUFC 7898
War War May Zin 1, Suradet Buttachon 1, Jamrearn Buaruang 2 and Anake Kijjoa 1
1 Instituto de Ciências Biomédicas Abel Salazar and CIIMAR, Universidade do Porto, Porto, Portugal
2 Division of Environmental Science, Faculty of Science, Ramkhamhaeng University, Bangkok, Thailand
In our ongoing search for new natural products with antibacterial activity produced by the marine-derived fungi of the genus Neosartorya [1,2], we have investigated the secondary metabolites of a Thai collection of Neosartorya takakii KUFC 7898, isolated from the marine alga Amphiroa sp.,
collected from Samaesarn Island in the Gulf of Thailand. The ethyl
acetate extract of its culture furnished so far, besides the indole
alkaloids aszonalenin (1a), acetylaszonalenin (1b), tryptoquivalines F (2a), H (2b), L (2c) and a new tryptoquivaline derivative which we have named tryptoquivaline U (3d), aszonapyrone A (3) and a dihydroisocoumarin derivative, 6-hydroxymellein (4).
The structures of the compounds were established by 1D and 2D NMR
spectral analysis and HRMS. All the compounds were evaluated for their
antibacterial activity against Gram positive (Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633) and Gram negative (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) bacteria as well as multidrug-resistant isolates from the environment.
Acknowledgments
This work was partially supported by the Project MARBIOTECH (reference NORTE-07-0124-FEDER-000047).
References
- Eamvijarn, A.; Nelson M. Gomes, N.M.; Dethoup, T.; Buaruang, J.; Manoch, L.; Silva, A.; Pedro, M.; Marini, I.; Roussis, V.; et al. Bioactive meroditerpenes and indole alkaloids from the soil fungus Neosartorya fischeri (KUFC 6344), and the marine-derived fungi Neosartorya laciniosa (KUFC 7896) and Neosartorya tsunodae (KUFC 9213). Tetrahedron 2013, 69, 8583–8591.
- Gomes, N.M.; Bessa, L.J.; Buttachon, S.; Costa, P.M.; Buaruang, J.; Dethoup, T.; Silva, A.M.S.; Kijjoa, A. Antibacterial and Antibiofilm Activities of Tryptoquivalines and Meroditerpenes Isolated from the Marine-Derived Fungi Neosartorya paulistensis, N. laciniosa, N. tsunodae, and the Soil Fungi N. fischeri and N. siamensis. Mar. Drugs 2014, 12, 822–839.
Anti-Inflammatory Activity of Tanzawaic Acid Derivatives from a Marine-derived Fungus Penicillium steckii 108YDC142
Chien Fang, Soo-Jin Heo, Hyi-Seung Lee, Yeon-Ju Lee, Jong Seok Lee and Hee Jae Shin
Korea Institute of Ocean Science and Technology, Ansan, Korea
Chemical investigation of a marine-derived fungus Penicillium steckii
108YDC142, isolated from a marine sponge sample collected at
Wangdolcho, East Sea, Korea, resulted in the discovery of a new
tanzawaic acid derivative (1), together with four known analogues, tanzawaic acids A (2), C (3), D (4), and K (5).
Their structures were determined by the detailed analysis of NMR and MS
data, along with chemical methods. These compounds significantly
inhibited the nitric oxide (NO) production and the new tanzawaic acid
derivative (1) inhibited the lipopolysaccharide
(LPS)-induced inducible nitric oxide synthase (iNOS) and
cyclooxygenase-2 (COX-2) proteins and mRNA expressions in RAW 264.7
macrophages. Additionally, compound 1 reduced the mRNA
levels of inflammatory cytokines, including tumor necrosis factor-α,
interleukin (IL)-1β, and IL-6. Taken together, the results of this study
demonstrate that the new tanzawaic acid derivative inhibits LPS-induced
inflammation.
Nocardiomycins A–C, New Cytotoxic Cyclic Depsipeptides Isolated from Marine Actinomycetes
Marta Pérez, Cristina Lillo, Rogelio Fernández, Librada Cañedo, Paz Zuñiga, Pilar Rodríguez
, Simon Munt and Carmen Cuevas
Pharmamar, Colmenar Viejo/Madrid, Spain
Cyclic
depsipeptides have emerged as a very important class of bioactive
compounds from marine derived bacteria. Several of these compounds have
been disclosed to have cytotoxic, antiviral and/or antifungal
properties. Specifically, rakicidins A, B and D [1–4] showed cytotoxic
activity against the murine carcinoma colon 26-L5 tumour cells, and
vinylamycin [5,6], exhibited antimicrobial activities against
Gram-positive bacteria including MRSA. In an effort to discover new
cytotoxic compounds from marine sources, three new lipopeptides named
nocardiomycin A (1), nocardiomycin B (2) and nocardiomycin C (3)
have been isolated from different marine bacteria belonging to the
order Actinomycetales. These compounds, structurally related to the
reported rakicidins, are 15-membered depsipeptides consisting of three
amino acids and a 3-hydroxyfatty acid. One of these amino acids
corresponds to the rare unusual 4-amino-2,4-pentadienoate, only found in
a few examples of secondary metabolites of Actinomycetes. Details of
the producer microorganisms, isolation and spectroscopic data leading to
the structure determination of these new cytotoxic compounds, as well
as their biological properties will be presented.
References
- McBrien, K.D.; Berry, R.L.; Lowe, S.E.; Neddermann, K.M.; Bursuker, I.; Huang, S.; Klohr, S.E.; Leet, J.E. Rakicidins, New Cytotoxic Lipopeptides from Micromonospora sp. Fermentation, Isolation and Characterization. J. Antibiot. 1995, 48, 1446–1452.
- Igarashi, Y.; Shimasaki, R.; Miyanaga, S.; Oku, N.; Onaka, H.; Sakurai, H.; Saiki, I.; Kitani, S.; Nihira, T.; Wimonsiravude, W. et al. Rakicidin D, an inhibitor of tumor cell invasion from marine-derived Streptomyces sp. J. Antibiot. 2010, 63, 563–565.
- Oku, N.; Matoba, S.; Yamazaki, Y.M.; Shimasaki, R.; Miyanaga, S.; Igarashi, Y. Complete Stereochemistry and Preliminary Structure-Activity Relationship of Rakicidin A, a Hypoxia-Selective Cytotoxin from Micromonospora sp. J. Nat. Prod. 2014, 77, 2561–2565.
- Sang, F.; Li, D.; Sun, X.; Cao, X.; Wang, L.; Sun, J.; Sun, B.; Wu, L.; Yang, G.; Chu, X.; et al. Total Synthesis and Determination of the Absolute Configuration of Rakicidin A. J. Am. Chem. Soc. 2014, 136, 15787–15791.
- Igarashi, M.; Shida, T.; Sasaki, Y.; Kinoshita, N.; Naganawa, H.; Hamada, M.; Takeuchi, T. Vinylamycin, a new depsipeptide antibiotic, from Streptomyces sp. J. Antibiot. 1999, 52, 873–879.
- Carr, G.; Poulsen, M.; Klassen, J.L.; Hou, Y.; Wyche, T.P.; Bugni, T.S.; Currie, C.R.; Clardy, J. Microtermolides A and B from Termite-Associated Streptomyces sp. and Structural Revision of Vinylamycin. J. Org. Lett. 2012, 14, 2822–2825.
New Cyclopeptides Isolated from Lissoclinum patella
Rogelio Fernández, Elena Gómez, Marta Pérez and Carmen Cuevas
Pharmamar, Colmenar Viejo/Madrid, Spain
Ascidians from the genus Lissoclinum
are a rich source of a variety of cytotoxic cyclic peptides
characterized by the presence of oxazole and thiazole moieties. Some of
these peptides contain threonine and serine residues whose side chains
have been modified as dimethylallyl ethers. They include the
cyclopeptides Nairaiamide, Patellin, Trunkamide A, Mollamide,
Hexamollamide and Comoramide [1]. In the course of our screening program
to isolate novel compounds with antitumor properties from marine
sources, we have isolated two new cyclopeptides, named Wetamide A and B
from the ascidian Lissoclinum patella collected off the coast
of Wetar in the Southwest islands of Indonesia. These compounds were
obtained by bioassay-guided fractionation of an organic extract of the
organism, using VLC RP-18 chromatography and reverse phase
semi-preparative HPLC. Structure elucidation of these new metabolites
was carried out by spectroscopic methods including MS, 1H, 13C
and 2D-NMR. The stereochemistry of the amino acids was determined by
hydrolysis followed by derivatization with Marfey’s reagent and
comparison with commercial standards by HPLC-MS [2].
Acknowledgments
Udayana
University of Bali. Indonesia. Sebastiano Gulinello (Expedition
Department Logistic Coordinator). Ministry of Marine Affairs and
Fisheries. Republic of Indonesia.
References
- Blunt, J.W.; Copp, B.R.; Keyzers, R.A.; Munro, M.H.G.; Prinsep, M.R. Marine natural products. Nat. Prod. Rep. 2015, 32, 116–211, and previous papers in this series.
- Marfey, P. Determination of d-amino acids. II. Use of a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. Carlsberg. Res. Commun. 1984, 49, 591–596.
Chemical Studies of a Sample of Hexadella sp. from the Arafura Sea (Indonesia)
Rogelio Fernández, Patricia Gema Cruz, Marta Perez and Carmen Cuevas
Pharmamar, Colmenar Viejo/Madrid, Spain
Marine
sponges of the order Verongida are a rich source of brominated tyrosine
metabolites, many of which have exhibited diverse biological activities
[1–4]. Chemical modification occurs both on the side chain and aromatic
ring of the brominated tyrosine precursors giving rise to a broad range
of biosynthetically related compounds. In the course of our screening
program to search for new antitumour compounds from marine organisms, we
have isolated two new bromotyrosine derivatives PM140657 (1) and PM140674 (2), as well as the known pseudoceratin A (3) [5], from a sample of Hexadella
sp. collected off the coast of Arafura (Indonesia). The structures of
all the compounds were elucidated by analysis of their 1D and 2D-NMR
spectra and comparison with data reported for other bromotyrosine
derivatives. PM140674 exhibits micromolar cytotoxicity against several
cell lines, including lung (A549), colon (HT29), breast (MDA-MB-231) and
pancreas (PSN1).
Acknowledgments
Udayana
University of Bali. Indonesia. Sebastiano Gulinello (Expedition
Department Logistic Coordinator). Ministry of Marine Affairs and
Fisheries. Indonesia.
References
- Blunt, J.W.; Copp, B.R.; Keyzers, R.A.; Munro, M.H.G.; Prinsep, M.R. Marine natural products. Nat. Prod. Rep. 2015, 32, 116–211, and previous papers in this series.
- Xu, M.; Andrews, K.T.; Birrell, G.W.; Tran, T.L.; Camp, D.; Davis, R.A.; Quinn, R.J. Psammaplysin H, a new antimalarial bromotyrosine alkaloid from a marine sponge of the genus Pseudoceratina. Bioorg. Med. Chem. Lett. 2011, 21, 846–848.
- Shaker, K.H.; Zinecker, H.; Ghani, M.A.; Imhoff, J.F.; Schneider, B. Bioactive metabolites from the sponge Suberea sp. Chem. Biodivers. 2010, 7, 2880–2887.
- Buchanan, M.S.; Carroll, A.R.; Wessling, D.; Jobling, M.; Avery, V.M.; Davis, R.A.; Feng, Y.; Hooper, J.N.A.; Quinn, R.J. Clavatadines C–E, Guanidine Alkaloids from the Australian Sponge Suberea clavata. J. Nat. Prod. 2009, 72, 973–975.
- Jang, J.-H.; van Soest, R.W.; Fusetani, N.; Matsunaga, S. Pseudoceratins A and B, antifungal bicyclic bromotyrosine-derived metabolites from the marine sponge Pseudoceratina purpurea. J. Org. Chem. 2007, 72, 1211–1217.
Bioactive Metabolites from Marine-Derived Actinobacteria from the East Mediterranean
Panagiota Georgantea 1, Leto-Aikaterini Tziveleka 1, Eleni Mavrogonatou 2, Eniko Rab 1,3, Dimitris Kekos 3, Harris Pratsinis 2, Dimitris Kletsas 2, Vassilios Roussis 1 and Efstathia Ioannou 1
1 Department of Pharmacognosy and Chemistry of Natural Products, Faculty of Pharmacy, University of Athens, Athens, Greece
2
Laboratory of Cell Proliferation and Aging, Institute of Biosciences
and Applications, National Centre for Scientific Research “Demokritos”,
Athens, Greece
3 Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Athens, Greece
The
screening of microbial natural products represents an important route
to the discovery of novel anticancer and antibiotic agents. Diverse
actinobacteria isolated from unique ecosystems have been shown to
produce bioactive compounds which exert their influence by processes
that are not compromised by existing multidrug-resistance pathways. In
order to obtain new strains likely to produce novel metabolites,
examination of samples from different habitats and extreme environments
is necessary. The East Mediterranean basin is a geomorphologically and
biologically unique marine ecosystem that has not been investigated so
far for its microbiota as producers of secondary metabolites. In search
of new bioactive secondary metabolites from marine microorganisms found
in the Greek seas, we have selectively isolated more than 900
actinobacterial strains from sediments and macroorganisms from the
Aegean and the Ionian Seas. On the basis of preliminary screening of the
chemical profiles of small scale liquid cultures of numerous
actinobacterial strains with LC-DAD-MS and NMR, in conjunction with
evaluation of their antibacterial and cytotoxic activities, strains
BI0048 and BI0383 were selected for further chemical investigation. The
large scale liquid cultures of strains BI0048 and BI0383 afforded two
crude extracts which were subjected to multi-step fractionations that
have led so far to the isolation of 24 and 16 compounds, respectively.
Among these, two polyketides are new natural products. The structure
elucidation and the assignment of the relative configurations of the
compounds were based on analyses of their spectroscopic data and
comparison with literature data. The isolated metabolites are currently
being evaluated for their antibacterial and cytotoxic activities.
Acknowledgments
This
work was supported by the project aristeia-2587 “biomaract”, which is
implemented under the “aristeia” action of the operational programme
“education and lifelong learning” and is co-funded by the European
Social Fund (ESF) and national resources.
Prevezanes and Related Diterpenes from Laurencia glandulifera and Evaluation of Their Anti-Inflammatory Activity
Maria Harizani 1, Gerasimos Konidaris 1, Maria Daskalaki 2, Sotirios Kampranis 3, Christos Tsatsanis 2, Vassilios Roussis 1 and Efstathia Ioannou 1
1 Department of Pharmacognosy and Chemistry of Natural Products, Faculty of Pharmacy, University of Athens, Athens, Greece
2 Laboratory of Clinical Chemistry, University of Crete Medical School, Heraklion, Greece
3 Mediterranean Agronomic Institute of Chania, Chania, Greece
Despite the extensive investigations on the chemical profile of numerous Laurencia
species, this cosmopolitan genus still represents a prolific source of
metabolites, often exhibiting antibacterial, antifungal, insecticidal
and/or cytotoxic activities. Laurencia biosynthesizes a wide spectrum of secondary metabolites, including sesquiterpenes, diterpenes, triterpenes and C15 acetogenins which are frequently characterized by the presence of halogen atoms. Previous investigations on Laurencia glandulifera
have led to the isolation of the brominated diterpene neorogioltriol
which has exhibited very significant levels of analgesic and
anti-inflammatory activity [1]. In search of new congeners, specimens of
L. glandulifera were collected in Kefalonia Island in the
Ionian Sea, Greece, at a depth of 1–2 m in April of 2014. Extraction of
the fresh algal tissues with mixtures of CH2Cl2/MeOH
afforded a residue that was subjected to a series of chromatographic
separations. Eight diterpenes of the prevezane skeleton have been
isolated until now. Among these, one metabolite features a new carbon
skeleton. The structures and relative configurations of the isolated
natural products were determined on the basis of extensive analysis of
their 1D and 2D NMR and MS data. The isolated metabolites are currently
being evaluated for their anti-inflammatory activity by a cell-based
assay which measures the extent of macrophage activation by bacterial
lipopolysaccharide (LPS).
Acknowledgments
This
work was supported by the project GSRT-EPANII-11ΣYN-3-770 “NRG”, which
is implemented under the “Cooperation 2011” Action of the Operational
Programme “Competitiveness and Entrepreneurship” and is co-funded by the
European Social Fund (ESF) and National Resources
Reference
- Chatter, R.; Kladi, M.; Tarhouni, S.; Maatoug, R.; Kharrat, R.; Vagias, C.; Roussis, V. Neorogioltriol: A brominated diterpene with analgesic activity from Laurencia glandulifera. Phytochem. Lett. 2009, 2, 25–28.
New Cytotoxic Sesquiterpenes from the Brazilian Red Alga Laurencia catarinensis
Miriam Falkenberg 1,2, Efstathia Ioannou 1, Cintia Lhullier 1,2, Tauana Wanke 1,2, Ana Claudia Philippus 2, Lucas F.O. Vieira 2, Panagiota Papazafiri 3 and Vassilios Roussis 1
1 Department of Pharmacognosy and Chemistry of Natural Products, Faculty of Pharmacy, Athens, Greece
2 Laboratory of Medicinal Chemistry and Natural Products, Federal University of Santa, Florianópolis, Brazil
3 Department of Animal and Human Physiology, Faculty of Biology, School of Science, Athens, Greece
The complex Laurencia (Rhodomelaceae)
includes species with a wide distribution throughout the world and
represents a prolific source of new secondary metabolites. Besides
fulfilling ecological needs of the seaweeds, the halogenated terpenes
and acetogenins that are frequently encountered in Laurencia species,
exhibit a wide range of pharmacological activities, e.g.,
antiinflammatory, cytotoxic, and antibacterial. Previous studies on Laurencia species
have indicated that the chemodiversity observed in the species level
may be affected by environmental factors. In the course of our ongoing
investigations towards the isolation of bioactive marine metabolites, we
have previously investigated the chemical composition of Laurencia catarinensis
Cordeiro-Marino & Fujii, collected off Ilha do Arvoredo, Santa
Catarina, Southern Brazil [1] and reported the isolation of seven new
and seven known metabolites, most of them structurally related to
caespitol. Recent collections of this species off Ilha do Xavier from
the state of Santa Catarina afforded a crude extract with a different
chemical profile. In order to study the magnitude of the difference in
the chemical profiles, extracts obtained from collections performed from
Ilha do Arvoredo and Ilha do Xavier were investigated in depth, leading
to the isolation of a number of minor metabolites. Herein, we report
the isolation and structure elucidation of nine new and 14 known
metabolites reported for the first time from L. catarinensis.
Among the two different populations, only four metabolites were found as
common constituents. The isolated compounds were evaluated for their in vitro cytotoxicity against three human tumor cell lines, namely HT29, MCF7, and A431, exhibiting variable levels of activity.
Reference
- Lhullier, C.; Falkenberg, M.; Ioannou, E.; Quesada, A.; Papazafiri, P.; Horta, P.A.; Schenkel, E.P.;Vagias, C.; Roussis, V. Cytotoxic halogenated metabolites from the Brazilian red alga Laurencia catarinensis. J. Nat. Prod. 2010, 73, 27–32.
Studies on the Red Sea Sponge Haliclona sp. for Its Chemical and Cytotoxic Properties
Ali El Gamal 1,2, Shaza Al-Massarani 1, Mansour Al-Said 1, Maged Abdel-Kader 3, Hazem Ghabbour 1, Hoong-Kun Fun 1, Wael Abdel-Mageed 1, Abdelkader Ashour 1, Ashok Kumar 1 and Adnan Al-Rehaily 1
1 King Saud University, Riyadh, Saudi Arabia
2 Mansoura University, El Mansoura, Egypt
3 Salman Abdulaziz University, Al-kharj, Saudi Arabia
The
Red Sea is characterized by a great diversity of living organisms [1].
Previous chemical studies of marine sponges belonging to the genus Haliclona
led to the isolation of a variety of bioactive secondary metabolites,
including alkaloids [2], macrolides [3], polyacetylenes [4], polyketides
[5] and peptides [6]. The total cytotoxic alcoholic extract of a sponge
belonging to genus Haliclona, collected from the eastern coast
of the Red Sea, Jeddah, Saudi Arabia, was subjected to intensive
chromatographic fractionation and purification guided by cytotoxic
bioassay toward various cancer cell lines. This investigation resulted
in the isolation of a new indole alkaloid, 1-(1H-indol-3-yloxy) propan-2-ol (1), and the previously synthesized pyrrolidine alkaloid, (2R,3S,4R,5R) pyrrolidine-(1-hydroxyethyl)-3,4-diol hydrochloride (4), isolated here naturally for the first time. In addition, six known compounds—tetillapyrone (2), nortetillapyrone (3), 2-methyl maleimide-5-oxime (5), Maleimide-5-oxime (6), 5-(hydroxymethyl) dihydrofuran-2(3H)-one (7), and Ergosta-5,24(28)-dien-3-ol (8)
were also identified. The structures of these isolated compounds were
elucidated based on extensive examination of their spectroscopic data
including 1D and 2D NMR. Compound 5 is reported here for the first time from the genus Haliclona, while compound 7
is not yet reported from a marine source. X-ray single-crystal
structure determination was performed to determine the absolute
configuration of compound 4. Unfortunately most of the
isolated compounds exhibited weak cytotoxic activity against HepG-2,
Daoy, and HeLa cancer cell lines.
References
- Lira, N.S.; Montes, R.C.; Barbosa-Filho, J.M. Brominated compounds from marine sponges of the genus Aplysina and a compilation of their 13C NMR spectral data. Mar. Drugs 2011, 9, 2316–2368.
- Rashid, M.A.; Gustafson, K.R.; Boyd, M.R. A new isoquinoline alkaloid from the marine sponge Haliclona species. J. Nat. Prod. 2001, 64, 1249–1250.
- Liu, Y.-H.; Wang, B.; Liu, D.-Y.; Li, L.-D.; Fei, L.N.C. Chemistry and Biological activities of marine sponge Halichlona. JTO 2008, 27, 70–82.
- Alarif, W.M.; Abdel-Lateff, A.; Al-Lihaibi, S.S.; Ayyad, S.E.N.; Badria, F.A. A New Cytotoxic Brominated Acetylenic Hydrocarbon from the Marine Sponge Haliclona sp. with a Selective Effect against Human Breast Cancer. Z. Naturforsch. C 2013, 68, 70–75.
- Kennedy, J.; Codling, C.E.; Jones, B.V.; Dobson, A.D.W.; Marchesi, J.R. Diversity of microbes associated with the marine sponge, Haliclona simulans, isolated from Irish waters and identification of polyketide synthase genes from the sponge metagenome. Environ. Microbiol. 2008, 10, 1888–1902.
- Rashid, M.A.; Gustafson, K.R.; Boswell, J.L.; Boyd, M.R. Haligramides A and B, Two New Cytotoxic Hexapeptides from the Marine Sponge Haliclona nigra. J. Nat. Prod. 2000, 63, 956–959.
Two New Xenicanes from the Soft Coral Clavularia sp.
Carlos Jiménez 2, Carlos Urda 1, Marta Pérez 1, Rogelio Fernández 1, Jaime Rodriguez 2 and Carmen Cuevas 1
1 PharmaMar S.A., Colmenar Viejo, Madrid, Spain
2 Universidade da Coruña, A Coruña, Spain
Soft
corals belonging to the order Alcyonacea (subclass Octocorallia), are
rich sources of xenicane-type compounds which are characterized by a
dihydropyran ring fused to nine-membered ring. Four families with
different functionalities have been identified within this structural
class and they are represented by the xenicins, xenialactols, and
xeniolides A and B [1–3]. In this communication, we describe the
isolation of two new xenicanes, named PM090004 and PM090082, from a soft
coral belonging to the genus Clavularia, collected off the
coast of Okuza (Tanzania). Only one xenicane has been reported from this
genus so far [4]. These new compounds were obtained by column
chromatography and semipreparative HPLC purification from an organic
extract of this organism. Their planar structures were determined by 1D
and 2D NMR and HRESIMS techniques while their relative stereochemistries
were elucidated by comparison of their chemical shifts and coupling
constants with the literature values of their congeners, as well as by
NOESY experiments. Both new xenicanes were moderately cytotoxic against a
panel of different tumour cell lines.
Acknowkedgments
P.
J. Ruysenaars (The Pemba channel fishing club Shimoni) and Ministry of
Livestock and Fisheries Development. Fisheries Department (Republic of
Tanzania).
References
- Ishigami, S.; Goto, Y.; Inoue, N.; Kawazu, S.; Matsumoto, Y.; Imahara, Y.; Tarumi, M.; Nakai, H.; Fusetani, N.; Nakao, Y. Cristaxenicin A, an Antiprotozoal Xenicane Diterpenoid from the Deep Sea Gorgonian Acanthoprimnoa cristata. J. Org. Chem. 2012, 77, 10962–10966.
- Lin, Y.-S.; Fazary, A.E.; Chen, C.-H.; Kuo, Y.-H.; Shen, Y.-C. Asterolaurins G – J, New Xenicane Diterpenoids from the Taiwanese Soft Coral Asterospicularia laurae. Helv. Chim. Acta 2011, 94, 273–281.
- Andrianasolo, E.H.; Haramaty, L.; Degenhardt, K.; Mathew, R.; White, E.; Lutz, R.; Falkowski, P. Induction of apoptosis by diterpenes from the soft coral Xenia elongate. J. Nat. Prod. 2007, 70, 1551–1557.
- Wang, S.; Huang, M.; Duh, C. Cytotoxic constituents from the formosan soft coral Clavularia inflata var. luzoniana. J. Nat. Prod. 2006, 69, 1411–1416.
Multiplicity Editing in Long-Range Heteronuclear Correlation NMR Experiments: Application to Natural Products
Josep Saurí 1, Eduard Sistaré 2, Michel Frederich 3, Alembert T Tchinda 4, Teodor Parella 2, R. Thomas Williamson 1 and Gary E. Martin 1
1 NMR Structure Elucidation, Process and Analytical Chemistry, Merck & Co. Inc., Rahway, NJ 07065, USA
2 Servei de Ressonància Magnètica Nuclear, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain
3 Laboratory of Pharmacognosy, Department of Pharmacy, CIRM, University of Liège, B36, 4000 Liège, Belgium
4
Center for Studies on Medicinal Plants and Traditional Medicine,
Institute of Medical Research and Medicinal Plants Studies (IMPM), P.O.
Box 6163, Yaoundé, Cameroon
Even C/CH2 and odd CH/CH3
carbon-multiplicity information can be directly distinguished from the
relative positive/negative phase of cross-peaks in a novel ME
(Multiplicity-Edited)-selHSQMBC experiment. The method can be extended
by a TOCSY propagation step, and is also fully compatible for the
simultaneous and precise determination of long-range heteronuclear
coupling constants. In addition, broadband homonuclear decoupling
techniques can also be incorporated to enhance sensitivity and signal
resolution by effective collapse of J(HH) multiplets.
Strychnine, taxol, staurosporine, and sungucine are utilized as model
compounds to demonstrate the usefulness of these techniques.
Homodecoupled 1,1- and 1,n-Adequate NMR Experiments: Application to the Structural Elucidation of Proton-Deficient Natural Products
Josep Saurí 1, Wolfgang Bermel 2, Alexei V. Buevich 1, Maged H.M. Sharaf 3, Paul L. Schiff 4, Teodor Parella 5, R. Thomas Williamson 1 and Gary E. Martin 1
1 NMR Structure Elucidation, Process and Analytical Chemistry, Merck & Co. Inc., Rahway, NJ 07065, USA
2 Bruker Biospin GmbH, Silberstreifen, 76287 Rheinstetten, Germany
3 American Herbal Products Association, Silver Spring, MD 20910, USA
4 Department of Pharmaceutical Sciences, School of Pharmacy University of Pittsburgh, Pittsburgh, PA 15261, USA
5 Servei de Ressonància Magnètica Nuclear, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain
Pure
shift NMR methods have recently been the subject of intense research
focus. By collapsing homonuclear proton-proton couplings, resolution and
experimental sensitivity both increase. Cryptospirolepine is the most
structurally complex alkaloid discovered thus far from any Cryptolepis.
Characterization of several degradants of the original sample a decade
after the initial report called the validity of the originally proposed
structure in question. We now wish to report the development of improved
homodecoupled variants of 1,1- and 1,n-ADEQUATE (HD-ADEQUATE)
and the utilization of these techniques in resolving long-standing
structural questions associated with crytospirolepine. In addition, we
evaluate the combination of NUS and homonuclear decoupling for the
acquisition of both 1JCC and nJCC homonuclear coupling constants in related J-modulated ADEQUATE experiments.
Extending Long-Range Heteronuclear NMR Connectivities by Modified HSQMBC Experiments
Josep Saurí 2, Núria Marcó 1, R. Thomas Williamson 2, Gary E. Martin 2 and Teodor Parella 1
1 Servei de Ressonància Magnètica Nuclear, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain
2 NMR Structure Elucidation, Process and Analytical Chemistry, Merck & Co. Inc., 126 E. Lincoln Avenue, Rahway, NJ 07065, USA
The detection of long-range heteronuclear correlations associated with J(CH)
coupling values smaller than 1–2 Hz is a challenge in the structural
analysis of small molecules and natural products. HSQMBC-COSY and
HSQMBC-TOCSY pulse schemes are evaluated as complementary NMR methods to
standard HMBC/HSQMBC experiments. The re-optimization of the interpulse
delay and the incorporation of an additional J(HH) transfer
step in the HSQMBC pulse scheme can favor the sensitive observation of
traditionally missing or very weak correlations and, in addition,
facilitates the detection of a significant number of still longer-range
connectivities to both protonated and non-protonated carbons under
optimal sensitivity conditions. A comparative 1H–13C study is performed using strychnine as a model compound and several examples are also provided including 1H–15N applications.
The Defensive Chemistry of the Irish Nudibranch Archidoris pseudoargus (Gastropoda Opisthobranchia)
Ryan Young 1,2 and Bill Baker 1,2
1 National University of Ireland, Galway, Co Galway, Ireland
2 University of South Florida, Tampa, FL, USA
Historically,
marine natural products from the Republic of Ireland have been greatly
underrepresented in the literature despite having a coastline of over
4500 miles. Archidoris pseudoargus is a soft-bodied, slow
moving Dorid nudibranch which inhabits the coastal waters of Ireland and
the United Kingdom. Nudibranchs are a good source of new chemical
diversity, employing these secondary metabolites to deter predation. In
this study we have identified new chemistry as well as used a
metabolomics approach to identify the origin of said chemistry. In early
Spring, mature adults come together to reproduce and shortly thereafter
to oviposit on the subtidal rocky shoreline. These egg sacs can be
brightly colored and exposed to predation, yet none of the many
surrounding predators appear to feed on these nutrient rich egg masses.
We have investigated whether the defensive chemistry of the parents is
responsible for protecting the egg masses.
Structural Elucidation of Secondary Metabolites in Laminaria digitata
Anne Vissers and Jean-Paul Vincken
Harry Gruppen Lab. Food Chemistry, Wageningen University, Wageningen, Gelderland, The Netherlands
For future protein supply, Laminaria digitata is a promising source due to its relatively high protein content (15% w/w
DM) and robustness in North-Sea growing conditions. In order to apply
the seaweed proteins in the feed industry, extraction is needed. The
first step in extraction of proteins requires cell rupture, as a result
of which cell walls and membranes are broken, allows proteins and
secondary defence metabolites to contact each other and interact.
Phlorotannins, polymers of 1,3,5-trihydroxy benzene are important
secondary metabolites exclusively found in brown seaweeds. The monomers
can connect to each other via carbon-carbon and via ether connections
and polymers ranging between 10 and 100 kDa are formed (Amsler 2008).
Due to the large size and abundance of hydroxyl groups, the molecules
can bind non-covalently to proteins via hydrogen bridges and hydrophobic
interactions with the tannin rings, and form phlorotannin-protein
complexes with limited solubility. An additional group of secondary
metabolites is represented by the terpenoids. In order to study the
phlorotannin-protein complexation potential during the process of
protein extraction, a detailed investigation on phlorotannins content,
degree of polymerisation and structure is needed. Chromatographic
analysis of phlorotannins forms a challenge due to the structural
diversity and similar polarities of these molecules. For investigation,
tannins were extracted from Laminaria digitata in 80% methanol
and partitioned with ethyl acetate. The ethyl acetate phase was
subjected to Reversed Phase flash chromatography to separate
co-extracted phlorotannins and terpenoids. Pools of phlorotannins up to a
degree of polymerisation of 15 subunits, and the terpenoid pool were
analysed using RP-UHPLC-MS. The fractionation by preparative
chromatography prior to RP-UHPLC analysis greatly facilitated
identification and quantification of the various phlorotannin and
terpenoid structures.
Initial Attempts in the Purification of the Mediterranean Sponge Crambe tailliezi
Siguara B. L. Silva 1,2, Erwan Poupon 2 and Olivier P. Thomas 1
1 Nice Institute of Chemistry-PCRE,
UMR 7272 CNRS, University Nice Sophia Antipolis, Parc Valrose, 28 Avenue
Valrose, 06108 Nice, France
2 BioCIS, Faculté de Pharmacie, University Paris-Sud, 5 rue J-B Clément, 92296 Châtenay-Malabry, France
First
described in 1982 by Vacelet and Boury-Esnault, Crambe tailliezi
(Crambeidae, Poecilosclerida, Demospongiae) is an encrusting cream
sponge found in the Mediterranean and the Macaronesian Sea where its
abundance seems to increase along the French Riviera (between 15 and 40
m). Even if the sister species C. crambe has been widely studied before, there is no work concerning the isolation and identification of chemicals from C. tailliezi. Considering the very interesting alkaloids isolated from Crambeidae sponges, such as the crambescins and crambecidins from C. crambe, we decided to investigate the chemodiversity of C. tailliezi.
The first results on the isolation and structure elucidation of the
major compounds present in this species were found to be extremely
complicated. However, we were able to recognize guanidine alkaloids
similar to the already described batzelladines. This would be the first
report of bioactive batzelladine derivatives in a Mediterranean sponge
as most of them were found in Caribbean sponges like from species of the
genus Batzella or Monanchora. The NMR and HRMS analyses also
allowed the identification of the already described compounds
crambescidin 816 and crambescidin 800. These compounds have been first
isolated from C. crambe and it is worth noticing that both C. crambe and C. tailliezi
share common secondary metabolites, what could raise interesting
questions about the divergent evolutionary history of metabolic pathways
in these two sister species.
Bioactive Compounds from Marine Bacteria Recovered from Sediments Collected at the St. Peter and St. Paul Archipelago, Brazil
Alison Batista Silva 1, Elthon G Ferreira 2, Karine Pires 2, Paula C Jimenez 3, Letícia Veras Costa-Lotufo 4, Otília Deusdência Pessoa 1, Edilberto R Silveira 1 and Maria Conceição M Torres 1
1 Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil
2 Instituto de Ciências do Mar, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil
3 Departamento de Ciências do Mar, Universidade Federal de São Paulo, Santos, São Paulo, Brazil
4 Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil
The
extension, localization and ecological aspects of the Brazilian coastal
zone makes it a remarkable reservoir of compounds with pharmacological
potential. Marine actonomycete have shown to be an attractive source of
structurally diversified bioactive compounds. The main purpose of this
study was to prospect antitumor compounds through a cytotoxicity-guided
fractionation approach of the actinomycete strains Salinispora arenicola
and Streptomyces sp. recovered from sediments collected at the
Saint Peter and Saint Paul’s Archipelago (SPSPA)—Brazil. Fractionation
of the ethyl acetate crude extract obtained from S. arenicola (IC50
0.55 μg/mL) resulted in two cytotoxic fractions. HR-LCMS dereplication
analysis of such fractions combined with comparison in the AntiMarin
database indicated the presence of staurosporine derivatives (1–4),
a well known group of anticancer compounds. Chromatographic separations
of the inactive fractions led to the isolation of a new rifamycin
derivative, 3-(2′-oxo-propyl)-rifamycin S (5), in
addition to five known metabolites, including 6-methoxy-1-methylisatin,
3-hydroxy-6-methoxy-1-methylindolin-2-one and three diketopiperazines.
The structures of these compounds were elucidated through a series of 1D
and 2D NMR experiments and HRMS. Bioassay-guided fractionation of the
ethyl acetate extract from Streptomyces sp. (IC50 of 2.27 μg/mL) afforded highly cytotoxic antibiotics piericidin A (6) and glycopiericidin A (7),
as well as three known diketopiperazines from the active fractions. The
identification of these compounds was carried out by analyses HR-LCMS,
followed by query to the AntiMarin data base. These results highlight
the biotechnological potential of the actonomycete strains recovered
from St. Peter and St. Paul Archipelago.
Secondary Metabolites and Their Biological Acitivity of the Marine-Derived Fungus Stemphylium globuliferum
Jan Schrör 1, Peter Hufendiek 1, Stefan Kehraus 1, Michael Gütschow 2 and Gabriele M. König 1
1 University of Bonn, Institute of Pharmaceutical Biology, Bonn, Germany
2 University of Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, Bonn, Germany
Fungi
are well known to be a rich source of structurally complex and
biologically active compounds. The aim of the current study was to
investigate metabolites of the marine-derived fungus Stemphylium
globuliferum. The strain was isolated from the brown alga Petalonia
zosterifolia collected in the Baltic Sea. Investigation of the extract
revealed several metabolites belonging to two structural classes, i.e.,
dialkylresorcinols and macrolides. Thus, after separation via vacuum
liquid chromatography and further purification using HPLC we gained the
resorcinol-type metabolite 4-butyl-3,5-dihydroxy-benzoic acid, a new
natural product. Another compound isolated was stemphol, a
2,5-dialkylresorcinol. This known metabolite showed activity against
multi-resistant Staphylococcus aureus, Enterococcus faecium as well as against Candida albicans.
In addition, a weak inhibitory activity toward the protease humane
leucocyte elastase (HLE) was found. A third metabolite was the
14-membered lactone coriolide, which was formerly reported from
butterflies. This macrolide was active against Bacillus megaterium.
Furthermore, coriolide displayed a specific inhibitory activity towards
HLE with an IC50 of 5 μg/mL, while its activity towards cathepsin B and
L was about 10-fold weaker. The isolated metabolites from the fungus Stemphylium globuliferum showed a promising antibiotic activity against selected microorganisms as well as towards the protease HLE.
Isolation and Structural Elucidation of Two Novel Pinnatifidenyne-Derived Acetogenins from Laurencia viridis
Adrián Morales Amador 1,2, Caterina Rodríguez de Vera 1,2, Olivia Márquez Fernández 3, Antonio Hernández Daranas 1,4, José Javier Fernández 1,2, Manuel Norte 1,2 and María Luis Souto 1,2
1
University Institute of Bio-Organic Chemistry “Antonio González”,
Center for Biomedical Research of the Canary Islands (CIBICAN),
University of La Laguna, La Laguna 39206, Canary Islands, Spain
2 Department of Organic Chemistry, University of La Laguna, La Laguna 39206, Canary Islands, Spain
3 Laboratorio de Alta Tecnología de Xalapa LATEX—Universidad Veracruzana, Xalapa, Veracruz, Mexico
4 Department of Chemical Engineering and Pharmaceutical Technology, University of La Laguna, La Laguna, Tenerife, Spain
The red alga of the genus Laurencia (Rhodomelaceae)
include many species widely distributed around the world, being the
main red algal genus chemically studied in the last 50 years [1]. They
are producers of a vast range of interesting halogenated secondary
metabolites, including sesquiterpenes, diterpenes, triterpenes, and
acetogenines. Many of these compounds are unique in terms of structural
or biological diversity, often exhibiting antibacterial, antifungal,
antiviral, antifeedant, antifouling, cytotoxic, antiproliferative,
anti-inflammatory, ichthyotoxic and insecticidal activity [2]. As part
of our continuing interest on the chemistry of the genus Laurencia, we report the isolation of two new C15 acetogenins from Laurencia viridis [3,4].
The structures were elucidated on the basis of detailed analysis of 1D
and 2D NMR data and revealed that these two compounds are interesting
variations on the pinnatifidenyne structure [5].
Acknowledgments
To
MINECO SAF2011-28883-C03-01 and FP7-REGPOT-2012-CT2012-316137-IMBRAIM
projects. C.R. de Vera to MINECO FPU program. The research group
acknowledge the financing granted to ULL by Agencia Canaria de
Investigación, Innovación y Sociedad de la Información, being 85%
cofinanced by the European Social Fund.
References
- Blunt, J.W.; Copp, B.R.; Keyzers, R.A.; Munro, M.H.G.; Prinset, M.R. Marine natural products. Nat. Prod. Rep. 2015, 32, 116–211, and earlier reviews in this series.
- Wang, B.G.; Gloer, J.B.; Ji, N.Y.; Zhao, J.C. Halogenated organic molecules of Rhodomelaceae origin: Chemistry and biology. Chem. Rev. 2013, 113, 3632–3685.
- Gutiérrez-Cepeda, A.; Daranas, A.H.; Fernández, J.J.; Norte, M.; Souto, M.L. Stereochemical Determination of Five-Membered Cyclic Ether Acetogenins Using a Spin-Spin Coupling Constant Approach and DFT Calculations. Mar. Drugs 2014, 12, 4031–4044.
- Gutiérrez-Cepeda, A.; Fernández, J.J.; Gil, L.V.; López-Rodríguez, M.; Norte, M.; Souto, M.L. Nonterpenoid C15 acetogenins from Laurencia marilzae. J. Nat. Prod. 2011, 74, 441–448.
- Norte, M.; Fernández, J.J.; Cataldo, F.; González, A.G. E-Dihydrorhodophytin, AC 15 acetogenin from the red alga Laurencia pinnatifida. Phytochemistry 1989, 28, 647–649.
Cytotoxic Anomoian B and Aplyzanzine B, New Bromotyrosine Alkaloids from Two Indonesian Sponges
Jaime Rodríguez 1, Guillermo Tarazona 2, Patricia G. Cruz 2, Rogelio Fernández 2, Marta Penas 2, Carmen Cuevas 2 and Carlos Jiménez 1
1 Departamento de Química
Fundamental, Facultade de Ciencias and Centro de Investigaciones
Científicas Avanzadas (CICA) Universidade da Coruña, 15071 A Coruña,
Spain
2 Medicinal Chemistry
Department, PharmaMar S.A.U., Pol. Ind. La Mina Norte, Avda. De los
Reyes, 1, 28770-Colmenar Viejo, Madrid, Spain
Marine
sponges belonging to the order Verongida are devoid of spicules [1],
and are difficult to characterize. As such, bromotyrosine derivatives
that are characteristic of these types of sponges have been used as
chemotaxonomic makers, and have proved a useful tool to facilitate
taxonomy identification [2]. Examples of such compounds that have been
used for taxonomic work are anomoian A [3] and aplyzanzine A [4]. In a
continuation of our investigation of new marine natural products, we now
report the isolation of two new bromotyrosine derivatives, anomoian B
and aplyzanzine B, that may also have utility as new chemotaxonomic
makers and that have been isolated from two different Verongida sponges,
collected off the coast of Indonesia. The structures of anomoian B and
aplyzanzine B were determined by 1D and 2D NMR experiments and confirmed
by high-resolution mass spectrometry. The stereochemistry of the new
molecules has been assigned by comparison with literature models [3].
Both compounds showed moderated cytotoxic activity against a panel of
different cancer cell lines [5], and their mechanism of action is
currently being studied.
References
- Ehrlich, H.; Ilan, M.; Maldonado, M.; Muricy, G.; Bavestrello, G.; Kljajic, Z.; Carballo, J.L. Three-dimensional chitin-based scaffolds from Verongida sponges (Demospongiae: Porifera). Part I. Isolation and identification of chitin. Int. J. Biol. Macromol. 2010, 47, 132–140.
- Gotsbacher, M.; Karuso, P. New Antimicrobial Bromotyrosine Analogues from the Sponge Pseudoceratina purpurea and Its Predator Tylodina corticalis. Mar. Drugs 2015, 13, 1389–1409.
- Kottakota, S.K.; Evangelopoulos, D.; Alnimr, A.; Bhakta, S.; McHugh, T.D.; Gray, M.; Groundwater, P.W.; Marrs, E.C.L.; Perry, J.D.; Spilling, C.D.; et al. Synthesis and biological evaluation of purpurealidin E-derived marine sponge metabolites: aplysamine-2, aplyzanzine A, and suberedamines A and B. J. Nat. Prod. 2012, 75, 1090–1101.
- Evan, T.; Rudi, A.; Ilan, M.; Kashman, Y. Aplyzanzine A, a new dibromotyrosine derivative from a Verongida sponge. J. Nat. Prod. 2001, 64, 226–227.
- Carter, D.C.; Moore, R.E.; Mynderse, J.S.; Niemczura, W.P.; Todd, J.S. Structure of majusculamide C, a cyclic depsipeptide from Lyngbya majuscule. J. Org. Chem. 1984, 49, 236–241.
Antimicrobial Metabolites from South African Laurencia spp.
Jameel Fakee 1, Kim A. Durrel 2, Marilize le Roes-Hill 2, John J. Bolton 3 and Denzil R. Beukes 4
1 Rhodes University, Grahamstown, South Africa
2 Cape Peninsula University of Technology, Bellville, South Africa
3 University of Cape Town, Rondebosch, South Africa
4 University of the Western Cape, Bellville, South Africa
The
rapid development of resistance against common antibiotics combined
with the slow pace of new antimicrobial drug discovery and development
present a significant health risk. We have therefore initiated a
programme to explore the antimicrobial potential of South African marine
organisms. A number of organic extracts from Laurencia spp. collected from the South African coast were screened against five biomedically relevant microorganisms, Acinetobacter baumannii, Enterococcus faecalis, Escherichia coli, Staphylococcus aureus subsp. aureus and Candida albicans. One of the more promising extracts was from the red alga, Laurencia corymbosa
which was selected for further studies. Some 30 metabolites were
isolated and characterized by spectroscopic methods. Two of the more
active metabolites include the chamigrane (1), which showed a minimum inhibitory activity (MIC) of 1 μg/mL against E. faecalis while the cuparene (2) showed the same level of activity against A. baumannii.
In Vitro Shistossomicial Activity of the Ascidians Botrylloides giganteum, Didemnum sp. and Trididemnum orbiculatum from Brazil
Luis Claudio Kellner Filho 1, Rita Cássia Nascimento Pedroso 1, Gustavo Muniz Dias 2, Lizandra Guidi Magalhães 1, Marcio Luis Andrade e Silva 1, Wilson Roberto Cunha 1, Patricia Mendonça Pauletti 1, Victoria Helen Woolner 3, Peter Thomas Northcote 3 and Ana Helena Januario 1
1 Universidade de Franca, Franca, São Paulo, Brazil
2 Universidade Federal do ABC, São Bernando do Campo, São Paulo, Brazil
3 Victoria University of Wellington, Wellington, New Zealand
The ascidians Botrylloides giganteum, Didemnum sp. and Trididemnum orbiculatum
marine species are widely distributed in the São Sebastião Channel, São
Paulo, Brazil. Ascidias (Tunicata, Ascidiacea) have yielded a variety
of structurally novel and pharmacologically interesting compounds
however, studies related to potential schistosomicidal about these
genera are non-existent. Schistosomiasis is one of the most significant
neglected diseases in the world. The aim of this research was to
evaluate the schistosomicidal potential of the crude methanolic extracts
of B. giganteum (BG), Didemnum sp. (DS) and T. orbiculatum (TO) against adult worms of Shistosoma mansoni.
The extracts were tested for viability and motor activity at the
concentrations 50 and 100 μg/mL. Regarding the mobility, TO extract
reduced on 100% of motor activity at 50 μg/mL after 72 h, while DS
extract decreased the motor activity on 100% of parasites at 100 μg/mL
after 24 h. In contrast, the BG extract was inactive. No extract
elicited death of the parasites. Despite the inactivity, the proton NMR
spectrum of a semi-purirfed fraction from the BO extract revealed
interesting signals which prompted further investigation. Combinations
of normal and reversed-phase column chromatography and HPLC-ELSD of BG
extract conduced the isolation of the aliphatic sulfated compound (3Z,6Z,9Z)-3,6,9-Dodecatrien-1-yl
hydrogen sulfate (1). The structural elucidation of 1 was established
by NMR spectroscopic and mass spectrometric analysis TOF-MS (−): m/z 259.1009 (C12H19O4S). This compound was previously isolated as a kairomone secreted by the crustacean Daphnia magna.
Search for New Natural Products in Marine Actinomycetales: First Insights into the Secondary Metabolites of Williamsia maris
Andrea Lubich 1, Liselotte Krenn 1, Martin Zehl 1,2 and Hanspeter Kaehlig 3
1 Department of Pharmacognosy, Faculty of Life Sciences, University of Vienna, Vienna, Austria
2 Department of Pharmaceutical Chemistry, Faculty of Life Sciences, University of Vienna, Vienna, Austria
3 Institute of Organic Chemistry, University of Vienna, Vienna, Austria
The
marine environment is recognized as the space with the highest
biodiversity on earth and is hence the richest source for new lead
structures in drug development. In this connection, microbes are
supposed to produce the largest number and variety of marine secondary
metabolites. Among microorganisms, bacteria of the order Actinomycetales
are most promising for the elucidation of new structures as 45% of all
previously discovered microbial secondary metabolites were isolated from
members of this order. This fact prompted us to focus our search for
new natural products on marine Actinomycetales. In our project, the
scarcely examined strain Williamsia maris, which was initially
isolated from the Sea of Japan, was obtained from DMSZ (Braunschweig,
Germany) and has been cultivated in a GYM-medium (0.4% glucose, 0.4%
yeast extract, 1% malt extract) for 14 days. By centrifugation, the
cells were removed and the resin Amberlite XAD-16 was added to the
fermentation broth to adsorb released metabolites. After two days of
incubation, the adsorbate was eluted from the resin with acetone to gain
1.5 g of dried crude extract. This extract was fractionated by SPE on
RP-18 cartridges into 18 fractions. Subsequent size-exclusion
chromatography led to the isolation of three compounds. Their structures
were determined by high-resolution MS and NMR spectroscopy as
lumichrome, indole-3-carbaldehyde and a 3-O-methyl mannose
polysaccharide. LC-MS analysis of another fraction of the extract
indicates the presence of two cyclic dipeptides, with the masses and
fragmentation patterns correlating with those of cyclo(Ile-Pro) and
cyclo(Leu-Pro). All five substances are reported in the strain Williamsia maris for the first time.
Industrial Biotechnology and Polymers and Biomolecules
Biocare Marine: Biomolecules of the Sea for Environmental Remediation and Healthcare
Christine Delbarre-Ladrat, Laetitia Kolypczuk, Delphine
Passerini, Frédérique Chevalier, Jacqueline Ratiskol, Corinne Sinquin,
Agata Zykwinska, Françoise Leroi and Sylvia Colliec-Jouault
Ifremer, Nantes, France
BioCare
marine was a two-year collaborative research project (2012–2014) funded
under the EU, Interreg 2-Seas programme. It aimed to isolate,
characterise and sustainably utilise marine biomolecules from seaweeds,
fish, oysters, shrimps and bacteria. The biomolecules were evaluated to
be integrated into functional products for human healthcare and
environmental applications. The ocean represents a vast and relatively
untapped resource where the organisms therein have evolved a myriad of
mechanisms to survive in this changing and demanding environment. These
include antimicrobial substances to help out-compete neighbouring
organisms, polysaccharides to prevent dehydration and provide structure
and defences against toxic metal poisoning. The scientific objectives of
Biocare Marine were to: (i) discover and functionalise new
antimicrobial compounds (ii) construct novel wound dressings and tissue
regeneration scaffolds using marine biomolecules that hold great promise
for the treatment of chronic wounds, and for the remodelling and
reconstruction of skin in burn victims (iii) utilise the specialised
polysaccharides produced by marine bacteria to construct heavy metal
capture-systems using an advanced technology platform. Toxic heavy
metals cause environmental and health damage and present a significant
threat to human wellbeing. Our consortium consisted of the University of
Brighton (UK), University of Gent (Gent, Belgium), Ifremer (a French
governmental laboratory, Plouzanr, France) and Polymaris (a marine
biotech company, Morlaix, France). The poster will be particularly
dedicated on bacteria and molecules from Ifremer collection.
Bioremediation of Heavy Metals Using Cyanothece sp. CCY 0110 Cultures or Its Released Polysaccharides (RPS)
Rita Mota 1,2, Federico Rossi 3, Sara B. Pereira 1, Ângela Brito 1,2, Roberto de Philippis 3,4, and Paula Tamagnini 1,2
1 i3S—Instituto de Investigação e
Inovação em Saúde & IBMC—Instituto de Biologia Molecular e Celular,
Universidade do Porto, Porto, Portugal
2 Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Porto, Portugal
3 Department of Agrifood Production and Environmental Sciences, University of Florence, Florence, Italy
4 Institute of Ecosystem Study (ISE), National Research Council (CNR), Sesto Fiorentino, Italy
Many
cyanobacteria can produce extracellular polymeric substances (EPS) that
can remain associated to the cell or be released into the environment
(RPS-released polysaccharides). The particular features of these
polymers, namely the presence of two different uronic acids, sulphate
groups and high number of different monosaccharides, makes them
promising for biotechnological applications such as metal removal from
polluted waters. Cyanothece sp. CCY 0110, a marine unicellular
cyanobacterium, is among the most efficient RPS-producers. The polymer
produced is remarkably thermostable, composed by nine different
monosaccharides, and contains sulfate groups and peptides [1]. The
effects of several heavy metals on the growth/survival, EPS production,
ultrastructure and protein profiles of Cyanothece were also
evaluated. The results showed that each metal affect the cells in a
particular manner, triggering distinctive responses [2]. For
optimization of the metal removal process, it is necessary to understand
the interactions between cells/EPS with metal ions. Therefore, the
affinity of various culture fractions for different metals in mono- and
multi-metal systems was assessed. Our results clearly showed that RPS
are the most efficient fraction in metal-adsorption. Moreover, an acid
or basic pre-treatment of RPS increased the specific metal removal.
Currently, major sites for metal binding are being identified by
physicochemical analyses.
References
- Mota, R.; Guimarães, R.; Büttel, Z.; Rossi, F.; Colica, G.; Silva, C.J.; Santos, C.; Gales, L.; Zille, A.; De Philippis, R.; et al. Production and characterization of extracellular carbohydrate polymer from Cyanothece sp. CCY 0110. Carbohydr. Polym. 2013, 92, 1408–1415.
- Mota, R.; Pereira, S.B.; Meazzini, M.; Fernandes, R.; Santos, A.; Evans, C.A.; De Philippis, R.; Wright, P.C.; Tamagnini, P. Effects of heavy metals on Cyanothece sp. CCY 0110 growth, extracellular polymeric substances (EPS) production, ultrastructure and protein profiles. J. Proteom. 2015, 120, 75–94.
Anti-Coagulant Potential of Polysaccharide-Rich Fractions of Macroalgae and Influence of Depolymerization and Sulfation
Amandine Adrien 1,2, Nicolas Bridiau 1, Delphine Dufour 2, Stanislas Baudouin 2 and Thierry Maugard 1
1 LIENSs Laboratory, UMR 7266 CNRS-University of La Rochelle, La Rochelle, France
2 SEPROSYS, Séparations Procédés Systèmes, La Rochelle, France
Macroalgae
have been used in traditional medicine for centuries and perceived as a
food with great health benefits. They are an important source of
polysaccharides, such as sulfated polysaccharides, which are responsible
for many of their potential properties that might find relevance in
nutraceutical, pharmaceutical and cosmeceutical applications. Nowadays,
to treat health troubles linked to thrombo-embolic complication, most of
the anticoagulant treatments are heparin-based. Despite its major
anticoagulant activity, heparin can cause serious adverse events.
Moreover, its low bioavailability makes such a treatment really
expensive. Given the risks and high costs of these treatments, there is a
compelling need for further investigation of new sources of
anticoagulants. To study the anticoagulant potential of sulfated
polysaccharides from macroalgae, different extracts were prepared using
various extraction processes leading to enriched polysaccharides
fractions. Extracts from six edible seaweeds, including brown (Laminaria digitata, Fucus vesiculosus, Himanthalia elongata, Ascophyllum nodosum), green (Ulva lactuca), and red (Chondrus crispus)
macroalgae, were prepared and the biochemical composition of each
extract was determined. The potential anticoagulant activity of each
extract was also investigated at different scales, from the specific
antithrombin-dependent pathway (anti-Xa and anti-IIa) to the intrinsic
and/or common (Activated Partial Thromboplastin Time), extrinsic
(Prothrombin Time) or common (Thrombin Time) anticoagulant pathways.
Furthermore, the extract anticoagulant properties were compared with
those of commercial anticoagulants: heparin and Lovenox®. To
have a better understanding of the structure-function relations for the
anticoagulant activity of sulfated polysaccharides, extracts from green
macroalgae (Ulvas) were also prepared and purified according to
a “green” industrial process using neither acid nor solvent. Those
polysaccharides were then submitted to chemical modifications
(sulfation) or physical modifications (depolymerization assisted by
ultrasounds or ion exchange resin).
Identification, Expression and Characterization of Marine Polysaccharide Degrading Enzymes from Novel Bacteria Isolated from Intertidal Biotopes
Varsha Kale 1,2, Olafur Fridjonsson 1, Sesselja Omarsdottir 2, Solveig Petursdottir 1, Bryndis Bjornsdottir 1, Brynjar Ellertsson 1, Solveig Olafsdottir 1 and Gudmundur Hreggvidsson 1,2
1 Matis ohf, Reykjavik, Iceland
2 University of Iceland, Reykjavik, Iceland
Intertidal
areas are often covered with algae containing a variety of complex
polysaccharides as well as invertebrates harbouring “unconventional”
structural polysaccharides. Organisms in these environments need to
tolerate extreme conditions, e.g., wave action, radiation and
fluctuating temperatures, osmolarity, and pH. Many of the adaptive
strategies adopted by the organisms depend on polysaccharides of
different structures and properties. Marine polysaccharides contain all
the lignocellulose sugars, but also “rare sugars”, e.g., deoxy sugars,
sugar acids and sugar alcohols, which are often complex, branched and
highly substituted. As the polysaccharides are an abundant source of
carbon, microbes from the unique intertidal biotopes are expected to be a
valuable source of scientifically interesting and industrially
applicable polysaccharide processing enzymes. A number of genomes from
novel marine bacteria, isolated from intertidal areas, including
geothermal biotopes, were sequenced. Following in-depth bioinformatic
analysis, selected genes encoding various carbohydrate enzymes were
expressed including enzymes capable of degrading complex marine
polysaccharides, e.g., chondroitin sulfate, chitin, laminarin, fucoidan
and alginate. Among those were the α-l-fucosidase
Aful2 from Litorilinea aerophila MAT4131; chondroitin lyase ChoA1 and
the sulfatase SulA2 from the Arthrobacter strain MAT3885. Both strains
were isolated from marine environments following enrichment on
chondroitin sulfate containing media. Functional analysis of the enzymes
revealed that the fucosidase AfuL2 was active on natural substrates
such as fucoidan from Laminaria digitata and Fucus vesiculosus.
Addition of the sulfatase SulA2 improved the breakdown of the
substrates. Furthermore, the release of fucose from sea cucumber derived
fucosylated chondroitin sulfate, induced by AfuL2, was aided by
co-incubation with SulA2 and the chondroitin lyase ChoA1. Accordingly,
the marine enzymes were capable of reacting with sulfated
polysaccharides in a concerted action.
Fucosterol from Padina Australis Protects Sh-Sy5y Cells against Amyloid-Induced Neurotoxicity by Enhancing the Expression of Neuroglobin
Li Zhe Wong 1, Sook Yee Gan 2 and Eng Lai Tan 2
1 School of Postgraduate Studies, International Medical University, Kuala Lumpur, Malaysia
2 Life Sciences, School of Pharmacy, International Medical University, Kuala Lumpur, Malaysia
Alzheimer’s
disease (AD) is a neurodegenerative disease that leads to progressive
loss of neurons which often results in deterioration of memory and
cognitive function. The development of AD is highly associated with the
formation of plaques within the dying cells in the brain and tangles
within the neurons. Recent evidences highlighted the importance of
neuroglobin (NGB) in ameliorating AD by protecting the neuronal cells.
However, the underlying protective mechanisms remain to be elucidated.
This study assessed the ability of fucosterol, a compound isolated from
Padina australis, in inducing the expression of NGB gene in
neuroblastoma cell line. SH-SY5Y cells were exposed to different
concentrations of fucosterol in the presence of beta-amyloid. The effect
on apoptosis was determined using Annexin V FITC staining and the
expression of NGB was determined using real-time PCR based on SYBR
Green. Flow cytometry confirmed the protective effects on SH-SY5Y cells
were significant when 5 μg/mL of fucosterol (7.47% double positive for
Annexin V and propidium iodide as compared to 9.88% in negative
control). Furthermore, results from both real-time PCR and western blot
showed that fucosterol significantly induced the expression of NGB in
SH-SY5Y with 4 fold increase of NGB expression as compared to the
negative control. Finally, we showed that inhibition of NGB through
aldrithiol significantly decreased the viability of SH-SY5Y with 41.51%
of cell populations under the late apoptosis. This study has thus
provided evidences that fucosterol was able to protect SH-SY5Y by
enhancing the expression of NGB.
Protein Isolation and Comparison from Various Green Sources
Emma Teuling 1, Peter Wierenga 1, Johan Schrama 2 and Harry Gruppen 1
1 Laboratory of Food Chemistry, Wageningen University, Wageningen, The Netherlands
2 Aquaculture and Fisheries Group, Wageningen University, Wageningen, The Netherlands
Green
sources like algae and leaves are gaining interest as alternative
protein sources for food and feed products. These green sources are
biologically diverse, ranging from microcellular marine organisms to
terrestrial multicellular organisms. The aim of this project is to get
more insight into the similarities and differences between green sources
and how these differences affect protein isolation and the
techno-functionalities of the protein isolates obtained. Protein sources
were selected from various unicellular photosynthetic phyla: Arthrospira sp. (cyanobacteria), Nannochloropsis gaditana (heterokontophyta) and Tetraselmis sp. (chlorophyta). The protein contents of these materials were found to be 37%, 42% and 57% for respectively Tetraselmis, Nannochloropsis and Arthrospira.
The total carbohydrate content was similar for all sources, ranging
from 8% to 11%. Total lipid contents ranged from 15% to 24%. Since all
three organisms were protein-rich but contained different types of
proteins and carbohydrates, they are good model organisms for comparing
protein isolation from unicellular, green sources. A mild protein
isolation process was developed, allowing application of the obtained
protein isolates in food and feed. After cell disruption 33%, 61% and
80% of the total biomass protein was extracted as soluble protein from Tetraselmis, Nannochloropsis and Arthrospira,
respectively. After further purification, the total yield (5%–9%) and
purity (66%–74%) of the protein isolates were similar. The microalgal
isolates obtained were colourless, while the cyanobacterial protein
isolate had an intense blue colour. To conclude, the developed isolation
process can be applied to obtain protein isolates from various sources
with similar purities and yields. The effects of the differences in
protein composition and gross composition of the isolates on the
techno-functional properties in food will be assessed by screening their
solubility, emulsifying, gelling and foaming properties. These results
will provide a better understanding concerning the differences and
similarities of proteins from various green sources.
Screening of New Anti-Microbials from Marine Bacteria
Laetitia Kolypczuk 1, Christine Delbarre-Ladrat 1, Michel Dion 2, Delphine Passerini 1, Frédérique Chevalier 1 and Françoise Leroi 1
1 IFREMER—BRM/EM3B, Nantes, France
2 University—UFIP, Nantes, France
Marine
environment is a rich and relatively little studied source of new
functional compounds as antimicrobials. Marine bacteria have developed a
range of molecules to survive and colonize this very competitive and
challenging environment. We have an important and original collection of
more than 4000 marine bacteria strains isolated from deep-sea
hydrothermal vents, from seafood products, from microalgae culture and
from an hypersaline lake of Kenya. The aim of our study is to screen our
collection to discover innovative anti-microbial activities to fight
the growing threat of broad spectrum antibiotic resistant infections in
human and animals health. All of our bacteria are screened against 15
selected targets bacteria, a range of Gram-positive and Gram-negative
bacteria, pathogens for human, for aquaculture organisms or involved in
food spoilage. Our first results are promising.
Collagen from Marine Sponges: Comparative Studies on Their Morphological and Biochemical Characterization
Leto-Aikaterini Tziveleka, Evangelia Foufa, Efstathia Ioannou and Vassilios Roussis
Department of Pharmacognosy and Chemistry of Natural Products, Faculty of Pharmacy, University of Athens, Athens, Greece
Marine
organisms have been proven an inexhaustible source of potent bioactive
compounds. Between the diverse and multiple pharmacological properties
of marine metabolites, bioactive natural macromolecules have gained
interest, since such biomacromolecules possess a wide range of
biomedical applications. Collagen is a ubiquitous high molecular weight
fibrous protein found in all multicellular organisms. Collagen
polypeptide chains are organized in three α-helices wrapping around one
another forming its characteristic triple helix tertiary structure. This
natural biomaterial has a wide range of applications in the
health-related fields. Three species of marine demosponges, Axinella cannabina, Suberites carnosus and Plakortis simplex,
all collected from the Aegean Sea, were comparatively studied for the
determination of their total collagen, intercellular collagen and
spongin contents. The isolated collagens were morphologically,
physicochemically and biochemically characterized. The yield of total
collagen, intercellular collagen and spongin was respectively, 12.6%,
0.1%, and 4.2% dry weight for A. cannabina and 5.0%, 0.4%, and 10.4% dry weight for S. carnosus, while for P. simplex only
0.8% dry weight of spongin was isolated. Light microscopy observations
showed fibrous structures, while scanning electron microscopy confirmed
the presence of collagen in the isolated samples. The measured IR
spectra were characteristic for proteins of the collagen class. The
acid-base properties of the material were investigated by titration,
which also showed that sponge collagen was insoluble in dilute acid
media, while dispersion of collagen was facilitated in dilute basic
media. Since collagen from cattle entails the risks of allergic
reactions and possible connective tissue disorders, such as arthritis
and lupus, and more seriously, of bovine spongiform encephalopathy and
transmissible spongiform encephalopathy, a safer source can be marine
sponges. Our results suggest that, if efficient aquaculture can be
established, A. cannabina and S. carnosus should be considered as alternative sources of collagen.
Comparison of Microwave- and Freeze-Drying of Sargassum muticum for Fucoxanthin Extraction
Elena M. Balboa 1,2, Noelia Flórez 1,2, Enma Conde 1,2, Andrés Moure 1,2 and Herminia Domínguez 1,2
1 Department of Chemical Engineering, Faculty of Sciences, University of Vigo-Campus Ourense As Lagoas s/n, 32004 Ourense, Spain
2 CITI-Universidade de Vigo, Parque Tecnolóxico de Galicia, Rúa Galicia n° 2, 32900 Ourense, Spain
Sargassum muticum (Yendo)
Fensholt is an invasive brown macroalga in European waters. In order to
control its expansion and because its eradication is not feasible, huge
amounts of algal biomass are collected from coastal waters with any
valuable destination but the direct application to agricultural lands. S. muticum
(Sm) biomass is available and susceptible to be valorized to obtain new
and active fractions and compounds for cosmetic, pharmaceutical and
food industries. As a brown macroalgae, Sm is a potential source for
carotenoids extraction, as fucoxathin. Several activities for this
pigment have been reported: antidiabetic, cholesterol-metabolism
control, anti-obesity, antioxidant, cancer cells anti-proliferation, and
anti-inflamatory [1]. Fucoxanthin-containing extracts have been
obtained by absolute ethanol extraction of freeze-dried Sm as previously
reported [2]. Recent data showed an increased availability of the
components of Sm algal material by microwave extraction, caused by cell
wall structure destruction [3]. Therefore, microwave extraction was
proposed as a preparative process before fucoxanthin extraction and as
alternative to freeze-drying. Sm was collected (July 2012, Praia
Mourisca, Pontevedra, Spain) washed and stored until use. A multimode
microwave extractor (NEOS-GR, MilestoneSrl, Italy) was used for algal
drying by using different two-steps combinations of time-power profiles
(5–10 min/200–800 W). Microwave-dried Sm samples were subjected to
ethanol extraction and resulting extracts were compared to those
obtained from freeze-dried Sm [2]. Differences on solubles and
fucoxanthin extraction yields were determined gravimetrically and by
HPLC using a fucoxanthin standard, respectively. Total phenolic content
was measured g gallic acid equivalents/g extract [4], and radical
scavenging capacity of extracts was expressed as g Trolox/g [5] extract
and as IC50, DPPH (g/L) [6]. Results are discussed
considering the moisture of initial Sm and the preferred use of cheaper
and efficient technologies and solvent reduction.
References
- Kanda, H.; Kamo, Y.; Machmudah, S.; Wahyudiono, E.Y.; Goto, M. Extraction of fucoxanthin from raw macroalgae excluding drying and cell wall disruption by liquefied dimethyl ether. Mar. Drugs 2014, 12, 2383–2396.
- Conde, E.; Moure, A.; Dominguea, H. Supercritical CO2 extraction of fatty acids, phenolics and fucoxanthin from freeze-dried Sargassum muticum. J. Appl. Phycol. 2015, 27, 957–963.
- Perez, L.; Conde, E.; Dominguea, H. Microwave hydrodiffusion and gravity processing of Sargassum muticum. Process Biochem. 2014, 49, 981–988.
- Singleton, V.L.; Rossi, J.A., Jr. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Viticult. 1965, 16, 144–158.
- Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 1999, 26, 1231–1237.
- Von Gadpw, A.; Joubert, E.; Hansmann, C.F. Comparison of the antioxidant activity of rooibos tea (Aspalathus linearis) with green, oolong and black tea. Food Chem. 1997, 60, 73–77.
Different Methods Fucoidan Extraction from Sargassum muticum
Noelia Flórez 1,2, María Parada 1,2, Elena M. Balboa 1,2, M. J. González-Muñoz 1,2 and Herminia Domínguez 1,2
1 University of Vigo, Ourense, Spain
2 Citi-university of Vigo, Ourense, Spain
Sargassum muticum
(Sm) is a marine brown seaweed that contains alginate, laminaran and
sulfated polysaccharides known as fucoidans. Fucoidans are found in the
cell walls but the amount varies seasonally and depends primarily on the
algae species. Studies have indicated that fucoidans are increasing
pharmaceutical interest due to their versatile bioactivity (1).
Anticoagulant, antitumor, antiviral, antiinflamatory activities have
been studied (2). Crude fucoidans can be extracted from brown algae with
different technologies; hydrothermal processing (3) and enzymatic
hydrolysis (4) are suitable for the fractionation of vegetal biomass.
During these processes, simultaneous extraction and depolymerisation of
the fucoidan take place. In this study, two aqueous extraction methods
have been proposed and compared for the solubilization and extraction of
polysaccharide components and fractions with antioxidant properties
from the invasive brown alga Sargassum muticum. Liquors obtained from autohydrolysis and enzymatic methods were analyzed and compared. S. muticum
was collected in Praia da Mourisca (Pontevedra, Spain) in June 2012,
washed with water and stored at −18 °C before use. Sm was mixed with
water at a liquid:solid ratio of 30:1 (w/w, dry basis)
and heated in a stainless steel reactor up to 170 °C (Parr Instr. Co.,
Moline, IL, USA), the reactor was quickly cooled and the liquid and
solid phases were separated by filtration. The residual alginate in
liquid phase was precipitated by adding 1% CaCl2 (3).
Enzymatic hydrolysis was performed with two different enzymes under
different conditions. Liquors from both processes were stored at −18 °C
until analysis. The extraction yield was gravimetrically measured and
the glucose, xylose, galactose, arabinose and fucose content was
determined by HPLC and sulfate content by gelatin-BaCl2 method.
Purification with Resins of the Phlorotannins in the Extracts Obtained by Microwave Hydrodiffusion and Gravity (Mhg) of Sargassum muticum
María Parada 1,2, Noelia Flórez 1,2, Elena M. Balboa 1,2 and Herminia Domínguez 1,2
1 University of Vigo, Ourense, Spain
2 Citi-University of Vigo, Ourense, Spain
Sargassum muticum
(Sm) is an invasive brown macroalgae in the Atlantic coasts. Different
aqueous based extraction processes have been tried to obtain soluble
fractions from this algae, including pressurized hot water extraction,
enzyme assisted aqueous extraction and microwave hydrodiffusion and
gravity (MHG). MHG was selected on the basis of its simplicity and the
possibility of using water as solvent or to perform a solvent free
process. In this study was to recover phenolic compounds with
antioxidant activity and a variety of biological actions from Sm, in a
MHG equipment and the selective recovery and concentration of the
phenolic compounds from the liquid phase separated has been proposed. Sm
was collected in Cape Estai (Pontevedra, Spain) in June 2012, was
washed with tap water, freezed and stored in closed plastic bags at −20
°C until use. Operation in microwave NEOS-GR extractor (Milestone
S.r.l., Italy) was carried out. Extraction was performed in the presence
of light in an open system at maximum temperature of 100 °C. Under
these conditions, the phenolic compounds could be stable, and it is also
expected that phloroglucinol derivatives would not be affected.
Combinations of irradiation power and time during operation, phenolic
compounds and radical scavengers were studied. Subsequent purification
in commercial food grade non ionic polymeric resins was accomplished.
Adsorption onto non-ionic food grade polymeric resins and further
desorption with 68% aqueous ethanol solutions were performed operating
under previously selected operational conditions. Several commercial
food grade resins were screened with the aim of selecting the most
suited for the practical recovery of phenolic compounds with radical
scavenging activity. Under the optimized adsorption-desorption
conditions a powdered yellowish product with 30% phologlucinol content,
expressed as g PGE/100 g extract, was obtained and the radical
scavenging capacity of one gram of product was equivalent to 1 g of
Trolox.
Multi Component Extraction from Scottish Macroalgae
Kirsty Black 1,2
1 Marine Biopolymers Limited, Ayrshire, UK
2 Strathclyde University, Glasgow, UK
Marine
Biopolymers Limited (MBL) is an SME founded in late 2009 specialising
in the extraction of components from indigenous Scottish seaweeds. MBL
has developed a disruptive process for the extraction of alginate,
typically the largest component in brown seaweeds, reducing
manufacturing time from 24 h or more in a traditional process to less
than 12 h. Although its initial main focus is alginate, MBL intends to
also bring a range of other, high value, seaweed components to the
market, including polysaccharides such as Laminarin, and Fucoidan. The
company is also involved in the EU FP7 project, SeaBioTech, and is
researching a range of microbial organisms and their metabolites as well
as other algae derived compounds with the aim of also commercialising
these into high value products.
Preparation of Semisynthetic Derivatives of Bromosphaerol and Evalution of Their Antifouling Activity
Kyriakos Proussis 1,2, Efstathia Ioannou 1, Theodora Calogeropoulou 2, Silvia Morgana 3, Veronica Piazza 3, Marco Faimali 3, Maria Protopapa 4, Skarlatos Dedos 4, George Verriopoulos 4 and Vassilios Roussis 1
1 Department of Pharmacognosy and Chemistry of Natural Products, Faculty of Pharmacy, University of Athens, Athens, Greece
2 Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, Athens, Greece
3 Institute of Marine Sciences (ISMAR), CNR, Genoa, Italy
4 Department of Zoology-Marine Biology, Faculty of Biology, University of Athens, Athens, Greece
Biofouling
represents one of the most serious problems in modern maritime
industries and its control is one of the biggest challenges of marine
biotechnology. In search of ecologically friendly, biodegradable
antifouling agents, a number of marine natural products were evaluated
for their inhibitory activity against the settlement of cyprids of Balanus amphitrite. Among them, the brominated diterpene bromosphaerol (1), isolated as the main constituent of the red alga Sphaerococcus coronopifolius,
exhibited prominent inhibitory activity coupled with negligible
toxicity against the barnacle nauplii [1]. To gain an insight on the
structural features responsible for the antifouling properties of
bromosphaerol and to further improve its selectivity index and effective
incorporation in marine paints, a number of structural analogues (2–12)
were designed and synthesized. The new derivatives are currently being
evaluated for their inhibitory activity against the settlement of
barnacle cyprids and for their toxicity against barnacle nauplii.
Acknowledgments
This
work was supported by the project GSRT-EPANII-11ΣYN-5-1274
“MARIPAINTS”, which is implemented under the “COOPERATION 2011” Action
of the Operational Programme “Competitiveness and Entrepreneurship” and
is co-funded by the European Social Fund (ESF) and National Resources.
References
- Piazza, V.; Roussis, V.; Garaventa, F.; Greco, G.; Smyrniotopoulos, V.; Vagias, C.; Faimali, M. Terpenes from the red alga Sphaerococcus coronopifolius inhibit the settlement of barnacles. Mar. Biotechnol. 2011, 13, 764–772.
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