Tuesday, 31 January 2017

Potential of marine natural products against drug-resistant fungal, viral, and parasitic infections

Antibiotics have revolutionised medicine in many aspects, and their discovery is considered a turning point in human history. However, the most serious consequence of the use of antibiotics is the concomitant development of resistance against them. The marine environment has proven to be a very rich source of diverse natural products with significant antibacterial, antifungal, antiviral, antiparasitic, antitumour, anti-inflammatory, antioxidant, and immunomodulatory activities. Many marine natural products (MNPs)—for example, neoechinulin B—have been found to be promising drug candidates to alleviate the mortality and morbidity rates caused by drug-resistant infections, and several MNP-based anti-infectives have already entered phase 1, 2, and 3 clinical trials, with six approved for usage by the US Food and Drug Administration and one by the EU. In this Review, we discuss the diversity of marine natural products that have shown in-vivo efficacy or in-vitro potential against drug-resistant infections of fungal, viral, and parasitic origin, and describe their mechanism of action. We highlight the drug-like physicochemical properties of the reported natural products that have bioactivity against drug-resistant pathogens in order to assess their drug potential. Difficulty in isolation and purification procedures, toxicity associated with the active compound, ecological impacts on natural environment, and insufficient investments by pharmaceutical companies are some of the clear reasons behind market failures and a poor pipeline of MNPs available to date. However, the diverse abundance of natural products in the marine environment could serve as a ray of light for the therapy of drug-resistant infections. Development of resistance-resistant antibiotics could be achieved via the coordinated networking of clinicians, microbiologists, natural product chemists, and pharmacologists together with pharmaceutical venture capitalist companies.


Infectious diseases continue to jeopardise the achievements of modern medicine for the past seven to eight decades.1 In particular, the development of antimicrobial drug resistance has imposed a major burden on global health and economics.2, 3, 4 Antimicrobial drug resistance could be defined as the ability of microbes to persist, multiply, and produce virulence, despite the administration of anti-infective drugs. Drug resistance might occur at drug doses equal to or higher than those recommended in clinical practice, but within the physiological tolerance of the patient. Even though antimicrobial drug resistance is conferred mainly because of acquisition of resistant mutations in micro-organisms, factors associated with antimicrobial usage, non-optimal prescriptions, incorrect dosage, and duration also serve as the aetiological parameters of antimicrobial resistance.2 Estimates suggest that antimicrobial resistance leads to about 25 000 deaths per year in the European Union (EU) and 23 000 deaths per year in the USA.5, 6, 7 The total economic cost (covering the health-care and productivity losses) of antimicrobial resistance is around €1·5 billion per year in the EU and is as high as US$20 billion per year in the USA to cover health-care costs. Additionally, costs for lost productivity in the USA run as high as US$35 billion per year.5, 6 WHO's Global Report on Surveillance (2014) outlines the magnitude of antibacterial, antifungal, antiviral, and antiparasitic drug resistance on a global scale.8 On the basis of the extent, spread, evolution, and impact of antimicrobial drug resistance described in the report, new drugs and vaccines are now urgently needed in the anti-infective drug discovery pipeline, to either enhance the efficacy of existing drugs or to remove them completely from the market.2, 8 Even though combination therapies (antimicrobial–antimicrobial combinations; antimicrobial–non-anti-microbial [adjuvant] combinations; drug-cocktails) are preferred over monotherapies to treat and prevent drug-resistant infections,9, 10, 11, 12, 13, 14 the resistance to one or both drugs in combination therapy has led to treatment failure in certain cases. Hence, novel drugs from new sources possessing innovative targets could provide effective prophylaxis and therapy of drug-resistant infections.15
The marine environment has proven to be a very rich source of diverse natural products with significant antibacterial, antifungal, antiviral, antiparasitic, antitumour, anti-inflammatory, antioxidant, and immunomodulatory activities.16, 17, 18 This Review recapitulates the potential of marine natural products (MNPs) in the treatment of infections caused by drug-resistant fungi (azole-resistant and amphotericin B-resistant Candida spp), viruses (amantadine-resistant and oseltamivir-resistant influenza strains; non-nucleoside reverse transcriptase inhibitor [NNRTI]-resistant HIV; aciclovir-resistant and phosphonoacetic acid-resistant herpes simplex virus [HSV]), and parasites (chloroquine-resistant and pyrimethamine-resistant Plasmodium falciparum) because of the current lack of effective medical strategies to combat those infections (figure 1). The discovery of MNPs against drug-resistant pathogens has consistently increased during the past 15 years (figure 1). Even though antibacterial resistance is an equally important and challenging issue, we will not discuss it in this Review, because use of MNPs against drug-resistant bacteria is vast and is reviewed extensively elsewhere.19, 20
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Figure 1

Marine natural products active against drug-resistant pathogens
(A) Percentage distribution (phylum-wise) of MNPs (active against drug-resistant pathogens including fungi, viruses and parasites) in the marine environment; (B) Discovery of MNPs against drug-resistant pathogens over the past 20 years. Data includes the compounds discovered until June, 2016. MNPs=marine natural products. *Drug-resistant fungi, viruses, and parasites.

Marine natural products with activity against drug-resistant fungi

The treatment of candidaemia among immunocompromised patients, whose numbers have increased—along with patients with AIDS, patients who receive chemotherapy, and recipients of organ transplants—remains a challenge despite the availability of an ever-widening choice of antifungal drugs. Prolonged therapy with antifungal agents has resulted in fast development of resistant Candida spp strains.21, 22 Resistance in Candida spp might develop during or after therapy with antifungal agents such as fluconazole, itraconazole, flucytosine, amphotericin B, and nystatin.23, 24 Resistance is often a result of expression of efflux pumps, modification of drug targets, modulation of transcription factors, and biofilm formation.25, 26 More often, drug resistance or drug insensitivity in fungi is a consequence of the membrane-associated efflux pumps within them. These efflux pumps reduce the activity of conventional antifungal agents such as fluconazole, itraconazole, and voriconazole, by exporting them outside the cell, which leads to a subsequent reduction in the intracellular accumulation of antibiotics. Blockage of these efflux pumps would increase the activity of the currently ineffective antibiotics in these fungal pathogens. Screening of MNPs for their ability to reverse drug resistance mediated by efflux pumps by using checkerboard assays in genetically engineered Saccharomyces cerevisiae (overexpressing the Candida albicans efflux pumps) and several clinical azole-resistant isolates of C albicans has the potential to provide novel drug leads to treat azole-resistant fungal infections. Furthermore, analysis of results obtained from checkerboard experiments using fractional inhibitory concentration (FIC) values and isobolograms (Cartesian coordinate plots used to study the nature of interaction [additivity, synergism, or antagonism] of two drugs at a given effect level), would provide an insight into the effect of such MNPs in combination treatments. On the basis of FIC values and isobolograms, it is possible to deduce whether the MNP is inactive or if it displays synergistic, additive, antagonistic effects with the actual antibiotics.
A new tetramic acid glycoside aurantoside K (appendix) was isolated from the Fijian marine sponge Melophlus spp Aurantoside K exhibited potent antifungal activity against wild-type C albicans (minimal inhibitory concentrations [MIC] of 2·6 μM) and amphotericin-resistant C albicans (MIC 42·05 μM), with no cytotoxicity in vitro against human colon cancer cell line, HCT-116.27 Liquid chromatography/mass spectrometry (LC/MS)-based metabolomics led to the discovery of the polyketide compound forazoline A from Actinomadura spp that was cultivated from the ascidian Ecteinascidia turbinata. Forazoline A exhibited promising in-vitro and in-vivo efficacies against C albicans K1. A study on the mechanism of action of forazoline A using yeast chemical genomics, revealed that forazoline A possibly acts by disrupting the membrane integrity by deregulating phospholipid homoeostasis. Furthermore, forazoline A displayed a synergistic antifungal effect with amphotericin B in vitro, suggesting a parallel or complementary mechanism of action.28 Additional marine natural products with activity against drug-resistant fungal infections have also been identified (appendix).
In some cases, natural products did not show any inherent antifungal activity when tested against drug-resistant fungal infections but rather enhanced the activity of azole compounds. Bioassay-guided fractionation led to the identification of an unusual epoxy sterol, 9α,11α-epoxycholest-7-ene-3β,5α,6α,19-tetrol 6-acetate (ECTA), from the marine sponge Dysidea arenaria. Checkerboard-type assays assessed the efficacy of ECTA in reversing the fluconazole resistance in a C albicans multidrug resistance 1 efflux pump overexpressing Saccharomyces cerevisiae. The antifungal activity of fluconazole was enhanced 35 times in combination with ECTA (3·8 μM). Evaluation of cytotoxicity against human ductal breast carcinoma T47D cell lines revealed that ECTA had no apparent cytotoxic effects up to concentrations of 10 μM. ECTA is the first identified MNP to reverse fluconazole resistance mediated by the C albicans MDR efflux pump.29
Bioassay-guided fractionation of the extract from the marine green alga Penicillus capitatus led to the identification of the cycloartanone triterpene sulphates capisterones A and B. Capisterones A and B enhanced the activity of fluconazole in efflux pump-overexpressing fluconazole-resistant S cerevisiae strains. The absence of cytotoxicity against several human cancerous and non-cancerous cell lines and the inherent antifungal activities by these compounds highlight the potential use of such MNPs as efflux pump inhibitors in combination therapies against fungal infections caused by azole-resistant strains.30 The cyclodepsipeptides unnarmicins A and C were identified from the marine gammaproteobacterium Photobacterium spp, strain MBIC06485. Unnarmicins A and C chemo-sensitised S cerevisiae strains hyper-expressing azole drug efflux pumps and azole-resistant C albicans to sub-MIC concentrations of fluconazole by inhibition of ATP-binding cassette (ABC) transporters/efflux pumps.31 Two sulphated sterols, geodisterol-3-O-sulphite and 29-demethylgeodisterol-3-O-sulphite, which exhibited reversal of multi-drug resistant 1 efflux pump-mediated fluconazole resistance in genetically-engineered fluconazole-resistant S cerevisiae and a clinical isolate of C albicans 1758, were identified from the marine sponge Topsentia spp. Geodisterol-3-O-sulphite and 29-demethylgeodisterol-3-O-sulphite enhanced the fluconazole susceptibility in these fluconazole-resistant strains. Both these sulphated sterols did not possess any antimicrobial or cytotoxic effects, implying the possible utility of such compounds to treat opportunistic fungal infections caused by azole-resistant C albicans.32

Marine natural products with activity against drug-resistant viruses

Marine natural products against drug-resistant influenza

MNPs have also shown efficacy against drug-resistant viruses (appendix). Two classes of antiviral agents are active against influenza viruses: the adamantanes and the neuraminidase inhibitors (NAIs). Of four developed NAIs, only oseltamivir phosphate and zanamivir are licensed worldwide. Peramivir and laninamivir are not licensed in the USA. However, all NAIs are active against both influenza A and B viruses.33 Amantadine and rimantadine hydrochloride are only active against influenza A viruses. In the past 10 years, widespread adamantane resistance among influenza A (H3N2) virus strains has made this class of medications less clinically useful. This resistance is caused by point mutations in the RNA-sequence encoding the M2 protein of influenza-A viruses. Resistance against the NAIs is much less frequent. This resistance can be based on mutations in neuraminidase as well as in haemagglutinin.34 However, during the 2008–09 season almost all influenza A 2007 H1N1-like strains from North America and Europe were oseltamivir resistant.35
A marine-derived fungus—Eurotium rubrum, F33—was found to exhibit an inhibitory effect against H1N1 virus, on the basis of its effects on cell proliferation, cytotoxicity, and damage to host cells during viral invasion using CellTitre-Glo assay (an assay to monitor cell proliferation; thereby to screen for and exclude compounds with significant cytotoxicity) and cytopathic effect (CPE) assay (an assay to measure the extent of damage to host cells during viral invasion). Chemical investigation led to the isolation of the prenylated indole diketopiperazine alkaloid neoechinulin B. Neoechinulin B displayed a strong inhibition against the H1N1 virus in infected MDCK cells, and also inhibited a panel of drug-resistant (resistant against amantadine, oseltamivir phosphate, and ribavirin) influenza clinical isolates. Further experiments revealed that neoechinulin B bound to the influenza envelope haemagglutinin and disrupted its interaction with the host sialic acid receptor and thus inhibited the attachment of influenza virus to the host cells. Multi-passage experiments and plaque formation assays showed that neoechinulin B displayed a diminished induction of drug resistance compared with amantadine. The absence of cytotoxicity, broad spectrum of action against drug-resistant clinical isolates together with the diminished induction of drug resistance, highlights the potential use of neoechinulin B to treat viral infections that are clinically resistant to commercially available antiviral influenza drugs.36
The cyclic tetrapeptide asperterrestide A was isolated from the fungus Aspergillus terreus SCSGAF0162, which was cultivated from the gorgonian coral Echinogorgia aurantiaca. Asperterrestide A exerted an inhibitory effect against the influenza strain A/WSN/33 H1N1 (an M2-resistant strain) and strain A/Hong Kong/8/68 H3N2 (an M2-sensitive strain) replication in MDCK cells. Asperterrestide A also exhibited cytotoxic effects on the human leukaemic monocyte lymphoma U937 and acute lymphoblastic leukaemia MOLT-4 cell lines.37, 38 Spiromastilactone D was isolated from a deep-sea derived fungus and inhibited a panel of amantadine and oseltamivir-resistant influenza strains. Niu and colleagues39 investigated the mechanism of action of spiromastilactone D and revealed that it has synergistic effects on viral entry and replication. Specifically, spiromastilactone D inhibits the attachment and entry of viruses into host cells by disrupting the haemagglutinin–sialic acid receptor interactions, and it also inhibits viral genome replication by targeting the viral ribonucleoprotein complex.39

Marine natural products with activity against drug-resistant HIV

HIV exhibits high genetic variability, and thus develops resistance to existing drugs and escapes host immune responses elicited by AIDS vaccine candidates. Around 26 individual anti-AIDS drugs have been approved, of which 13 target the reverse transcriptase of this retrovirus. The remaining drugs target key steps in the viral lifecycle.40 Nucleoside reverse transcriptase inhibitors (NRTIs) do not directly inhibit reverse transcriptase but terminate the elongation of the DNA primer. Resistance to nucleoside analogue drugs (eg, zidovudine) primarily appear along the dNTP-binding track of the reverse transcriptase.41 Non-NRTIs (NNRTIs) inhibit reverse transcriptase directly by binding to a hydrophobic pocket and allosterically inhibit DNA polymerisation. Primary NNRTI-resistance appears around the NNRTI pocket.40 The high replication rate, together with the low fidelity of the viral reverse transcriptase, predicts that every possible change will occur every day at every position of the viral genome in a patient with active viral replication.42 Therefore, resistance can develop against every anti-HIV drug.
The phenylspirodrimane stachybotrin D was isolated from the marine sponge-associated fungus Stachybotrys chartarum MXH-X73 along with ten other compounds. Testing of the compounds for anti-HIV activity revealed that only stachybotrin D inhibited HIV-1 replication without any cytotoxicity. Furthermore, time of addition and reverse transcriptase activity assays proved that the anti-HIV activity of stachybotrin D was due to its inhibitory action on reverse transcriptase, which led to inhibition of HIV-1 replication. Additionally, assessment of stachybotrin D for its anti-HIV activity against drug-resistant strains revealed that it displayed similar inhibitory effects on HIV-1 replication of wild type and several NNRTI-resistant HIV-1 strains.43

Marine natural products with activity against drug-resistant herpes simplex viruses

Emergence of aciclovir-resistant herpes simplex viruses (HSVs) is not only observed in immunocompromised patients but also in cases of neonatal herpes and HSV infections at immune-privileged locations such as the central nervous system and the cornea. HSV can acquire resistance to aciclovir by alterations in the viral thymidine kinase gene UL23, or nucleotide substitutions in the viral DNA polymerase gene UL30, or both. Drug resistance in HSV has been observed against drugs used in first-line (aciclovir), second-line (penciclovir, foscarnet sodium), and third-line (cidofovir) treatments.44 Chronic HSV infections of HIV-positive individuals, solid organ transplant recipients, and cancer patients require prolonged antiviral treatment, which substantially increases the risk of selection for drug-resistant viruses.45
MNPs have been a template for many of the commercially available antiviral drugs. In fact, most of the currently available antiviral drugs are synthetic derivatives of MNPs (eg, the antiviral drug adenine arabinoside, or vidaribine, a synthetic analogue of the marine sponge-derived nucleoside spongouridine). Even though the marketing of vidaribine has been currently discontinued owing to the availability of better drugs, reports suggest it has the potential to inhibit aciclovir-resistant HSV and varicella zoster virus infections.46 methanol extract of the marine algae Symphyocladia latiuscula displayed substantial antiviral activities against aciclovir and phosphonoacetic acid-resistant HSV-1, thymidine kinase-deficient HSV-1, and wild-type HSV-1 strains in vitro without apparent cytotoxicity. Fractionation of this extract led to isolation of the active components 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether (TDB) and TDB alcohol. Furthermore, evaluation of the therapeutic efficacy of methanol extract and TDB in a mouse HSV-1 infection model revealed that oral administration of these compounds impeded the development of skin lesions and suppressed the virus yields in the brain and skin without any toxic effects. The antiviral effect against aciclovir-resistant strains further suggested that TDB acts via an alternate mechanism that is probably different from the mechanism of action of aciclovir.47, 48 Thus, marine algal extracts and their bromophenol constituents could be used for treatment of aciclovir-resistant herpes infections.

Marine natural products against drug-resistant malaria

Malaria is caused by the primary species Plasmodium falciparum and Plasmodium vivax. Although most (around 90%) malaria cases occur in Africa, certain regions of southeast Asia and Latin America are also at risk, but to a lesser extent.13, 49, 50, 51 Anti-malarial drugs have been developed against specific targets (table). The 2015 Nobel prize in physiology or medicine was awarded to Youyou Tu of the China Academy of Traditional Chinese Medicine for her work on the antimalarial drug artemisinin, which saves millions of lives every year.52 Muangphrom and colleagues53 extensively discussed the application of biotechnology to production of artemisinin and the multitude of genes and proteins affected in the malarial parasite, upon treatment with artemisinin.


List of known anti-malarial drugs and their modes of action
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Resistance has developed to different degrees against all three substance classes.54, 55 Parasites become resistant to antifolate drugs by point mutations in target enzymes, or gene amplification of the first enzyme in the parasite folate synthesis pathway (ie, GTP-cyclohydrolase), or both.56 Resistance against chloroquine, the cheapest and safest among all other antimalarial drugs57, 58 is mediated by mutations in PfMDR1 (P falciparum multidrug resistance transporter)59, 60 and PfCRT (P falciparum chloroquine resistance transporter).61, 62 Artemisinin resistance has emerged in southeast Asia and now poses a threat to the control and elimination of malaria.63 The key mediator of artemisinin resistance is the P falciparum phosphatidylisonsitol-3-kinase (PFPI3K) in the early ring stages. However, dihydroartemisinin, the active form of all artemisinins, blocks production of phosphatidylisonsitol-3-phosphate (PI3P) by interfering with PfKelch13, which contains the Cys580Tyr mutation in artemisinin-resistant P falciparum.64 MNPs have diverse potential to treat drug-resistant P falciparum (appendix).
Haliclonacyclamine A was isolated from marine sponge Haliclona spp and exhibits antiplasmodial activity in vitro against chloroquine-sensitive (3D7; IC50 0·7 μM) and chloroquine-resistant strains of P falciparum (FcB1) (IC50 0·11 μM), respectively.65 Furthermore, haliclonacyclamine A showed low cytotoxicity on the breast cancer cell line MCF7 (IC50 5·5 μM) and showed antiplasmodial activity in vivo on mice infected with Plasmodium vinckei petteri by reducing the mortality over an 18-day period.65 Bioassay-guided fractionation led to the isolation of a new bispyrroloiminoquinone alkaloid, tsitsikammamine C, and the previously known makaluvamines J, G, and L from the Australian marine sponge Zyzzya spp. These compounds exhibited potent antiplasmodial activities against chloroquine-sensitive (3D7) and chloroquine-resistant and mefloquine-resistant (Dd2) strains of P falciparum with IC50 values less than 100 nM and displayed moderate cytotoxicity on the human embryonic kidney cell line HEK293 with IC50 in the range of 1·1–3·6 μM. Furthermore, tsitsikammamine C inhibited both ring and trophozoite stages of the malaria parasite life cycle and displayed selectivity indices of more than 200 against HEK293 cells. Makaluvamine G suppressed parasite growth in P berghei-infected mice after subcutaneous administration of 8 mg/kg per day.66, 67
Novel thiazine-derived alkaloids thiaplakortones A-D were isolated from the Australian marine sponge Plakortis lita. Thiaplakortones A–D exhibited striking antimalarial activities against chloroquine-sensitive (3D7) and resistant (Dd2) strains of P falciparum (IC50 <651 nM) with moderate cytotoxicity against HEK293 cells. Thiaplakortone A was found to be the most active compound against chloroquine-sensitive (3D7; IC50 51 nM) and chloroquine-resistant (Dd2; IC50 6·6 nM) strains of P falciparum.68 The pentacyclic ingamine alkaloid ingamine A was isolated along with two new pentacyclic ingamine alkaloids—namely, 22(S)-hydroxyingamine A and dihydroingenamine D—from the marine sponge Petrosid Ng5 Sp5. An antimalarial lactate dehydrogenase assay revealed that alkaloid ingamine A exhibited strong antiplasmodial activity against chloroquine-sensitive (D6; IC50 0·2 μM) and chloroquine-resistant (W2; IC50 0·174 μM) strains of P falciparum; and dihydroingenamine D exhibited similarly strong antiplasmodial activity against D6 (IC50 0·17 μM) and W2 (IC50 0·13 μM) P falciparum strains; whereas 22(S)-hydroxyingamine A was found to be less active against D6 (IC50 0·43 μM) and W2 (IC50 0·30 μM) P falciparum strains. Measurement of cytotoxicity against a panel of cancerous and non-cancerous cell lines revealed that the compounds, all three pentacyclic ingamine alkaloids, were devoid of in vitro cytotoxicity.69 These pentacyclic ingamine alkaloids represent a novel antiplasmodial pharmacophore devoid of the β-carboline ring, which is ascribed to the cytotoxicity of the manzamine class of marine alkaloids.
The ascidian metabolite 1, 14-sperminedihomovanillamide (orthidine F) was identified as a non-toxic inhibitor of dual drug-resistant strain P falciparum K1 (IC50 0·89 μM) in an antiparasitic screening with synthesised and isolated MNPs. Investigations of preliminary structure–activity associations identified the requirement of the spermine polyamine core and the 1,14-disubstitution for potent antiplasmodial activity. An analogue of orthidine F, 1, 14-spermine-di-(2-hydroxyphenylacetamide), displayed antiplasmodial activity (IC50 8·6 nM) that was increased by two orders of magnitude without in vitro cytotoxicity.70 The polyether metabolite that was isolated from the marine sediment-derived Streptomyces spp, H668, displayed in vitro antiplasmodial activities against chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of P falciparum with IC50 values in the range of 0·145–0·29 μM and no cytotoxicity towards Vero cells.71
Antimalarial screening using a SYBR green assay of marine sediment-derived actinomycete extracts revealed the antiplasmodial activity of the extract from the marine actinomycete Salinispora tropica. Furthermore, assessment of the cytotoxic component salinisporamide A from this bacterium against the plasmodial growth demonstrated that salinisporamide A has potent antimalarial activities against chloroquine-sensitive and resistant clones (FCB) of P falciparum. Salinisporamide A was also shown to inhibit the erythrocytic stages of the parasite life cycle. Results from biochemical experiments and crystal structure modeling suggested that salinisporamide A probably acts through inhibition of the 20S parasitic proteasome. Administration of low doses of salinisporamide A (130 μg/kg) significantly protected mice from malaria infection, highlighting salinisporamide as a novel candidate for antimalarial treatments to target 20S parasitic proteasomes.72, 73
Antiplasmodial screening of a library of enriched marine natural product fractions led to identification of the bis(indolyl)imidazole alkaloid nortopsentin A. Nortopsentin A displayed substantial antimalarial activities against chloroquine-sensitive (3D7; IC50 460 nM) and chloroquine-resistant (Dd2; IC50 580 nM) strains of P falciparum, with no notable cytotoxicity. Nortopsentin A was found to inhibit the parasitic growth more specifically at the trophozoite stage.74 Eight compounds, including three tyramine derivatives, two steroidal pregnane glycosides, and three sesquiterpenoids, were isolated from the octocoral Muricea austere. Seven of the eight compounds exhibited activities against a drug-resistant P falciparum strain (W2, IC50 36–80 μM), although one sesquiterpenoid was inactive. The antiprotozoal activities of synthetic glycosides and tyramine derivatives of these compounds were also assessed in vitro against a drug-resistant P falciparum strain.75
Antimalarial screening of a bacterially derived prefractionated library of MNPs against a chloroquine-resistant W2 strain of P falciparum led to the isolation of the antimalarial long-chain bicyclic phosphotriester compounds salinipostins A-K from the marine-sediment-derived Salinospora spp. Salinipostins A-K exhibited strong inhibition of a chloroquine-resistant W2 strain of P falciparum with EC50 values in the range of 50 nM–50 μM. Among the compounds isolated, salinipostin A exhibited the strongest antimalarial effect (EC50 50 nM) and displayed no apparent cytotoxic effects on mammalian cell lines tested (EC50 >50 μM). A combination of microscopy and resistance selection assays revealed that salinipostin A exhibited growth-stage-specific effects (that differ from compounds that inhibit haem-polymerisation) and was less susceptible to the development of resistance.76
The polyhydroxymacrolide bastimolide A isolated from the tropical marine cyanobacterium Okenia hirusta, displayed potent antimalarial activities against four multidrug-resistant strains of P falciparum (IC50 80–270 nM). Bastimolide A displayed moderate cytoxic effects on MCF-7 (IC50 3·1 μM) and Vero cells (IC50 2·1 μM).77
The rare bicyclic phosphotriester backbone among natural products, lack of structural similarity to existing antimalarials, together with the reduced possibility to develop resistance, highlight the potential efficacy of compounds that possess this scaffold to tackle antimalarial drug resistance. Although natural product scaffolds continue to be the basis of antimalarial medicines, studies on the antimalarial potential of MNPs are evidently still in their infancy.78 Novel chemical motifs, sub-micromolar potency and mode of action, the absence of host cell cytotoxicity, and less possible induction of drug resistance are important factors to be addressed in antimalarial drug discovery.
Cervantes and colleagues79 developed a simple, semi-automated RNA fluorescence-based high-content live cell-imaging (HCLCI) assay that linked the complex biology of malaria parasites with drug discovery. This HCLCI platform was used to discover antimalarial MNPs and to study their cellular phenotypic effects in P falciparum and the cytotoxic effects on host red blood cells. In this HCLCI screening platform, the MNPs library is briefly subjected to a primary antimalarial screen using the standard high-throughput SYBR Green I based assay. From the results obtained with the primary screen, extracts showing parasite inhibition at lower concentrations are further projected to a secondary HCLCI screen, whereby the drug effects on parasite morphology, transcriptional activity, and toxicity on host red blood cells are assessed based on visualisation of the live parasites in the host red blood cells and transmitted light images. The hits obtained from the secondary screen were then subjected to purification steps to identify the antimalarial compound, and the method could then be finally verified with the purified compound.79 Thus, such novel high-content screening platforms based on live cell imaging could augment the discovery of potent parasite inhibitors against drug-sensitive and drug-resistant strains as well as provide a clue about their possible modes of action, along with their effects on red blood cell integrity and cytotoxicity.

Analysis of drug-like physicochemical properties

The physical properties (molecular weight, log P, hydrogen bond donor, hydrogen bond acceptor, rotatable bond, and topological polar surface area) of the 74 MNP compounds highlighted in this Review (appendix) were calculated using the Chemaxon toolkit of the molecules (appendix) and projected onto a drug-like cutoff threshold of Lipinski's rules80 (figure 2) and Veber's oral bioavailabilty rule (figure 3).81 Derived from the 90th percentile of drug candidates that reached phase 2 clinical trials, Lipinski's rule of five is an algorithm consisting of four rules (based on molecular weight, log P, hydrogen bond donor, hydrogen bond acceptor). According to Lipinski's rule, to be drug-like, a candidate should have a molecular weight of less than 500 Da, a partition coefficient log P of less than 5, fewer than five hydrogen bond donors, and fewer than ten hydrogen bond acceptors. Lipinski's rule of five has been widely used to design small molecular libraries for screening. Veber's oral bioavailability physicochemical parameters (rotatable bonds and polar surface area) have also been used to predict favourable drug metabolism and pharmacokinetic outcomes. Compounds that have ten or fewer rotatable bonds and a polar surface area equal to or less than 140 × 10−10/m2 will have a high probability of good oral bioavailability in rats.
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Figure 2

Distributions of Lipinski descriptors for drug-likeness prediction
Analysis of physicochemical properties for marine natural product antifungals, antivirals, and antimalarials by (A) MW, (B) calculated log P, (C) HBD, (D) HBA, and (E) compliance with Lipinski's rule of five. The green line (indicates the maximum desirable value for oral bioavailability defined by Lipinski's rule of five: MW<500 Da; log P<5, HBD<5, and HBA<10. The cyan area is the Lipinski-compliant region with 0 or 1 violation, and the grey area is the Lipinski-non-compliant region (>1 violation). MW=molecular weight. MNP=marine natural products. MW=molecular weight. HBD=hydrogen bond donors. HBA=hydrogen bond acceptors.
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Figure 3

Distributions of Veber descriptors for oral bioavailability prediction
Bioavailability physicochemical values of marine natural product antifungals, antivirals, and antimalarials as assessed by (A) number of rotatable bonds, (B) TPSA, and (C) overall oral bioavailability prediction. The cyan area indicates a good oral bioavailability prediction, and the grey area indicates an unfavourable oral bioavailability prediction. MNP=marine natural products. TPSA= topological polar surface area.
47 of the 74 MNP compounds were molecular weight-compliant with Lipinski's rules. 17 of the 20 compounds that had a molecular weight greater than 500 Da and 11 with a log P less than 5 were antifungals, which corresponds with the observation of other studies that anti-infective drugs are biased towards higher molecular weight and polar chemistry space in physicochemical space to allow drugs to penetrate into nonhuman cells.82, 83 Potent antifungals theopapuamides A–D and theonellamide G, which did not score favourably by Lipinski's rule, had a large molecular weight (1500–1700 Da), but very low calculated log P values (−9·7 to 4·3). The log P profile of the 74 compounds (figure 2B) showed a wide range from −9·7 to 8·4. The distribution of hydrogen bond donors and hydrogen bond acceptors showed that all antivirals and most of the antimalarials (>90%) were within the Lipinski-compliant region. Overall, 27 of the 74 compounds did not violate Lipinski's rule of five, and 31 compounds had one violation. More than 78% of the 74 compounds (17 antifungals, seven antivirals, and 34 antimalarials) showed drug-like potential (figure 2E and appendix).
Analysis of the 74 compounds in terms of rotatable bonds and polar surface area showed that 42 compounds (18 antifungals, seven antivirals, 24 antimalarials) were in the favourable oral bioavailability space (figure 3). According to this prediction, compounds with a molecular weight higher than 500 Da (eurysterols A and B, melearoride B, capisterone B, geodisterol-3-O-sulphite, and 29-demethylgeodisterol-3-O-sulphite) can be orally bioavailable if their molecular flexibility (number of rotatable bonds) is kept under control. Geodisterol-3-O-sulphite, and 29-demethylgeodisterol-3-O-sulfite, which had more than one Lipinski violation, showed a good oral bioavailability prediction. Polyketide PF1163D, unnarmicin A, thiaplakortones C-D, 1,14-spermine-di-(2-hydroxyphenylacetamide, tyramine derivatives from otocoral Muricea austera, and salinipostins A-K had one Lipinski violation but obtained an unfavourable prediction on oral bioavailability due to their high rotatable bond values. The main potential reason for predicted bioavailability problems in 30 of the compounds is their high conformational flexibility.

Vaccines and antimicrobial resistance

The most powerful tools to prevent infectious diseases are vaccines. By contrast with antibiotics, efficacious vaccines have never been shown to elicit vaccine-resistant strains. This difference might be due to the presence of multiple immunogenic epitopes in a vaccine, so that several mutations need to accumulate before resistant strains can actually emerge. Furthermore, by preventing infections, vaccines do not allow pathogens to replicate in the host, limiting the selection process of variants to the initial phases of infection. Finally, unlike antibiotics, vaccines do not pose massive selective pressure in the environment. The reduction in transmission levels of pathogens could be achieved by establishment of herd immunity with use of vaccines. With this approach, antibiotic therapy could be minimised and the consequent problem of antimicrobial resistance could be curtailed.84, 85 Because of reverse vaccinology, novel adjuvants, and omics science, it is now possible to have vaccines against virtually every pathogen.86

Conclusion and outlook

The upsurge in the global burden of drug resistance, together with a sharp falloff in the discovery of new anti-infectives, has intensified the need to develop drugs against resistant pathogens. The marine environment harbours a huge repertoire of yet undiscovered drugs possessing broad-spectrum clinical applications. Several MNP-based anti-infectives have already entered phase 1, 2, and 3 clinical trials, with six approved for usage by the US Food and Drug Administration and one by the EU.87, 88 The effective translation of MNPs from the drug discovery pipeline to pharmaceutical markets suffers from a number of challenges associated with biodiversity (eg, efficient sampling approaches, accessibility of marine resources, taxonomical expertise, and variability or identification of producers), supply, and technology (eg, sustainable supply of marine compounds on an industrial scale, rediscovery of known compounds, toxicity, and efficacy studies), and the market (eg, market needs and academic partnerships, process costs, regulatory requirements, and intellectual property status). The huge time and cost required to reach the market, difficulty in isolation and purification procedures, toxicity associated with the active compound, ecological impacts on natural environment, and insufficient investments by pharmaceutical companies are some of the clear reasons behind market failures and a poor pipeline of MNPs available to date.89, 90, 91, 92, 93 However, the success stories of clinically approved MNP-based drugs make it clear that strategies such as advancements in screening procedures (involving functional assays in conjunction with phenotypic screens), the optimisation of structural modifications, the synthetic supply of unmodified MNPs, and the optimisation of drug formulations could take drugs from mere bioprospecting to real pharmaceutical markets and overcome the mentioned bottlenecks.89, 90, 91, 92, 93
On the basis of the existing literature, the diverse natural abundance of products in the marine environment could evidently serve as a ray of light in the therapy of drug-resistant fungal, viral, and parasitic infections, and the massive biological and chemical diversity enclosed in the marine environment could be translated to novel biomedicines. The extreme conditions offered by the oceans (ie, owing to differences in the temperature, light intensity, pH, pressure, and presence of abiotic chemicals) could be a potential reason for the presence of diverse antimicrobial compound sources in the marine environment. Nutritional scarcity and chemical defence are other possible reasons that are linked to production of anti-infectives by marine symbionts.94 Marine invertebrates and microbes, particularly marine sponges and actinomycetes, appear to be among the most abundant reserves of MNPs that are active against drug-resistant pathogens. Exploitation of these metabolically rich phyla using specific approaches could enhance the rates of drug discovery. Even though the large-scale production of drugs from these invertebrates remains a daunting challenge, strategies such as the identification of the pharmacophore, coupled with chemical synthetic and metabolomics approaches, could augment the scale-up of drugs from these marine prototypes.95, 96, 97 An alternate strategy is the large-scale fermentation of marine-derived microbes with and without certain metabolic induction schemes.98 Furthermore, the large fraction of uncultivable microbes represents a major bottleneck in the latter strategy that could be resolved by identification of biosynthetic gene clusters via metagenomics-based approaches that could assist in the discovery of MNPs from these uncultured microbes.99, 100, 101
Microbes that are insensitive to MNPs could emerge in the future. Therefore, the search for new MNPs should continue. However, only if we preserve the source of MNPs—the marine environment—will researchers be able to make use of this enormous resource. The preservation of the natural environment will need to include stopping climate change, development of sustainable development goals, and in a more holistic view—care about planetary health—which was defined by the Rockefeller Foundation–Lancet Commission on planetary health as “the health of human civilisation and the state of the natural systems on which it depends”.102 Therefore, efforts for environmental sustainability are social and ethical imperatives to maintain the natural environment for MNPs.
A way forward in the development of resistance-resistant antibiotics could be achieved through several approaches:103, 104, 105, 106 discovery of new antimicrobials with new possible single or multiple modes of action (polypharmacology) with less possible development of resistance; discovery of antivirulent drugs (and not antibacterial drugs), and drugs which boost innate immunity, thereby imposing less selective pressure on the pathogen to develop resistance; drug repurposing or repositioning that aims to spot new uses for already existing drugs; finally, multifaceted efforts involving the coordinated networking of clinicians, microbiologists, natural product chemists, and pharmacologists together with pharmaceutical venture capitalist companies could push the MNPs towards clinical applications and bolster the treatment of patients experiencing drug-resistant infections. Hence, efficient MNP screening platforms together with novel strategies for identification of the possible development of resistance could strengthen the drug development pipeline for antibiotics that do not induce resistance.

Search strategy and selection criteria

We searched PubMed and Google Scholar for English language publications published without start date restrictions up to June, 2016, with the terms “drug-resistance”, “marine natural products”, “marine natural products against drug-resistant Candida”, “marine natural products against drug-resistant influenza”, “marine natural products against drug-resistant HIV”, “marine natural products against drug-resistant HSV”, “marine natural products against drug-resistant malaria”, and “vaccines and antimicrobial resistance”. Cross referencing and “related articles” functions were used to expand the search criteria. Searches were further supplemented with publicly available information and reports from US Centers for Disease Control and Prevention and WHO websites.
URA initiated the review. URA, SB, TAO, TG, and NBP developed the scope of the manuscript. SB, TG, and NBP did the literature search and prepared the first draft. URA, SB, and NBP designed the figures and interpreted the findings. URA, TAO, UH, and RJQ critically reviewed the data and draft. All authors subsequently modified the manuscript jointly. URA is the guarantor of the final version, which was read and approved by all the authors.
Declaration of interests
We declare no competing interests.
We acknowledge the pioneering spirit and continuous support by G Bringmann (University of Würzburg). Financial support to UH and TAO was provided by the Deutsche Forschungsgemeinschaft (SFB 630 TP A5 and Z1) and by the European Commission within its FP7 Programme, under the thematic area KBBE.2012.3.2–01 with Grant Number 311932 (SeaBioTech). SB was supported by a fellowship of the German Excellence Initiative to the Graduate School of Life Sciences, University of Würzburg. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organisations imply endorsement by the US Government.

Supplementary Material

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