J Biol Chem. 2017 Jan 6;292(
1):112-120. doi: 10.1074/jbc.M116.750299. Epub 2016 Nov 21.
Harshbarger W1,
Gondi S1,
Ficarro SB2,
Hunter J1,
Udayakumar D1,
Gurbani D1,
Singer WD1,
Liu Y1,
Li L1,
Marto JA2,
Westover KD3.
- 1From
the Departments of Biochemistry and Radiation Oncology, The University
of Texas Southwestern Medical Center, Dallas, Texas 75390 and.
- 2the Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
- 3From
the Departments of Biochemistry and Radiation Oncology, The University
of Texas Southwestern Medical Center, Dallas, Texas 75390 and
kenneth.westover@utsouthwestern.edu.
Abstract
Glutathione S-transferase pi 1 (GSTP1) is frequently overexpressed in cancerous tumors and is a putative target of the plant compound piperlongumine
(PL), which contains two reactive olefins and inhibits proliferation in
cancer cells but not normal cells. PL exposure of cancer cells results
in increased reactive oxygen species and decreased GSH. These data in
tandem with other information led to the conclusion that PL inhibits
GSTP1, which forms covalent bonds between GSH and various electrophilic
compounds, through covalent adduct formation at the C7-C8 olefin of PL,
whereas the C2-C3 olefin of PL was postulated to react with GSH.
However, direct evidence for this mechanism has been lacking. To investigate, we solved the X-ray crystal structure of GSTP1 bound to PL and GSH at 1.1
Å resolution to rationalize previously reported structure activity
relationship studies. Surprisingly, the structure showed that a
hydrolysis product of PL (hPL) was conjugated to glutathione
at the C7-C8 olefin, and this complex was bound to the active site of
GSTP1; no covalent bond formation between hPL and GSTP1 was observed.
Mass spectrometry (MS) analysis of the reactions between PL and GSTP1
confirmed that PL does not label GSTP1. Moreover, MS data also indicated
that nucleophilic attack on PL at the C2-C3 olefin led to PL
hydrolysis. Although hPL inhibits GSTP1 enzymatic activity in vitro,
treatment of cells susceptible to PL with hPL did not have significant
anti-proliferative effects, suggesting that hPL is not
membrane-permeable. Altogether, our data suggest a model wherein PL is a
prodrug whose intracellular hydrolysis initiates the formation of the
hPL-GSH conjugate, which blocks the active site of and inhibits GSTP1
and thereby cancer cell proliferation.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
KEYWORDS:
GSTP1; cancer; cancer therapy; crystal structure; inhibition; inhibition mechanism; natural product; piperlongumine; structure; therapeutic
