title: Structural and Biochemical Analyses Reveal the Mechanism of Glutathione S-Transferase Pi 1 Inhibition by the Anti-cancer Compound Piperlongumine.
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.
authors.affiliation: From the Departments of Biochemistry and Radiation Oncology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390 and.
authors.forename: Wayne
authors.initials: W
authors.lastname: Harshbarger
authors.affiliation: From the Departments of Biochemistry and Radiation Oncology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390 and.
authors.forename: Sudershan
authors.initials: S
authors.lastname: Gondi
authors.affiliation: the Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
authors.forename: Scott B
authors.initials: SB
authors.lastname: Ficarro
authors.affiliation: From the Departments of Biochemistry and Radiation Oncology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390 and.
authors.forename: John
authors.initials: J
authors.lastname: Hunter
authors.affiliation: From the Departments of Biochemistry and Radiation Oncology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390 and.
authors.forename: Durga
authors.initials: D
authors.lastname: Udayakumar
authors.affiliation: From the Departments of Biochemistry and Radiation Oncology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390 and.
authors.forename: Deepak
authors.initials: D
authors.lastname: Gurbani
authors.affiliation: From the Departments of Biochemistry and Radiation Oncology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390 and.
authors.forename: William D
authors.initials: WD
authors.lastname: Singer
authors.affiliation: From the Departments of Biochemistry and Radiation Oncology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390 and.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: From the Departments of Biochemistry and Radiation Oncology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390 and.
authors.forename: Lianbo
authors.initials: L
authors.lastname: Li
authors.affiliation: the Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
authors.forename: Jarrod A
authors.initials: JA
authors.lastname: Marto
authors.affiliation: From the Departments of Biochemistry and Radiation Oncology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390 and kenneth.westover@utsouthwestern.edu.
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Westover
mesh_terms: D049109:Cell Proliferation / Q000187:drug effects*; D018360:Crystallography, X-Ray; D004148:Dioxolanes / Q000494:pharmacology*; D005978:Glutathione / Q000378:metabolism*; D051549:Glutathione S-Transferase pi / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D010190:Pancreatic Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology*; D011485:Protein Binding; D011487:Protein Conformation; D014407:Tumor Cells, Cultured; D010881:Piperidones
chemical_list: D004148:Dioxolanes; D005978:Glutathione; D051549:Glutathione S-Transferase pi; C496556:GSTP1 protein, human; C498077:piperlongumine; D010881:Piperidones
keywords: GSTP1; cancer; cancer therapy; crystal structure; inhibition; inhibition mechanism; natural product; piperlongumine; structure; therapeutic