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.

Keywords: GSTP1; cancer; cancer therapy; crystal structure; inhibition; inhibition mechanism; natural product; piperlongumine; structure; therapeutic.

FIGURE 1.

Hydrolyzed piperlongumine inhibits GSTP1. A, structure of PL and hPL. B, architecture of GSTP1 dimer. Protomer A is colored brown, and protomer B is gray. The hPL-GSH complex is shown as green sticks, and the glutathione (G-site) and hydrophobic (H-site) binding sites are each labeled. C, 2F_o − F_c map for hPL-GSH complex is shown in green and scaled to 1σ. D, binding of the hPL-GSH complex (green sticks) to GSTP1. Residues that make notable hydrogen-bonding or van der Waals interactions are shown as gray sticks.

FIGURE 2.

Surface representation of GSTP1 bound by inhibitors: hPL-GSH (A); chlorambucil-GSH (B); NBDHEX-GSH (C); and ethacrynic acid-GSH (D). Inhibitor structures are shown as green sticks. The red arrow indicates the portion of the hPL-GSH structure that extends deeper into the binding cleft when compared with the other inhibitor-bound structures shown.

FIGURE 3.

PL does not label GSTP1. A and B, purified GSTP1 was incubated at 10 μm without (A) or with (B) PL and GSH. Deconvoluted electrospray mass spectra (A and B, upper panels) detected GSTP1 (23,349 Da; A and B; green circles correspond to full-length protein, while yellow circles indicate loss of N-terminal methionine) but not GSTP1+PL (expected molecular weight 23,666 Da). C–E, PL reacts with cysteine thiols and readily forms hydrolysis products. A model peptide, FGLCSGPADTGR, was incubated with PL and GSH, and the reaction mixture was analyzed by mass spectrometry. Prominent peaks corresponding to [M+H]+ of FPEP-PL hydrolysis product (peak 1), FPEP-PL (peak 2), FPEP-PL-GSH (peak 3), GSH-PL (peak 4), GSH-PL hydrolysis product (peak 5), PL (peak 6), and GSH (peak 7) were observed (C and D). MALDI-MS/MS analysis confirmed labeling of the FPEP cysteine residue (E). Ions of type y are marked by red circles, water loss is denoted by *, and C(*) corresponds to the cysteine-PL adduct. An ion at m/z 1277, colored green, corresponds to the indicated PL hydrolysis product (indicated by (A) in panel F). F, proposed PL, GSH, and FPEP reaction scheme. MW, molecular weight.

FIGURE 4.

PL requires cell entry prior to being hydrolyzed to active form. A, cell proliferation in the presence of PL versus hPL. Exposure of cancer cell lines previously shown to be sensitive to PL (PANC1, HELA, SW620) shows impaired growth in the micromolar range when exposed to PL (blue hue curves) consistent with prior studies (1, 12). Cells are unaffected at similar concentrations by hPL (red hue curves). Error bars indicate ± S.E. B, dose-response curve shows the inhibition of GSTP1 activity by hPL. C, schematic of the entry of PL, activation by free thiols, and inhibition of GSTP1, leading to reduced cellular glutathione, increased ROS, and cell death. TMCA, trimethoxycinnamic acid.