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.
Highlights
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
The structure showed that a hydrolysis product of PL 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
Coupled with in vitro kinetic data, which show a concentration-dependent inhibition of GSTP1 by hPL, our data suggest that PL is a prodrug, which becomes activated by hydrolysis to hPL, resulting in the formation of an hPL-GSH conjugate and inhibition of GSTP1
Summary
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. The anti-cancer effects of PL are associated with an elevation of reactive oxygen species (ROS) and a decrease in cellular GSH [11] Consistent with those observations, a leading cellular target was identified as glutathione S-transferase-Pi 1 (GSTP1), based on stable isotope labeling [1]. Structural information is available for many of these bound to their respective GST targets, and in each case, GSH resides in the glutathione binding site, or G-site, while the inhibitor occupies the hydrophobic region of the active site adjacent to GSH, or the H-site Several of these inhibitors have been shown to exhibit anti-tumor effects, general toxicity has prevented them from moving forward in human trials [1, 26]. Coupled with in vitro kinetic data, which show a concentration-dependent inhibition of GSTP1 by hPL, our data suggest that PL is a prodrug, which becomes activated by hydrolysis to hPL, resulting in the formation of an hPL-GSH conjugate and inhibition of GSTP1
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