Determining the Effect of Dithiolethione Compounds on the Activity of Human Glyceraldehyde-3-Phosphate Dehydrogenase

Abstract

Dithiolethione compounds, like anethole dithiolethione (ADT) have chemopreventive, cytoprotective, and antimitogenic effects. The chemopreventive effects are thought to be mediated by the activation of antioxidant response elements and of the tumor repressor PP2a. We have shown that ADT interacts with the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Cancer cells usually exhibit increased glycolysis (Warburg effect) that generates ATP to meet their energy needs. Mounting evidence suggests that inhibition of glycolysis in cancer cells severely depletes ATP and may be an effective strategy in chemoprevention. We hypothesize that the chemopreventive effects of ADT may be in part due to inhibition of glycolysis via inhibition of GAPDH. Dithiolethiones can directly or indirectly modify cysteine residues in protein targets. GAPDH contains three cysteine residues that are susceptible to thiol-modifying agents. Therefore, we set out to examine how ADT affects GAPDH activity. Human GAPDH was overexpressed in bacterial cells and purified by ion-exchange and affinity chromatography. We measured the catalytic activity of GAPDH in the absence and the presence of ADT and showed a concentration-dependent inhibition of GAPDH catalytic activity by ADT. In order to determine the exact mechanism of ADT modification of GAPDH we are coupling site-directed mutagenesis with enzymatic assay, and mass-spectrometry.This investigation was sponsored in part by NIH/NIGMS MARC U∗STAR T34 08663 National Research Service Award to UMBC and also by the Howard Hughes Medical Institute's Precollege and Undergraduate Science Education Program (JC) and UMBC DRIF SRAIS (EG).