The long term objective of this project is to determine whether inhibitors of the glyoxalase enzyme system, composed of the isomerase glyoxalase I (GlxI) and the thioester hydrolase glyoxalase II (GlxII), can be developed into effective antitumor agents in whole animals. This general strategy is potentially important because (a) it does not directly target nucleic acid metabolism and, therefore, might not give rise to the side effects commonly associated with most cancer chemotherapies, and (b) uses hydrophilic enzyme inhibitors that might be less susceptible to multidrug resistance once delivered into cancer cells as lipophilic prodrugs. In support of the feasibility of this anticancer strategy, we recently synthesized four enediol analogs that are the strongest inhibitors of human GlxI yet reported: GSC(O)N(OH)R, where GS = glutathionyl, R = CH3(l), C6H5(2), C6H4Cl(3), C6H4Br(4). Preliminary in vitro studies are consistent with the idea that these compounds are potential tumor- selective anticancer agents when delivered into human leukemia cells as the lipophilic [glycyl, gamma-glutamyl] diethyl esters. The immediate objective of this revised proposal is to further examine the in vitro and in vivo antitumor properties of these compounds in a murine model system, and to better understand the molecular basis of tight-binding inhibition of GlxI by the enediol analogs.
The specific aims are as follows: (l) To examine the mechanism by which the diethyl esters of the enediol analogs (1-4) are transported into mouse leukemia L1210 cells in vitro. (2) To evaluate the cytotoxicities of the enediol analogs (1-4) and their ethyl esters toward L1210 cells in vitro. (3) To determine the pharmacokinetic properties of the enediol analogs (1-4) and their ethyl esters in serum esterase deficient mice. (4) To test the in vivo efficacy of the enediol analogs and their ethyl esters in serum esterase-deficient mice bearing L1210 murine leukemia and in C.B-l7 SCID mice bearing human tumor xenografts, androgen-independent human prostate cancer (PC-3), and human colon adenocarcinoma (HT-29). (5) To test the hypothesis that stabilization of the enediol(ate) intermediate, formed along the reaction pathway of GlxI, is due to the movement of a flexible (TIM-like) peptide loop near the active site. (6) To test the hypothesis that during the GlxI reaction an active site glutamic acid residue catalyzes the proton transfer associated with isomerization.
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