In this renewal application we propose to continue the development of novel new classes of antitumor agents that function by either reversibly or irreversibly inhibiting the enzymes of the glyoxalase pathway. In the previous funding period, we have demonstrated that enediol analog inhibitors (GSC(O)N(OH)R, where GS = glutathionyl) of the first enzyme in the pathway, glyoxalase I, inhibit the growth of murine and human tumors in vitro and in vivo. Growth inhibition appears to be due to the buildup of cytotoxic methylglyoxal, the substrate for the glyoxalase pathway. We have also discovered an efficient 'sulfoxide prodrug' for rapidly generating the enediol analog inside tumor cells. In addition, we have made major advances in our understanding of the structural biochemistry and mechanisms-of-action of glyoxalase I and the second enzyme in the pathway, glyoxalase II. We will now use this information to develop more potent antitumor agents.
The specific aims are: 1.) To synthesize the sulfoxide prodrugs CH3(CH)nS(O)C(O)N(OH)C6H4Cl (n = 1,5,8,10), designed to rapidly diffuse into tumor cells and give rise to the enediol analogs via an acyl-interchange reaction with intracellular glutathione. 2.) To test for correlations between the structures of the sulfoxides and their ability to deliver enediol analog into L1210 leukemia and B16 melanoma cells, and to inhibit the growth of these tumors, in culture. 3.) To evaluate the pharmacokinetics and antitumor properties of the most promising sulfoxide prodrugs in mice bearing B16 melanotic melanoma. 4.) To determine the molecular basis of active-site directed irreversible inactivation of GlxI by S-(4-bromo-2,3-dioxobutyl)glutathione. 5.) To synthesize affinity labels of human GlxI, which target either Glu172 or Cys60 in the active site. Some of the affinity labels are designed to be hydrolyzed by GlxII, an enzyme activity that is high in normal cells but low in tumor cells.
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