The long-term goal of this project is to understand the molecular determinants of substrate specificity and the enzymatic mechanism of enzymes involved in the activation of nucleoside prodrugs. Nucleoside prodrugs, major therapeutic agents for the treatment of HIV and cancer, are administered as uncharged nucleoside analogs to achieve effective intracellular concentrations. Once inside the cell, these prodrugs must be phosphorylated by cellular enzymes to become the active drug. Examples are AZT against HIV infections, which is activated by thymidylate kinase, and 6thiopurine, which is activated by guanylate kinase, against cancer. A major limitation to the efficacy of such prodrugs results from their poor activation by the human kinases. We are focusing on two families of nucleoside analogs, those that are activated by thymidylate kinase (TMPK) and those activated by guanylate kinase (GMPK).
The specific aims of this proposal are: 1. Delineate the role of conserved active site residues in catalysis and in achieving substrate specificity in the human thymidylate kinase. 2. Determine the effects of 3'-substitutions of TMP analogs on the phosphorylation rate by TMPK. 3. Elucidate the determinants of substrate specificity and the catalytic mechanism of kinases involved in the phosphorylation of guanine-based prodrugs. Our primary technology will include X-ray crystallography, complemented by other biochemical, biophysical and genetic approaches that include steady state kinetics, stopped-flow kinetics, and site-directed mutagenesis. Successful completion of this work will yield detailed information on catalysis and substrate recognition by nucleoside and nucleotides kinases. In addition, the information relating to the phosphoryl transfer reaction, a common and significant reactions in biological systems, will be generated. Collectively, this new information is expected to facilitate the development of new nucleoside prodrugs for the treatment of a spectrum of diseases including AIDS, cancer, and other viral infections.
