Combined Substrate Polymerase Inhibitors

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DESCRIPTION (provided by applicant): Nucleotides are natural products that are essential for all processes involving gene replication, maintenance, expression and protein synthesis (in transcription factor cycling), not to mention their importance in energy storage, group transfer reactions, chemical coupling, and signal transduction (e.g., G-proteins). The main goals of our research on modified nucleotides are: 1) the design, synthesis, and kinetic analysis of combined substrate, also called template-competitive, inhibitors of bacterial and viral DNA polymerases and viral reverse transcriptases. We propose in this application to expand our studies to inhibitors of cloned viral RNA-Dependent RNA polymerases (RDRP). The goal of these studies is to maximize inhibition by modifications in nucleotide, sugar (new to this application), side chain moieties, and phosphates (new to this application); 2) to determine the activity and selectivity of these inhibitors over a range of cloned bacterial and viral targets; 3) to expand the nucleotide analog libraries to study the binding and inhibition of modified nucleotides as combined substrate inhibitors of inosine monophosphate dehydrogenase (IMPDH), a target applicable to tissue rejection, cancer, bacterial, and parasite treatments (new to this application); and 4) to develop cloning methods for rapid expression and purification of the targets needed for these studies (new to this application). These studies offer numerous opportunities for undergraduate training, including modified nucleoside, NMP and NTP synthesis and purification using standard and combinatorial methods; development of continuous kinetic methods for analysis of polymerase and IMPDH activity with analysis of inhibition patterns; PCR for cDNA production, cloning, transformation and selection, bacterial cell growth, harvesting, and breakage, and enzyme purification, including affinity and ion-exchange chromatographies. These methods cover a wide variety of techniques in experimental design, execution, data collection, and analysis that can benefit undergraduates in chemistry and biology seeking post-baccalaureate education for careers in the biomedical sciences. Combined substrate inhibitors concept is a type of drug design that is being applied to find new entities that might be used to treat cancer and bacterial, parasite, and viral infections. The compounds specifically targets proteins that are key in the replication of these cell or particle types. These targets include DNA polymerase, reverse transcriptases, viral RNA dependent DNA polymerases, and inosine monophosphate dehydrogenase, a control enzyme in the cell synthesis of GMP.