In 1992 the FDA approved ddC (zalcitabine) for the treatment of AIDS, the first drug to receive approval under the new accelerated review process but it is now known that ddC has severe toxic side effects and is rarely used today. In a 1993 hepatitis B clinical trial, five patients died from toxic side effects of another analog, FIAU. Based upon the discoveries we made during the past grant period we can now identify those compounds with the highest potential for mitochondrial toxicity before they go into the clinic. The toxicity of nucleoside analogs is correlated with their rate of incorporation by the mitochondrial DNA polymerase and their rate of removal by the proofreading exonuclease. Direct rate measurements indicate greater than a 100,000-fold variation in toxicity among the seven nucleoside/nucleotide analogs currently approved for the treatment of AIDS. Although other sites of action may lead to some observed toxic side effects, this work provides an easily quantifiable system to assess the potential for mitochondrial toxicity and provides an assay to screen for potential toxicity of new inhibitors of HIV RT or other viral polymerases. The goal of this research is to establish the structural and mechanistic rules governing nucleotide specificity for incorporation and removal so that we can predict which analogs are likely to be the least toxic. In particular, new features that may stimulate removal by the proofreading exonuclease could lead to even more effective and less toxic drugs. The structural and mechanistic basis for nucleotide selectivity will be examined using a combination of rapid reaction kinetic studies, mutation analysis, and structure/function studies on the mitochondrial polymerase. The rapid reaction kinetic analysis will allow quantification of the incorporation of natural and unnatural nucleotides to map empirically the structural constrains imposed upon the nucleotides during incorporation and excision reactions. Analysis of mutant enzymes and structure/function studies on the enzyme will afford exploration of the enzymatic structural basis for nucleotide selectivity. In addition, we will examine the roles of the mitochondrial DNA polymerase in base excision repair and its response to oxidatively damaged DNA. It is anticipated that this work will provide the basis for the design and testing of new nucleoside analogs with lower human toxicity to be used in the treatment of viral infections as well as providing basic knowledge of the reactions governing the replication of mitochondrial DNA.
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