The goal of this proposed research is to promote through studies of its metabolism the utility in human disease of aphidicolin, a specific DNA polymerase Alpha inhibitor and possible antitumor and antiviral agent, and to develop new chemotherapeutic agents structurally related to aphidicolin. Aphidicolin has witnessed considerable application in identifying the functional roles of eukaryotic DNA polymerases and its specific and unique mode of action have encouraged clinical trials. However, aphidicolin, at levels active in inhibiting DNA synthesis, is rapidly """"""""inactivated"""""""" by hepatic enzymes (S-9 fraction) of rats, mice and presumably humans. Due to the rate of inactivation and to the great variability in the levels of many liver enzymes involved in xenobiotic metabolism (due to sex, age and induction), the metabolism of aphidicolin could severely limit its employment in certain disease states. We propose an integrated set of investigations through which a detailed knowledge of aphidicolin metabolism will be obtained and applied to the development of rationally defined aphidicolin analogs and specific enzyme inhibitors. These investigations include metabolic studies of aphidicolin which will determine the chemical structures of the metabolites generated, the specific enzymes responsible for its metabolism in microsomes (and cytosol) isolated from rat liver, and the inducibility of the responsible enzymes by chemical agents including aphidicolin. All new compounds isolated will be assayed for DNA polymerase Alpha inhibitory activity. Through a knowledge of the structures of the metabolites and their DNA polymerase inhibition activities, vastly improved structure-activity relationships for this unique compund will be obtained. In addition, """"""""active"""""""" metabolites with functionalized sites (eg. hydroxylation at saturated positions) may assist in the drug formulation of this lipophilic compound. Specific inhibitors of the enzymes involved in the metabolism will be pursued (eg. inhibitors of specific cytochrome P-450 isozymes), if such inhibitors would not prove lethal. An enantioselective, direct and versatile synthetic entry into the aphidicolin framework will be developed which is capable of synthesizing aphidicolin and generating aphidicolin analogs with specific site modifications (which are designed to inhibit enzymatic biotransformation).
