This Phase II proposal is for a collaborative effort between Dr. Richard Weinshilboum of the Mayo Institute and BellBrook Labs, LLC to translate basic pharmacogenetics research on conjugative drug metabolizing enzymes into molecular screening assays that can be applied in preclinical drug development. Drug metabolism problems such as drug interactions and production of toxic metabolites cause many of the adverse reactions that contribute to the extremely high failure rate of clinical trials. The most promising approach for effectively managing individual differences in drug response is the implementation of pharmacogenetic strategies for predicting drug metabolism in each patient. However, critical information on which enzymes are metabolizing drug candidates and how enzyme function is affected by genetic variation is lacking because in vitro assays for profiling compound metabolism with individual drug metabolizing enzymes are not available. Sulfation is a major route of drug metabolism carried out by a family of 12 enzymes in humans, and recently an additional 25 sulotransferase allozymes were identified, many with functional phenotypes. In Phase I, we developed a homogenous, fluorescent high throughput screening (HTS) method called donor product fluorescence polarization immunoassay (DP-FPIA) that provides a universal screening platform for measuring sulfation of any compound by any SUIT isoform. In Phase II, we will optimize the performance of the assay for pharma HTS isolating a highly selective monoclonal antibody against a nucleotide and synthesizing conjugates of red fluors-to' the same nucleotide. In addition a full panel of 20 human sulfotransferases - including several important genetic variants - will be expressed, and purified in parallel using automated, multfwell methods. We will then validate the assay for high throughput screening and kinetic analysis using prototypic substrates, drugs, and hormones screened against the full panel of sulfotransferases. The sulfotransferase DP-FPIA platform will be marketed in a kit format to pharmacuetical companies for preclinical metabolism studies and lead optimization. The unique capabilities of the DP-FPIA and the availability of a panel of recombinant sulfotransferases will enable a systematic delineation of human drug sulfation capacity and how it is affected by genetic variation.
