Fluorescent HTS Assays for Human Sulfotransferases

描述（由申请人提供）：该II期提案是由Mayo研究所的Richard Weinshilboum博士和BellBrook Labs， LLC合作进行的，旨在将结合药物代谢酶的基础药物遗传学研究转化为可用于临床前药物开发的分子筛选分析。药物代谢问题，如药物相互作用和有毒代谢物的产生引起许多不良反应，导致临床试验的失败率极高。有效管理药物反应个体差异的最有希望的方法是实施药物遗传学策略来预测每个患者的药物代谢。然而，关于哪些酶代谢候选药物以及酶的功能如何受到遗传变异的影响的关键信息是缺乏的，因为没有用于分析单个药物代谢酶的化合物代谢的体外分析。硫酸化是人类药物代谢的主要途径，由12种酶组成的家族进行，最近又发现了25种硫转移酶同酶，其中许多具有功能表型。在第一阶段，我们开发了一种均匀的荧光高通量筛选（HTS）方法，称为供体产物荧光偏振免疫测定（DP-FPIA），它提供了一个通用的筛选平台，用于测量任何化合物被任何SUIT异构体的硫酸化。在第二阶段，我们将优化制药HTS检测的性能，分离针对核苷酸的高选择性单克隆抗体，并合成红色荧光-相同核苷酸的偶联物。此外，20种人类硫转移酶——包括几种重要的遗传变异——将被表达，并使用自动化的多孔方法并行纯化。然后，我们将使用针对全组硫转移酶筛选的原型底物、药物和激素来验证高通量筛选和动力学分析。巯基转移酶DP-FPIA平台将以试剂盒形式销售给制药公司，用于临床前代谢研究和先导物优化。DP-FPIA的独特功能和一组重组硫转移酶的可用性将能够系统地描述人类药物硫酸化能力及其如何受到遗传变异的影响。

DESCRIPTION (provided by applicant): 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.