Development of HTS assay and screening paradigm to discover new kinase inhibitors

描述(由申请人提供)：该项目的总体目标是提供一种HTS筛查试验和范例，以确定导致人类非洲锥虫病(HAT)或昏睡病的布鲁氏锥虫蛋白激酶抑制剂。HAT在撒哈拉以南非洲流行，每年夺走约30000人的生命，使约6,000万人面临感染风险。HAT不是制药业的优先事项，因此NIH和WHO将其归类为一种被忽视的热带病(NTD)。迫切需要新的、有效的化疗。为了识别新的化疗药物，Kojo Wilmot-Mensa博士(UGA)使用了一种使用激酶抑制剂拉帕替尼的化学蛋白质组策略来识别寄生虫所必需的激酶。他确定了三种具有额外基因验证的激酶。在前期工作中，表达、纯化了这些酶，并建立了显示活性的酶分析。为了实现发现HAT新药的目标，已经建立了一个多学科团队来开发这些激酶的HTS分析和筛选范例。该团队将采用一种策略，利用表型和分子方法进行药物发现，从而通过以下方式增加成功的可能性：1)使用三种布鲁氏锥虫激酶，2)全长蛋白质和底物的分析形式，以捕捉3)不同的分子作用机制，如缓慢解离、ATP非竞争性抑制和阻断激酶激活。识别出的分子将被聚集在一起，并在表型寄生检测中评估代表的活性。我们的多学科方法融合了寄生虫生物学和化学蛋白质组学(Kojo Wilmot-Mensa博士，UGA)、药物发现和激酶酶学(David Swinney博士，非营利性稀有和被忽视疾病药物发现研究所)、激酶HTS分析开发和药物发现(Chakk Ramesha博士，Medhus Bio博士)以及蛋白质表达和生产(David Chereau博士，96种蛋白质)的互补技能。在为期三年的资助期结束时，我们预计将提供经过验证的HTS分析和筛选范例，以提供具有成为药物的质量的化合物。我们预计，通过这一倡议开发的化验将提交给HTS设施，我们预计将利用HTS的结果作为基础，从适当的实体寻求额外资金用于铅化合物开发。

DESCRIPTION (provided by applicant): The overall objective of the project is to provide a HTS screening assay and paradigm to identify protein kinase inhibitors for Trypanosoma brucei that causes Human African trypanosomiasis (HAT), or sleeping sickness. HAT is endemic in sub-Saharan Africa, claiming the lives of about 30000 people every year and putting approximately 60 million people at risk of infection. HAT is not a priority for the pharmaceutical industry and thus the NIH and WHO categorized it as a neglected tropical disease (NTD). The need for new, effective chemotherapy is urgent. In order to identify new chemotherapy Dr Kojo Wilmot-Mensa (UGA) has used a chemical proteomic strategy using the kinase inhibitor, lapatinib, to identify kinases essential to the parasite. He identified three kinases that have additional genetic validation. In preliminary work these kinases were expressed, purified and enzyme assays established that demonstrated activity. Toward the goal of discovering new medicines for HAT, a multidisciplinary team has been established to develop an HTS assay and screening paradigm for these kinases. The team will employ a strategy to take advantage of both phenotypic and molecular approaches to drug discovery and therefore increase the probability of success through the 1) use of three T. brucei kinases, 2) full-length protein and substrates in assay formats that will capture 3) different molecular mechanisms of action, such as slow-dissociation, ATP noncompetitive inhibition and blocking kinase activation. The molecules identified will be clustered and representatives evaluated for activity in phenotypic parasitic assays. Our multidisciplinary approach merges the complementary skills of parasite biology and chemical proteomics (Dr Kojo Wilmot-Mensa, UGA), drug discovery and kinase enzymology (Dr David Swinney, non-profit Institute for Rare and Neglected Diseases Drug Discovery), kinase HTS assay development and drug discovery (Dr Chakk Ramesha, Medhus Bio) and protein expression and production (Dr David Chereau, 96 Proteins). At the end of the three-year funding period we expect to deliver a validated HTS assays and screening paradigm to deliver compounds with the quality to become medicines. We anticipated that assays developed through this initiative will be submitted to HTS facilities and we expect to use the results from HTS as the basis for seeking additional funding from appropriate entities for lead compound development.