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HTS筛选试验和范例，以鉴定引起人类非洲锥虫病（HAT）或昏睡病的布氏锥虫的蛋白激酶抑制剂。HAT在撒哈拉以南非洲地区流行，每年夺去约3万人的生命，并使约6000万人面临感染风险。HAT不是制药行业的优先事项，因此NIH和WHO将其归类为被忽视的热带病（NTD）。对新的、有效的化疗的需求是迫切的。为了鉴定新的化疗药物，Kojo Wilmot-Mensa博士（UGA）使用了一种化学蛋白质组学策略，使用激酶抑制剂拉帕替尼来鉴定寄生虫所必需的激酶。他发现了三种具有额外遗传验证的激酶。在初步工作中，这些激酶被表达、纯化，并建立了证明活性的酶测定。为了发现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测定和筛选范式。该研究小组将采用一种策略，利用表型和分子方法来发现药物，因此通过1）使用三个T。布氏杆菌激酶，2）全长蛋白和底物，以测定形式捕获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活化。将对鉴定的分子进行聚类，并在表型寄生虫试验中评价代表性分子的活性。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（大卫Swinney博士，非营利研究所罕见和被忽视的疾病药物发现），激酶HTS检测开发和药物发现（Chakk Ramesha博士，Medhus Bio）以及蛋白质表达和生产（大卫Chereau博士，96蛋白质）的互补技能。在三年资助期结束时，我们希望提供经过验证的HTS检测和筛选模式，以提供具有药物质量的化合物。我们预计，通过这一举措开发的检测试剂盒将提交给HTS设施，我们预计将使用HTS的结果作为基础，从适当的实体寻求额外的资金，用于先导化合物的开发。

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.