Inhibitors Against Trypanosomatid Pyruvate Kinases

描述（由申请人提供）：该项目的目标是从两种原生寄生虫中发现并优化丙酮酸激酶（PYK）的选择性抑制剂：克氏锥虫在整个美洲引起恰加斯病，包括德克萨斯州和美国其他南部地区，而布鲁氏锥虫的两个亚种在撒哈拉以南非洲地区引起昏睡病。锥虫家族的寄生虫总共感染了全世界3000万人（约5亿人生活在流行地区），每年造成约13万人死亡。鉴于其使人衰弱和普遍存在的性质，它们给主要是发展中世界的全体人民造成了沉重的医疗、经济和社会负担。在过去的50年里，现有的治疗方法进展甚微，目前的药物毒性高，疗效差，这进一步加剧了这个问题。糖酵解在克氏体和布鲁氏体的感染阶段是必不可少的，因此是一个有希望的药物靶点。因此，糖酵解酶抑制剂如PYK可作为开发新药的先导化合物。此外，TbPYK已被RNAi验证为药物靶点，并获得了详细的结构信息。此外，锥虫的PYKs与人类的PYKs表现出显著的差异，特别是在变构特性方面。本研究的具体目的有：(1)建立并验证NCGC高通量筛选平台上克氏体和布鲁氏体PYKs的qHTS检测方法。(2)探索一种含有激活剂F16BP的检测HTS的新方法，从而确保靶酶处于激活构象中。效应结合（或r态）构象不仅与体内的主要构象相对应（例如，果糖二磷酸水平在布鲁氏t细胞中通常是饱和的），而且也是一种更刚性的结构，其结果是与r态结合位点结合的抑制剂可能在熵上更有利。(3)利用这些方法筛选含有超过30万个小分子的分子库（Molecular Library Small Molecule Repository， MLSMR）。与相应的人类酶相比，锥虫PYK的独特结构特征将被用于发现这些酶的选择性抑制剂。(4)通过一组二次命中验证试验确认这些化合物的效价，并在动力学和选择性试验中测试它们的特异性，并进一步提高通过基于结构的方法、类似物合成和药物化学原理获得的最有希望的分子的效价。(5)通过酶分析、蛋白质晶体学和生物物理测量来确定最有希望的分子的作用模式。(6)测试化合物在抑制培养的代表寄生虫致病阶段的锥虫细胞生长的能力方面表现出最高的效力，以及对培养的人类细胞缺乏毒性。

DESCRIPTION (provided by applicant): The goal of this project is to discover and optimize selective inhibitors of pyruvate kinase (PYK) from two protist parasites: Trypanosoma cruzi causes Chagas' disease throughout the Americas, including Texas and other southern regions of the United States, whereas two subspecies of Trypanosoma brucei cause sleeping sickness in areas of sub-Saharan Africa. Collectively parasites of the trypanosomatid family infect 30 million people worldwide (~500 million live in endemic areas), and account for ~130K deaths annually. Given their debilitating and prevalent nature, they impose a heavy medical, economic and social burden on entire populations primarily in the developing world. The problem is further exacerbated by the fact that existing treatments have progressed little in the past 50 years, and current drugs show high toxicity and poor efficacy. Glycolysis is essential in the infective stages of T. cruzi and T. brucei, and is therefore a promising drug target. Inhibitors of glycolytic enzymes such as PYK may thus serve as lead compounds for the development of new drugs. Furthermore, TbPYK has been validated as a drug target by RNAi, and detailed structural information is also available. In addition, trypanosomatid PYKs show significant differences from their human counterparts, especially with regard to allosteric properties. The proposed research has as specific aims: (1) To develop and validate qHTS assays mounted on the NCGC high-throughput screening platform for T. cruzi and T. brucei PYKs. (2) To explore a novel approach to HTS with assays containing the activator F16BP, thereby ensuring that the target enzyme will be in its activated conformation. The effector-bound (or R-state) conformation not only corresponds to the predominant conformation in vivo (for example, fructose bisphosphate levels are normally saturating in T. brucei cells), but is also a more rigid structure, with the consequence that inhibitors binding to R-state binding sites are likely to be entropically favoured. (3) To use these assays to screen the Molecular Library Small Molecule Repository (MLSMR) containing more than 300,000 small molecules. Unique structural features of trypanosomatid PYK (compared with the corresponding human enzymes) will be exploited for the discovery of selective inhibitors of these enzymes. (4) To confirm the potency of these compounds in a panel of secondary hit validation assays and to test their specificity in kinetic and selectivity assays, and to further improve the potency of the most promising molecules thus obtained by structure-based methods, analogue synthesis and medicinal chemical principles. (5) To determine the mode of action of the most promising molecules by enzyme assays, protein crystallography and biophysical measurements. (6) To test compounds displaying the highest potency for their ability to inhibit growth of cultured trypanosomatid cells representing pathogenic stages of the parasites, as well as for lack of toxicity toward cultured human cells.