Inhibitors Against Trypanosomatid Pyruvate Kinases

描述(申请人提供)：这个项目的目标是从两种原生寄生虫中发现和优化丙酮酸激酶(PYK)的选择性抑制剂：克氏锥虫在整个美洲引起查加斯病，包括德克萨斯州和美国其他南部地区，而布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5亿人生活在流行区)，每年造成约13万人死亡。鉴于其衰弱和普遍的性质，它们给主要是发展中世界的所有人口造成了沉重的医疗、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，而且目前的药物显示出高毒性和低疗效，这进一步加剧了这个问题。糖酵解在克鲁兹毛滴虫和布氏毛滴虫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，PYK等糖酵解酶的抑制剂可以作为新药开发的先导化合物。此外，TbPYK已被RNAi证实为药物靶点，并且还可以获得详细的结构信息。此外，锥虫PYK与它们的人类同行显示出显著的差异，特别是在变构性质方面。拟议的研究有以下具体目标：(1)开发和验证安装在NCGC高通量筛查平台上的克鲁兹毛滴虫和布氏毛滴虫PYK的QHTS检测方法。(2)探索利用含有激活剂F16BP的检测来检测HTS的新方法，从而确保目标酶将处于其激活的构象。效应器结合的(或R状态)构象不仅对应于体内的主要构象(例如，在布氏毛滴虫细胞中果糖二磷酸水平通常是饱和的)，而且也是一种更刚性的结构，结果是与R状态结合部位结合的抑制剂可能在信息论上更受青睐。(3)利用这些方法筛选包含30多万个小分子的分子文库小分子文库(MLSMR)。锥虫PYK的独特结构特征(与相应的人类酶相比)将被用来发现这些酶的选择性抑制剂。(4)在一组二次HIT验证分析中确认这些化合物的效力，并在动力学和选择性分析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前途的分子的效力。(5)通过酶分析、蛋白质结晶学和生物物理测定，确定最有希望的分子的作用方式。(6)测试显示最高效力的化合物，其抑制代表寄生虫致病阶段的培养的锥虫细胞的生长，以及对培养的人类细胞没有毒性。

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.