Inhibitors Against Trypanosomatid Pyruvate Kinases

锥虫丙酮酸激酶抑制剂

• 批准号: 8304823
• 负责人: Malcolm Douglas Walkinshaw
• 金额: $ 2.7万
• 依托单位: UNIVERSITY OF EDINBURGH
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-22 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8304823
• 关键词: Accounting; Africa South of the Sahara; African Trypanosomiasis; Americas; Area; Binding; Binding Sites; Biological Assay; Calorimetry; Cells; Cessation of life; Chagas Disease; Chemicals; Consumption; Crystallization; Crystallography; Databases; Detection; Development; Disease; Drug Delivery Systems; Economic Burden; Ensure; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Family; Firefly Luciferases; Fructose; Glycolysis; Goals; Growth; Human; Institutes; Kinetics; Lead; Leishmania; Leishmania mexicana; Life; Ligands; Measurement; Medical Economics; Metabolic; Methods; Mining; Molecular Bank; Molecular Biology; Molecular Conformation; Molecular Models; NADH; Nature; Parasites; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phosphotransferases; Population; Poverty; Procedures; Property; Protein Isoforms; Proteins; Public Health; Pyruvate Kinase; RNA Interference; Reading; Research; Resistance; Risk; Roentgen Rays; Screening procedure; Site-Directed Mutagenesis; Specificity; Staging; Structure; Structure-Activity Relationship; Surface Plasmon Resonance; Testing; Texas; Titrations; Toxic effect; Trypanosoma brucei brucei; Trypanosoma cruzi; United States; Universities; Validation; analog; base; chemical synthesis; fructose-1,6-diphosphate; health economics; high throughput screening; improved; in vivo; inhibitor/antagonist; light scattering; molecular modeling; novel strategies; nutrition; preclinical study; programs; relational database; repository; response; scaffold; small molecule; social; structural biology; three dimensional structure; virtual

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.

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。锥虫科寄生虫总共感染全世界 3000 万人（约 5 亿人生活在流行地区），每年造成约 13 万人死亡。鉴于其使人衰弱且普遍存在的性质，它们给主要是发展中国家的全体人民带来了沉重的医疗、经济和社会负担。由于现有的治疗方法在过去 50 年来几乎没有取得任何进展，而且现有药物毒性高、疗效差，这一事实进一步加剧了这一问题。糖酵解在克氏锥虫和布氏锥虫的感染阶段至关重要，因此是一个有前途的药物靶点。因此，PYK 等糖酵解酶抑制剂可以作为新药开发的先导化合物。此外，TbPYK 已被 RNAi 验证为药物靶点，并且还提供了详细的结构信息。此外，锥虫 PYK 与其人类对应物表现出显着差异，特别是在变构特性方面。拟议的研究有以下具体目标：（1）开发和验证安装在 NCGC 高通量筛选平台上的 T. cruzi 和 T. brucei PYK 的 qHTS 检测方法。 (2) 探索一种新的 HTS 方法，通过含有激活剂 F16BP 的检测，从而确保目标酶处于激活构象。效应器结合（或 R 态）构象不仅对应于体内的主要构象（例如，T. brucei 细胞中的二磷酸果糖水平通常是饱和的），而且还是一种更刚性的结构，因此与 R 态结合位点结合的抑制剂可能会受到熵的青睐。 (3) 使用这些检测方法筛选包含超过300,000个小分子的分子库小分子存储库(MLSMR)。锥虫 PYK 的独特结构特征（与相应的人类酶相比）将被用于发现这些酶的选择性抑制剂。 (4) 在一系列二次命中验证试验中确认这些化合物的效力，并测试其在动力学和选择性试验中的特异性，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力。 (5) 通过酶测定、蛋白质晶体学和生物物理测量确定最有希望的分子的作用方式。 (6) 测试化合物在抑制代表寄生虫致病阶段的培养锥虫细胞生长的能力方面表现出最高效力，并且对培养的人类细胞缺乏毒性。