Selective inhibitors of phosphoglycerate mutase and phosphoglycerate kinase as le

描述(申请人提供)：本项目的目标是发现和优化锥虫寄生虫(布氏锥虫、克鲁兹锥虫和利什曼原虫)的磷酸甘油酸变位酶(PGAM)和磷酸甘油酸激酶(PGK)的选择性抑制剂。这些病原体在热带和亚热带国家造成严重的、往往是致命的人类疾病，如昏睡病、恰加斯病和黑热病，主要是在非洲、中美洲和南美洲以及亚洲，这些国家有3000多万人受到感染，每年约有13万人死亡。可悲的是，目前治疗这些疾病的药物并不令人满意，因为它们有毒，对某些形式的疾病无效，而且耐药性正变得越来越普遍。糖酵解在布氏毛滴虫(TB)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，糖酵解酶的抑制剂，如PGAM和PGK，可能成为开发新药的先导化合物。PGAM是一个特别有吸引力的靶标，因为相应的宿主酶不是同源的，也没有与寄生虫酶的共同特征。TbPGAM已被RNAi证实为药物靶点，TbPGK已被抑制剂研究证实。关于这两种酶的详细结构信息已经可用。本研究的具体目标是：(1)建立和验证安装在NCGC高通量筛查平台上的新型正反向检测锥虫PGAM和PGK的方法。这四种分析方法将作为确认活性物质的初级分析和正交分析。(2)用这四种方法筛选包含30多万个小分子的分子文库小分子文库(MLSMR)。锥虫PGAM和PGK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，最初来自布鲁氏锥虫和墨西哥锥虫。(3)比较正反两个方向的激活剂集合。对TbPGK的初步检测已经表明，在这两个方向上进行检测时，都有可能获得不同化学类型的抑制剂和激活剂。(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。 公共卫生相关性：撒哈拉以南非洲的昏睡病、中美洲和南美洲的恰加斯病以及世界热带和亚热带地区的黑热病及相关疾病给已经陷入贫困、营养不良和疾病的悲惨循环的人们造成严重的公共卫生和经济负担。全世界有数百万人感染了这些潜在的致命疾病，数亿人处于危险之中。在过去的50年里，现有的治疗方法开发得很少，而且存在毒性、低效和耐药性；该项目的目标是开发适合进入临床前试验的先导药物。

DESCRIPTION (provided by applicant): The goal of this project is to discover and optimize selective inhibitors of phosphoglycerate mutase (PGAM) and phosphoglycerate kinase (PGK) of trypanosomatid parasites (Trypanosoma brucei, T. cruzi and Leishmania species). These pathogens cause serious, often fatal diseases of humans such as sleeping sickness, Chagas' disease and kala-azar in tropical and subtropical countries primarily in Africa, Central and South America, and Asia where over 30 million people are infected, with approximately 130,000 deaths annually. Tragically, current drugs for their treatment are unsatisfactory because they are toxic and ineffective against some forms of the diseases, and resistance is becoming increasingly common. Glycolysis is essential in the infective stage of T. brucei (Tb) and therefore a promising drug target. Inhibitors of glycolytic enzymes such as PGAM and PGK may thus serve as lead compounds for the development of new drugs. PGAM is a particularly attractive target because the corresponding host enzyme is not homologous and has no features in common with the parasite enzyme. TbPGAM has been validated as a drug target by RNAi, and TbPGK by inhibitor studies. Detailed structural information is already available for both enzymes. The proposed research has as specific aims: (1) To develop and validate novel forward and reverse assays mounted on the NCGC high-throughput screening platform for trypanosomatid PGAM and PGK. The four assays will serve as both primary and orthogonal assays for confirmation of actives. (2) To use the four assays to screen the Molecular Library Small Molecule Repository (MLSMR) containing more than 300,000 small molecules. Unique structural features of trypanosomatid PGAM and PGK (compared with the corresponding human enzymes) will be exploited for the discovery of selective inhibitors of these enzymes initially from T. brucei and L. Mexicana. (3) To compare sets of actives in the forward and reverse directions. Preliminary assays on TbPGK have already shown the possibility to obtain inhibitors and activators of different chemotypes when assayed in the two directions. (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. PUBLIC HEALTH RELEVANCE: Sleeping sickness in sub-Saharan Africa, Chagas' disease in Central and South America, and kala-azar and related diseases in tropical and subtropical regions of the world cause severe public health and economic burdens on populations that are already caught in a tragic cycle of poverty, poor nutrition and disease. Millions of people worldwide are infected by these potentially fatal diseases and hundreds of millions are at risk. Existing treatments have developed little in the past 50 years, and suffer from toxicity, inefficiency and resistance; the goal of this project is to develop lead drugs that will be suitable for entry into pre-clinical trials.