Inhibitors of Trypanosomatid Phosphoglycerate Mutase and Phosphoglycerate Kinase

项目描述（由申请人提供）：本项目旨在发现并优化锥虫寄生虫（布氏锥虫、克氏锥虫和利什曼原虫）磷酸甘油酸变化酶（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（主要是非洲、中美洲和南美洲以及亚洲）造成昏睡病、恰加斯病和黑热病等严重的、往往是致命的人类疾病，这些国家有3 000多万人感染，每年约有13万人死亡。可悲的是，目前用于治疗这些疾病的药物并不令人满意，因为它们对某些形式的疾病有毒且无效，而且耐药性正变得越来越普遍。糖酵解在布鲁氏t （Tb）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PGAM和PGK可以作为新药开发的先导化合物。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，与寄生虫酶没有共同的特征。TbPGAM已被RNAi证实为药物靶点，TbPGK已被抑制剂研究证实为药物靶点。这两种酶的详细结构信息已经可用。本研究有以下几个具体目标：(1)在NCGC高通量筛选平台上开发和验证新型的锥虫PGAM和PGK正向和反向检测方法。这四种试验将作为确认活性的主要试验和正交试验。(2)利用4种检测方法筛选含有30万以上小分子的Molecular Library Small Molecule Repository （MLSMR）。将利用锥虫PGAM和PGK的独特结构特征（与相应的人类酶相比），首先从T. bruei和L. Mexicana中发现这些酶的选择性抑制剂。(3)比较正向和反向的活性集。对TbPGK的初步分析已经表明，在两个方向上进行分析时，有可能获得不同化学型的抑制剂和激活剂。(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 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.