Identification of selective inhibitors of phosphofructokinase as lead compounds a

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）磷酸果糖激酶（PFK）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）引起严重的、往往是致命的人类疾病，如昏睡病、恰加斯病和黑热病，这些国家有数百万人生活在这些疾病流行的地区。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。糖酵解在布鲁氏杆菌的感染阶段是必需的，因此是一个有希望的药物靶点。因此，糖酵解酶抑制剂如PFK可作为开发新药的先导化合物。提出的研究有以下具体目标：(1)利用锥虫PFK的独特特征（其详细的结构信息已经可用，并已被RNAi验证为药物靶点），通过高通量筛选含有超过30万个小分子的分子库小分子库（MLSMR），发现布鲁氏锥虫PFK的选择性抑制剂（TbPFK）；(2)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；(3)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；(4)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以及对培养的人类细胞缺乏毒性方面表现出最高的效力。

DESCRIPTION (provided by applicant): The goal of this project is to discover and optimize selective inhibitors of phosphofructokinase (PFK) 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 many millions live in areas where the diseases are endemic. 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 and therefore a promising drug target. Inhibitors of glycolytic enzymes such as PFK may thus serve as lead compounds for the development of new drugs. The proposed research has as specific aims: (1) To exploit unique features of trypanosomatid PFK (for which detailed structural information is already available and which has been validated as a drug target by RNAi) for the discovery of selective inhibitors of PFKs from T. brucei (TbPFK) through high-throughput screening of the Molecular Library Small Molecule Repository (MLSMR) containing more than 300,000 small molecules; (2) 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; (3) To determine the mode of action of the most promising molecules by enzyme assays, protein crystallography and biophysical measurements; and (4) 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 40 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.