M tuberculosis Membrane Protein Pharmaceutical Targets

描述(申请人提供)：本建议旨在解决：1)一种需要新药的重要疾病--40年来没有治疗结核分枝杆菌的新药，耐多药菌株和极端耐药菌株正变得越来越常见；2)缺乏整个药物靶标的结构信息，膜蛋白-不到1%的已知蛋白质结构是膜蛋白，而大多数生物基因组的25%至30%编码膜蛋白-此外，膜蛋白是比水溶性蛋白更有效的药物靶标；3)有效靶点的生物学、功能和结构特征--我们将只对那些对结核分枝杆菌生长至关重要的蛋白质进行特征分析，通过靶向膜蛋白，特别是外膜蛋白，药物靶标的获取将不需要通过细菌的膜运输。4)筛选技术上的差距-将专门针对膜蛋白开发基于溶液和固态核磁共振波谱的新的小分子筛选技术。我们已经根据初步结果和关于基本基因、毒力因子、外膜蛋白鉴定和对特定潜在靶点的大量个人研究的文献，制定了初步的靶标清单。其中一些蛋白质已经被确认为高潜力的药物靶标，这些蛋白质形成了一个优先目标清单，将允许所有项目和核心在资助的第一天开始努力。从生物功能(项目1)到分析开发(项目2)再到结构表征(项目3)，这些活动将密切合作。与分子结构相结合的分析将有助于建立结构-活性-关系。检测技术的发展将使对小分子的筛选成为可能，这些小分子对了解功能很重要，对结构研究可能也很重要。我们开发的分析方法和我们确定的配体将为旨在了解结核分枝杆菌生命和感染周期的生物学实验提供动力。这些配体将在药物发现中作为先导化合物有用，虽然这超出了本计划的范围，但该团队将为那些可能想要为这些膜蛋白靶标开发药物的人保护知识产权。为了实现这些目标，一个独特的研究团队被聚集在一起，他们拥有广泛的知识：结核分枝杆菌、基本结核分枝杆菌基因、膜蛋白生理学、分子生物学、生物物理学和结构表征。该计划提供了世界上两个一流的核磁共振设施及其在方法和技术开发方面的专业知识，此外，该团队还带来了独特的Mtb膜蛋白表达文库以及伯纳姆研究所的精湛筛选设施和专业知识。 项目1：确定目标(密苏里州尼德韦斯) 项目1说明(申请人提供)：超过60%的药物靶点是膜蛋白。然而，目前的抗结核药物中只有一种是乙胺丁醇，它针对的是结核分枝杆菌的膜蛋白，这表明这种巨大的潜力在很大程度上尚未被结核分枝杆菌所利用。最近发现的一种针对结核分枝杆菌ATP合成酶的二芳基喹啉新药，其活性超过了两种主要的结核病药物异烟肼和利福平，这证明了这一假设是正确的。因此，我们从大约800种可能的结核分枝杆菌内膜蛋白中选择了11种，它们对结核分枝杆菌在体外或在小鼠体内的生长是必不可少的。这些蛋白质被选择来涵盖广泛的功能，如必要营养物质的运输、细胞分裂、蛋白质和细胞壁的输出以及脂肪的生物合成。此外，它们足够小，可以适用于当前的固态核磁共振方法。外膜蛋白作为药物靶点提供了巨大的优势，抑制剂可能不需要穿过外膜，而外膜是分枝杆菌极其有效的渗透屏障。此外，它们很可能代表新的药物靶点，因为它们似乎与其他蛋白质没有任何相似之处。因此，我们在我们的初始靶点列表中包括了7个假定的外膜蛋白，其中6个功能未知。该项目的目标是验证18个选定的膜蛋白作为药物靶点的潜力，阐明这6个未知同源蛋白的生理功能，并确定其中一些蛋白可能的相互作用伙伴。

DESCRIPTION (provided by applicant): This Proposal is designed to address: 1) an important disease that needs novel drugs - no new drugs for Mycobacterium tuberculosis in 40 years and multi-drug resistant strains as well as extreme drug resistant strains are becoming more common; 2) the lack of structural information for an entire class of drug targets, the membrane proteins - less than 1% of known protein structures are membrane proteins, while 25 to 30% of the genome of most organisms code for membrane proteins - in addition, membrane proteins are more frequently effective drug targets than water-soluble proteins; 3) biological, functional and structural characterization of validated targets - we will characterize only those proteins that are essential for Mtb growth and by targeting membrane proteins, especially the outer membrane proteins, access to the drug targets will not require transport across the bacterial membranes. 4) a gap in screening technology - new small molecule screening technologies based on solution and solid state NMR spectroscopy will be developed specifically for membrane proteins. We have developed an Initial Target List from preliminary results and from literature on essential genes, virulence factors, identification of outer membrane proteins and numerous individual studies on specific potential targets. Some of these proteins are already validated as high potential pharmaceutical targets, these form a Prioritized Target List that will allow all of the Projects and Cores to initiate their efforts on the first day of funding. From biological function (Project 1) to assay development (Project 2) to structural characterization (Project 3) these activities will work closely together. Assays coupled with molecular structure will help establish structure-activity-relationships. Assay development will enable screening against small molecules important for understanding function and potentially important for structural studies. The assays we develop and the ligands we identify will fuel biological experiments designed to understand the life and infection cycle of Mtb. These ligands will be useful as lead compounds in drug discovery, and while this is beyond the scope of this Program, this team will protect the intellectual property for those who may want to pursue the development of drugs for these membrane protein targets. To accomplish these goals a unique team of investigators has been brought together with extensive knowledge of: Mycobacterium tuberculosis, essential Mtb genes, membrane protein physiology, molecular biology, biophysics, and structural characterization. The Program offers access to two of the premier NMR facilities in the world along with their expertise in methodology and technology development, in addition, the Team brings with it unique expression libraries of Mtb membrane proteins and the superb screening facilities and expertise of the Burnham Institute. PROJECT 1: TARGET IDENTIFICATION (NIEDERWEIS, M) PROJECT 1 DESCRIPTION (provided by applicant): More than 60% of all drug targets are membrane proteins. However, only one of the current antituberculous drugs, ethambutol, targets membrane proteins of M. tuberculosis, indicating that this vast potential is largely untapped for M. tuberculosis. The recent discovery of a new diarylquinoline drug that targets the ATP synthase of M. tuberculosis and exceeds the activities of the two main TB drugs isoniazid and rifampin provides proof of principle that this assumption is correct. Therefore, we have selected 11 out of approximately 800 putative inner membrane proteins of M. tuberculosis, which are essential for growth of M. tuberculosis in vitro or in mice. These proteins were selected to cover a broad variety of functions, such as transport of essential nutrients, cell division, export of proteins and cell wall and lipid biosynthesis. Furthermore, they are sufficiently small to be amenable to current solid-state NMR methods. Outer membrane proteins offer the tremendous advantage as drug targets that inhibitors may not need to cross the outer membrane which is an extremely efficient permeability barrier in mycobacteria. In addition, they are likely to represent novel drug targets because they do not appear to show any similarity to other proteins. Therefore, we included seven putative outer membrane proteins in our list of initial targets, six of which have no known function. The goal of this project is to validate the potential of the 18 selected membrane proteins as drug targets, to elucidate the physiological function of the six proteins with no known homologs and to identify putative interaction partners for some of these proteins.