Drug Discovery from Slow Growing and Rare Microbial Species

描述（由申请人提供）：本项目的总体目标是发现新型抗生素，以对抗重要的耐药病原体。我们正在用尽对病原体的治疗选择，如S。金黄色葡萄球菌MRSA、万古霉素耐药肠球菌（VRE）、多重耐药铜绿假单胞菌、A.鲍曼不动杆菌、产超广谱β-内酰胺酶肠杆菌和产金属β-内酰胺酶肠杆菌;结核在过去的30年中，只有3种新型抗生素被引入-利奈唑胺，达托霉素和非达霉素。利奈唑胺和非达霉素在60年代被发现，但当时并没有足够的吸引力。随着这种发现的步伐，耐药性的上升也就不足为奇了。越来越明显的是，抗生素发现的瓶颈是缺乏良好的起始化合物。没有一种药物来自合成化合物库的HTS。放线菌产生的次级代谢产物一直是抗生素的主要来源，但这一资源被过度开发。与此同时，还有一个潜在的非常大的未开发的天然产品来源-以前未培养的细菌，占所有细菌物种的绝大多数。生长缓慢的物种需要几个月的时间才能在培养皿上形成菌落，这是大多数物种的重要组成部分。我们推断，缓慢生长的细菌实际上可能代表了休眠形式的细菌，并且在重新接种后会迅速生长。大多数生长缓慢的菌株确实可以在重新接种后快速培养，许多分离株代表以前未知的种和属。在第一阶段，我们开发了一种方法，通过将单个细胞置于微量滴定板的威尔斯孔中，同时分离和培养缓慢生长的细胞。筛选这些分离株中的5，000株产生了3种新的抗菌化合物，包括特异性抗M的Novo 23。结核Novo 23的靶点是基本分枝杆菌ClpP蛋白酶的ClpC 1亚基。Novo 23在小鼠中具有低细胞毒性、良好的耐受性和血液水平。我们将检查Novo 23在结核病小鼠模型中的疗效。我们三种新型抗菌药物的进一步开发是第二阶段的主要重点。然而，我们认识到，只有一小部分铅使其成为药物。因此，我们还将进行大规模的发现工作，以确定将进入验证的其他抗菌药物， 它们变得可用。将检查新化合物的光谱、效价、耐药性发展、稳定性、作用机制和结构新奇。出现的线索将在小鼠感染模型中进行测试。第二阶段的最终结果将是三种先导化合物在动物模型中显示出疗效。这将使随后的临床前开发能够实现IND、临床研究和FDA批准新的治疗药物。我们相信这种策略-推进线索，同时用发现管道支持它们-大大增加了项目成功的机会。

DESCRIPTION (provided by applicant): The overall goal of this project is to discover novel antibiotics to combat important drug-resistant pathogens. We are running out of treatment options for pathogens such as S. aureus MRSA, vancomycin-resistant Enterococci (VRE), multidrug-resistant P. aeruginosa, A. baumannii, ESBL and New Delhi metallo-b- lactamase-producing Enterobacteriaceae, and M. tuberculosis. Only 3 novel antibiotics have been introduced in the past thirty years - linezolid, daptomycin, and fidaxomicin. Linezolid and fidaxomicin were discovered in the 60s, but did not appear sufficiently attractive at the time. With this pace of discovery, it is not surprising that resistance is on the rise. It is becoming increasingly apparent that the bottleneck in antibiotic discovery is the lack of good starting compounds. Not a single drug came out of HTS of synthetic compound libraries. Secondary metabolites produced by actinomycetes have been the main source of antibiotics, but this resource was over mined. At the same time, there is a potentially very large untapped source of natural products - previously uncultured bacteria that make up the vast majority of all bacterial species. Slow- growing species that require months to form colonies on a Petri dish are an important component of this majority. We reasoned that slow growers may actually represent dormant forms of bacteria, and will rapidly grow upon reinoculation. The majority of slow growers can indeed be rapidly cultured upon reinoculation, and many of the isolates represent previously unknown species and genera. In Phase I, we developed a method to simultaneously isolate and culture slow growers by placing individual cells in wells of a microtiter plate. Screening 5,000 of these isolates produced 3 new antimicrobial compounds, including Novo23 that acts specifically against M. tuberculosis. The target of Novo23 is the ClpC1 subunit of the essential mycobacterial ClpP protease. Novo23 has low cytotoxicity, favorable tolerability and blood levels in mice. We will examine efficacy of Novo23 in mouse models of tuberculosis. Further development of our three novel antibacterials are a major focus of Phase II. However, we recognize that only a small fraction of leads makes it to a drug. Thus, we will also undertake a large-scale discovery effort to identify additional antibacterials which will enter validation as they become available. Novel compounds will be examined for spectrum, potency, resistance development, stability, mechanism of action, and novelty of structure. Leads that emerge will be tested in mouse models of infection. The end result of Phase II will be three lead compounds showing efficacy in animal models. This will enable subsequent preclinical development towards an IND, clinical studies, and FDA approval of a new therapeutic. We believe this strategy - advancing leads while backing them with a discovery pipeline - greatly increases the chances for the project's success.