Drug Discovery from Slow Growing and Rare Microbial Species

描述(申请人提供)：这个项目的总体目标是发现新的抗生素来对抗重要的耐药病原体。我们对金黄色葡萄球菌、耐万古霉素肠球菌(VRE)、耐多药铜绿假单胞菌、鲍曼不动杆菌、超广谱β-内酰胺酶和新德里产金属β-内酰胺酶肠杆菌以及结核分支杆菌等病原体的治疗选择正在耗尽。在过去的30年里，只有3种新的抗生素被引入--利奈唑胺、达托霉素和非达克西星。利奈唑胺和非达克索米星是在60年代发现的，但当时似乎没有足够的吸引力。随着发现的步伐加快，阻力正在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。合成化合物库中没有一种药物是从HTS中出来的。放线菌产生的次生代谢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天然产品来源--以前未培养的细菌，它们构成了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群体，这是大多数物种的重要组成部分。我们推断，生长缓慢的细菌实际上可能代表了休眠的细菌形式，重新种植后会迅速生长。大多数生长缓慢的植物在重新接种后确实可以快速培养，其中许多分离物代表了以前未知的物种和属。在第一阶段，我们开发了一种方法，通过将单个细胞放置在微滴定板的孔中来同时分离和培养慢速生长菌。对这些分离物中的5000株进行筛选，产生了3种新的抗微生物化合物，其中包括专门针对结核分枝杆菌的Novo23。Novo23的靶标是基本分枝杆菌ClpP蛋白酶的ClpC1亚单位。Novo23具有低细胞毒性、良好的耐受性和小鼠的血药浓度。我们将检验Novo23在小鼠结核病模型中的疗效。我们的三种新型抗菌药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先导能用于药物。因此，我们还将进行一项大规模的发现工作，以确定其他将进入验证的抗菌药 它们变得可用。将对新化合物的光谱、效力、抗药性发展、稳定性、作用机制和结构新颖性进行检查。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有效性。这将使随后的临床前开发转向IND、临床研究和FDA批准一种新的治疗方法。我们相信，这一战略--在推进线索的同时以发现管道为后盾--极大地增加了该项目成功的机会。

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.