Selective agents against C. difficile infection

描述(申请人提供)：这个项目的总体目标是开发一种针对艰难梭菌的选择性抗生素，艰难梭菌是导致抗生素引起的腹泻和结肠炎的主要病原体。这种病原体可以在健康的人身上携带，并通过肠道共生体被挡在门外。抗生素治疗会抑制正常的菌群，但艰难梭菌的孢子会存活、萌发并致病。使用甲硝唑、万古霉素或非达克索米星治疗不仅会杀死病原体，还会杀死共生菌，这可能会导致复发。理想情况下，人们希望有一种治疗药物，这种药物对艰难梭菌有选择性，因为它 在治疗病原体的过程中让正常的菌群恢复，防止复发。根据基因组研究，细菌共享一个约200个基因的核心，除此之外，可能有多达100-200个特定物种/属的必需基因。我们推测，大量特定靶点的存在为发现对艰难梭菌具有选择性作用的抗菌剂提供了机会。病原体是厌氧菌，而高温超导通常需要好氧条件。因此，目前治疗这种感染的疗法来自其他项目，并且没有选择性。我们开发了一种厌氧高温反应，并对艰难梭菌进行了第一次直接筛查。考虑到从天然产品中发现抗生素的历史上的高成功率，我们筛选了一个土壤细菌提取物库。这个文库来自一种未开发的资源，未培养的细菌，并含有丰富的新化合物。该文库对金黄色葡萄球菌和大肠杆菌进行了筛选，命中率分别为30%和0.5%。我们推测，剩下的“非活性”菌株可能含有对初始筛查中遗漏的特定物种具有活性的化合物。对艰难梭菌进行约5,000种提取物的中试筛选，结果符合率为1.3%。初步分析 在这些提取物中，有两种对艰难梭菌具有选择性，当与一小部分肠道共生菌进行测试时，并含有具有新质量的化合物。在拟议的项目中，我们将跟进这些热门歌曲，并筛选出50,000个额外的摘录 图书馆。点击率将由LC/MS取消复制，这将表明新颖性的程度，并进行测试 针对肠道共生菌的主要群体的代表。通过去复制的梭状芽孢杆菌选择性化合物将被测试效力、耐药频率、细胞毒性和对肠道上皮细胞的渗透。未被吸收并因此保留在感染部位的化合物将被优先考虑。结构测定将确认化合物的新颖性，并提供关于化合物是否适合进一步开发的信息。耐药突变株的全基因组测序将表明可能的目标。阿司匹林的最大耐受量和生物利用度 将在仓鼠身上确定先导化合物，这将为艰难梭菌疗效研究的设计提供信息。第一阶段的目标是在艰难梭菌感染的仓鼠模型中确定两到三种有效的先导化合物。这将为第二阶段的应用奠定坚实的基础，该阶段的应用旨在向IND的临床前发展。

DESCRIPTION (provided by applicant): The overall goal of this project is to develop a selective antibiotic against Clostridium difficile, the major agent responsible for antibiotic-induced diarrhea and colitis. The pathogen can be carried in healthy individuals, and is kept at bay by gut symbionts. Antibiotic treatment suppresses the normal flora, but spores of C. difficile survive, germinate and cause disease. Treatment with metronidazole, vancomycin, or fidaxomicin kills not only the pathogen, but also the symbionts, which may result in relapse. Ideally, one would like to have a therapeutic, which is selective against C. difficile, as it would allow the normal flora to restore in the course of treating the pathogen, preventing relapse. Based on genomic studies, bacteria share a core of ~200 genes, and in addition to those, there may be up to 100-200 essential genes specific to a given species/genus. We reasoned that the existence of a large number of specific targets presents an opportunity to discover antimicrobials acting selectively against C. difficile. The pathogen is an anaerobe, while HTS normally requires aerobic conditions. As a result, current therapeutics for treating the infection come from other programs, and are not selective. We developed an anaerobic HTS and performed a first direct screen against C. difficile. Given the historically high success rate of discovering antibiotics from natural products, we screened a library of extracts from soil bacteria. This library comes from an untapped resource, uncultured bacteria, and is rich in novel compounds. The library had been screened against S. aureus and E. coli, producing hit rates of 30% and 0.5%, respectively. We reasoned that the remaining "inactive" strains may harbor compounds active against particular species that were missed in the primary screen. A pilot screen of ~5,000 extracts resulted in a hit rate of 1.3% against C. difficile. Preliminary analysis of these extracts showed that two were selective against C. difficile when tested against a small panel of gut commensals, and contained compounds with novel masses. In the proposed project, we will follow up on these hits, and screen 50,000 additional extracts from the "inactive" library. Hits will be de-replicated by LC/MS, which will indicate the degree of novelty, and tested against representatives of the main groups of gut symbionts. Clostridium-selective compounds passing dereplication will be tested for potency, resistance frequency, cytotoxicity and penetration into intestinal epithelial cells. Compounds that are not absorbed and thus retained at the site of infection will be given priority. Structure determination will confirm novelty and provde information on the suitability of compounds for further development. Whole genome sequencing of resistant mutants will indicate the likely target. Maximum tolerated dose and bioavailability of leads will be determined in hamsters, which will inform the design of a C. difficile efficacy study The goal of Phase I is to identify two to three lead compounds with efficacy in a hamster model of C. difficile infection. This will form a solid basis for a Phase II application aimed at preclincal development towards an IND.