Selective agents against C. difficile infection

描述（由申请人提供）：本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，艰难梭菌是导致腹泻和结肠炎的主要病原体。这种病原体可以携带在健康的个体中，并被肠道共生体阻止。抗生素治疗抑制了正常植物群，但C.艰难梭菌存活、发芽并引起疾病。用甲硝唑、万古霉素或非达霉素治疗不仅可以杀死病原体，还可以杀死共生体，这可能导致复发。理想情况下，人们希望有一种治疗剂，它对C有选择性。很难，因为它会 在治疗过程中恢复正常植物群，防止复发。 基于基因组研究，细菌共有约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。我们推断，大量特异性靶标的存在为发现选择性抗C的抗菌剂提供了机会。很难病原体是一种厌氧菌，而HTS通常需要有氧条件。因此，目前用于治疗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HTS，并进行了第一次直接筛选C。很难鉴于从天然产物中发现抗生素的历史性高成功率，我们筛选了一个土壤细菌提取物库。这个库来自一个未开发的资源，未培养的细菌，并富含新的化合物。对该文库进行了S. aureus和E.大肠杆菌，产生的命中率分别为30%和0.5%。我们推断，剩余的“非活性”菌株可能含有对初级筛选中遗漏的特定物种有活性的化合物。对约5，000种提取物的中试筛选导致对C的命中率为1.3%。很难初步分析 结果表明，其中2种提取物对C.当针对一小组肠道寄生虫进行测试时，它很难被发现，并且含有具有新质量的化合物。在拟议的项目中，我们将跟踪这些点击，并从“非活动”中筛选50，000个额外的摘录 图书馆命中将通过LC/MS进行去重复，这将表明新奇的程度，并进行测试 主要肠道共生体的代表。将测试通过去复制的梭菌选择性化合物的效价、耐药频率、细胞毒性和对肠上皮细胞的渗透。未被吸收并因此保留在感染部位的化合物将被优先考虑。结构测定将确认新奇并提供化合物进一步开发的适用性信息。抗性突变体的全基因组测序将表明可能的靶点。最大耐受剂量和生物利用度 将在仓鼠中确定先导物，这将为C.艰难梭菌功效研究I期的目标是鉴定两到三种在C.艰难感染这将为旨在临床前开发IND的II期申请奠定坚实的基础。

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.