Selective agents against C. difficile infection

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，艰难梭菌是主要的 抗生素引起的腹泻和结肠炎的负责人。这种病原体可在健康人群中携带。 个体，并被肠道共生体隔绝。抗生素治疗会抑制正常的菌群，但孢子 艰难梭菌存活、萌发和致病。甲硝唑、万古霉素或非达霉素治疗 不仅能杀死病原体，还能杀死可能导致复发的共生体。理想情况下，人们希望有一个 一种对艰难梭菌有选择性的治疗方法，因为它可以使正常的菌群在治疗过程中恢复 治疗病原体，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，可能还有 多达100-200个特定于给定物种/属的必需基因。我们认为一个大型星系的存在 大量的特定靶点提供了发现选择性作用于C。 艰难抉择。病原体是厌氧菌，而高温超导通常需要好氧条件。因此，当前 治疗感染的治疗方法来自其他项目，并且不是有选择性的。我们开发了一种 并对艰难梭菌进行了首次直接筛查。鉴于历史上的高成功率 在从天然产品中发现抗生素的过程中，我们筛选了土壤细菌提取物的文库。这个图书馆 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。图书馆一直是 对金黄色葡萄球菌和大肠杆菌进行筛选，命中率分别为30%和0.5%。我们推论说 剩下的“非活性”菌株可能含有对特定物种有活性的化合物，而这些化合物在 主屏幕。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%。 对这些提取物的初步分析表明，其中两种提取物对艰难梭菌具有选择性。 肠道共生物的小面板，包含具有新奇质量的化合物。在拟议的项目中，我们将 跟进这些点击，并筛选出50,000个额外的摘录从“不活跃的”图书馆。点击率将会是 由LC/MS分离，这将表明新颖性的程度，并与 肠道共生菌的主要类群。通过去复制的梭状芽孢杆菌选择性化合物将进行测试 效力、耐药频率、细胞毒性和对肠上皮细胞的渗透。这些化合物是 未被吸收并因此保留在感染现场的将被优先考虑。结构确定将确认 新颖性，并提供关于化合物是否适合进一步开发的信息。全基因组 对抗药性突变体的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 将在仓鼠身上确定铅含量，这将为艰难梭菌疗效研究的设计提供依据。的目标是 第一阶段是在艰难梭菌感染的仓鼠模型中鉴定两到三种有效的先导化合物。这 将为旨在临床前开发IND的第二阶段应用奠定坚实的基础。

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 fidaxomycin 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 dereplicated 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 provide 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 preclinical development towards an IND.