Selective agents against C. difficile infection

针对艰难梭菌感染的选择性药物

• 批准号: 8713336
• 负责人: Dallas Hughes
• 金额: $ 29.97万
• 依托单位: NOVOBIOTIC PHARMACEUTICALS, LLC
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8713336
• 关键词: Actinobacteria class; Address; Aerobic; Anaerobic Bacteria; Antibiotic Therapy; Antibiotics; Bacteria; Bacterial Genome; Biological Assay; Biological Availability; Biological Factors; Cells; Clostridium; Clostridium difficile; Colitis; Collection; Communities; Development; Diarrhea; Disease; Epidemic; Epithelial Cells; Escherichia coli; Essential Genes; Evaluation; Fractionation; Frequencies; Genes; Genome; Genomics; Goals; Growth; Hamsters; Healthcare; Individual; Infection; Intestines; Lead; Libraries; Maximum Tolerated Dose; Metronidazole; Modeling; Oral; Penetration; Pharmaceutical Preparations; Phase; Production; Relapse; Reproduction spores; Resistance; Resources; Site; Soil; Solid; Staphylococcus aureus; Structure; Testing; Therapeutic; Vancomycin; antimicrobial; base; cytotoxicity; design; drug candidate; experience; follow-up; genome sequencing; in vitro testing; in vivo; killings; liquid chromatography mass spectrometry; microbial; mutant; novel; pathogen; potency testing; prevent; programs; public health relevance; scale up; screening; small molecule; success

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.

该项目的总体目标是开发针对艰难梭菌的选择性抗生素，这是主要的 负责抗生素诱导的腹泻和结肠炎的代理。病原体可以在健康中携带 个人，并由肠道共生体避开。抗生素治疗抑制了正常的菌群，但孢子 艰难梭菌生存，发芽并引起疾病。用甲硝唑，万古霉素或虚拟霉素治疗 不仅杀死病原体，还杀死可能导致复发的共生体。理想情况下，一个人想拥有 一种对艰难梭菌有选择性的治疗性，因为它可以使正常的菌群在此过程中恢复 治疗病原体，防止复发。 基于基因组研究，细菌具有约200个基因的核心，除此之外，可能还有 最多针对给定物种/属的100-200个必需基因。我们认为很大的存在 特定目标的数量为发现有选择性地针对C的抗菌剂提供了机会。 艰难的。病原体是厌氧菌，而HTS通常需要有氧条件。结果，当前 治疗感染的治疗剂来自其他程序，并且不是选择性的。我们开发了一个 厌氧HTS并对艰难梭菌进行了第一个直接筛选。鉴于历史上很高的成功率 通过从天然产物中发现抗生素，我们从土壤细菌中筛选了一个提取物库。这个库 来自未开发的资源，未培养的细菌，并具有丰富的新颖化合物。图书馆曾经是 针对金黄色葡萄球菌和大肠杆菌进行筛选，分别产生30％和0.5％的命中率。我们认为这一点 其余的“无活性”菌株可能会在特定物种上具有活跃的化合物。 主要屏幕。约5,000个提取物的试点屏幕对艰难梭菌的命中率为1.3％。 对这些提取物的初步分析表明，对A进行测试时，两种是针对艰难梭菌的选择性 肠道的小面板，并包含具有新颖质量的化合物。在拟议的项目中，我们将 跟进这些命中，并从“非活动”库中屏幕50,000个提取物。命中将是 由LC/MS解换，这将指示新颖的程度，并对代表的代表进行了测试 肠道共生体的主要群体。梭状芽胞杆菌选择性化合物通过消除的化合物将进行测试 效力，电阻频率，细胞毒性和渗透到肠上皮细胞中。化合物是 未吸收并因此在感染部位保留，将获得优先级。结构确定将确认 新颖性并提供有关化合物进一步开发的适用性的信息。全基因组 抗性突变体的测序将指示可能的目标。最大耐受剂量和生物利用度 铅将在仓鼠中确定，这将为艰难梭菌疗效研究的设计提供信息。目标 第一阶段将在艰难梭菌感染的仓鼠模型中识别两到三种具有功效的铅化合物。这 将为旨在朝着IND的临床前开发的II期应用构成坚实的基础。