NOVEL METHODS FOR DISCOVERY OF ANTI-MICROBIALS

描述(由申请者提供)：我们的长期目标是发现广谱抗生素对潜在的生物药剂起作用。这一探索性项目的目标是开发一种全面的方法来绕过现有的药物发现障碍，这将包括将活性化合物输送到病原体中，识别新的抗菌剂类别，以及有效地评估毒性/疗效。 刘易斯实验室之前的研究表明，植物合成多药耐药外排泵的抑制剂，可以促进抗菌剂向微生物病原体的输送。奥苏贝尔实验室独立开发了一种致病模型，涉及到人类微生物病原体杀死线虫秀丽线虫。因此，线虫可以作为初步筛选抗微生物药物的动物模型。该提案描述了旨在合并这两个实验室开发的互补技术的实验，以产生一种新的抗微生物药物发现方法。具体目标是： 1.利用感染不同NIAID A和B组制剂的线虫，开发高通量全动物筛选新的抗菌剂。这些将包括致泻性大肠杆菌、肠杆菌和一种革兰氏阳性模式病原菌粪肠球菌。我们还将确定线虫是否感染炭疽杆菌和图拉氏杆菌。抗菌试验的基本原理是通过测试化合物来监测感染人类病原体的蠕虫的治愈情况。使用绿色荧光蛋白标记的线虫的液体分析将发展成为高通量的自动化分析。 2.将在体外和体内对感染的线虫进行多药耐药抑制剂和新的抗菌药的筛选。一个商业合成化合物文库，以及NCI的萃取物集合将被筛选。体外和体内筛选的比较将确定只在体内发挥作用的可能化合物(前药、针对毒力的化合物或体内生存所必需的其他成分，以及先天免疫的刺激物)。初步结果表明，NCI文库对所有测试的病原体都具有直接和多药耐药抑制活性。我们将专注于获得针对革兰氏阴性病原体的新型抗菌药和多药耐药抑制剂。这种多药耐药抑制剂与抗菌化合物的组合将产生广谱抗生素。在这项初步研究中，我们计划筛选30,000种化合物/提取物。 3.进行抗菌化合物的提纯和鉴定。活性提取物将被用来分离纯净的物质。一种足够纯净的化合物将被用来与病原体小组一起测定最低抑菌浓度，而那些高效力的化合物将被进一步研究。化学结构的确定将通过MS和核磁共振方法的组合进行。

DESCRIPTION (provided by applicant): Our long-term goal is to discover broad-spectrum antibiotics acting against potential biowarfare agents. The goal of this exploratory project is to develop a comprehensive approach to bypass the existing obstacles to drug discovery, which will include delivering active compounds into the pathogen, identifying new classes of antimicrobials, and efficient evaluation of toxicity/efficacy. Previous research in the Lewis laboratory showed that plants synthesize inhibitors of multi-drug resistant efflux pumps that can facilitate delivery of antimicrobials into microbial pathogens. Independently, the Ausubel laboratory developed a pathogenesis model that involves the killing of the nematode worm Caenorhabditis elegans by human microbial pathogens. The nematode can therefore be used as an animal model for primary screening of antimicrobials. This proposal describes experiments designed to merge the complementary technologies developed in these two laboratories to produce a novel approach to antimicrobial drug discovery. The Specific Aims are: 1. High throughput whole-animal screen for novel antimicrobials will be developed using C. elegans infected with a variety of NIAID group A and B agents. These will include diarrheagenic E. coli, S. enterica, and a model gram-positive pathogen E. faecalis. We will also establish whether C. elegans is infected with B. anthracis and F. tularensis. The rationale of the antimicrobial assay is to monitor curing of worms infected with human pathogens, by test compounds. A liquid assay using GFP-labeled C. elegans will be developed into a high-throughput automated assay. 2. Screening for MDR inhibitors and new antimicrobials will be performed in vitro, and in vivo with infected C. elegans. A commercial synthetic compound library, and the NCI collection of extracts will be screened. Comparison of the in vitro and in vivo screens will identify possible compounds that only act in vivo (prodrugs, compounds targeting virulence or other components necessary for in vivo survival, and stimulators of innate immunity). Preliminary results show that the NCI library has hits for both direct and MDR inhibitory activity against all pathogens tested. We will focus on obtaining novel antimicrobials and MDR inhibitors acting against gram-negative pathogens. A combination of such an MDR inhibitor with an antimicrobial compound will produce a broad-spectrum antibiotic. We plan to screen 30,000 compounds/extracts in this pilot study. 3. We will purify and identify antimicrobial compounds. Active extracts will be used to isolate a pure substance. A sufficiently pure compound will be used to determine MIC with the pathogen panel, and those with high potency will be studied further. Determination of chemical structure will be performed by a combination of MS and NMR methods.