Inhibitors of isoprenoid synthesis for antibacterial therapy

用于抗菌治疗的类异戊二烯合成抑制剂

• 批准号: 8602834
• 负责人: Donald T Moir
• 金额: $ 30万
• 依托单位: MICROBIOTIX, INC
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-07 至 2015-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8602834
• 关键词: Acinetobacter; Address; Anabolism; Animal Model; Anti-Bacterial Agents; Antibiotics; Bacteremia; Bacteria; Biochemical; Biochemical Reaction; Biological Assay; Biological Factors; Burkholderia; Burkholderia pseudomallei; Burn injury; Cell Extracts; Cell Survival; Cell Wall; Cell physiology; Cells; Cellular Assay; Cephalosporinase; Chemicals; Colistin; Collection; Cytoplasm; Cytosol; Drug Efflux; Drug Targeting; Drug resistance; Electron Transport; Enzymes; Exhibits; Fatty Acids; Goals; Growth; HIV; Human; Immune; In Vitro; Infection; Inhibitory Concentration 50; Lead; Libraries; Malignant Neoplasms; Mammalian Cell; Mechanical ventilation; Medical; Membrane; Multi-Drug Resistance; Nosocomial Infections; Operon; Pathway interactions; Patients; Pharmaceutical Preparations; Phase; Pneumonia; Polymyxins; Proteins; Pseudomonas aeruginosa; Qualifying; Radiolabeled; Reaction; Reporter; Reporting; Research; Resort; Role; Specificity; Stenotrophomonas; Structure; Toxic effect; Toxicity Tests; Urinary tract infection; Virulent; Xenorhabdus luminescens; analog; base; combat; cystic fibrosis patients; cytotoxicity; drug development; drug resistant bacteria; efficacy testing; efflux pump; enzyme pathway; experience; improved; inhibitor/antagonist; innovation; inorganic phosphate; isoprenoid; mevalonate; novel; novel strategies; pathogen; promoter; public health relevance; radiotracer; resistance mechanism; screening; small molecule

DESCRIPTION: Pseudomonas aeruginosa is a common and extremely virulent cause of serious infections in immune- compromised/suppressed patients (e.g., HIV and cancer), cystic fibrosis patients, and those on mechanical ventilation or with burn wounds. Related clinically important Gram-negative [Gr(-)] nonfermenters include species of the genera Burkholderia, Acinetobacter, and Stenotrophomonas, which are prevalent in severe nosocomial infections, such as pneumonia, bacteremia and urinary tract infections. Multidrug resistance is increasing among Gr(-) nonfermenters, and as a last resort, polymyxins such as colistin have been revived for use against these infections. The discovery of new chemical entities that are not subject to existing target- based resistance mechanisms is an important strategy to address this unmet need, and screening for inhibitors of new or under-exploited targets is a useful approach. The overall goal of this research is discover a novel class of drugs that target the bacterial isoprenoid biosynthetic pathway for therapy of Gr(-) nonfermenter infections. Isoprenoids are essential for electron transport and cell wall biosynthesis in bacteria. Many bacterial species, including the Gr(-) nonfermenters, utilize an alternate isoprenoid synthesis pathway, the 2-C- methyl-D-erythritol 4-phosphate (MEP) pathway, which is quite distinct from that found in humans, the mevalonate (MVA) pathway, and provides a high likelihood of selectivity for inhibitors. While screens for MEP inhibitors have been reported, this remains an underexploited pathway, in part because availability of substrates for research is quite limited and because most previous strategies have relied on biochemical enzymatic screens, yielding inhibitors with poor cellular activity. The novel approach of this study is to screen for P. aeruginosa MEP pathway inhibitors with a sensitive cellular bioluminescent reporter assay and to profile resulting hits with biochemical assays to validate compounds as specific MEP inhibitors and to identify the precise targets. This is feasible because of the experience of the team with building and utilizing these types of screens and because of the unique access of the team to synthesized substrates of each of the seven enzymatic reactions in the pathway. In Phase I, we will develop and apply a P. aeruginosa MEP pathway cellular reporter screen to a diverse library of >300,000 discrete small molecules. Hits will be confirmed in the screening assay and evaluated for specificity for the MEP pathway by differential growth inhibition of strains carrying MEP vs. MEP+MVA pathway enzymes. The specific targeted MEP reaction will be identified for pathway-validated non-cytotoxic hits in biochemical assays utilizing bacterial cytosol and radiolabeled substrates for each enzymatic step. Inhibitors will be evaluated for potency, mode of inhibition and spectrum in biochemical and MIC assays and prioritized by their antibacterial spectrum. In Phase II, we will examine analogs of the highest priority hits and chemically optimize the most promising of these structures to develop lead compounds for efficacy and toxicity testing in animal models.

描述：铜绿假单胞菌是免疫受损/抑制的患者（例如HIV和癌症），囊性纤维化患者以及机械通气或机械通气或烧伤伤口的常见且极具危险的原因。相关临床上重要的革兰氏阴性[GR（ - ）]非侵蚀者包括Burkholderia属的物种，动杆菌和稳定性菌群，这些物种在严重的医生感染中普遍存在，例如肺炎，细菌和尿道感染。在GR（ - ）非发酵剂中，多药的耐药性正在增加，作为最后的度假胜地，诸如colistin之类的多粘蛋白已恢复为用于这些感染。发现不受现有基于目标的抗药性机制的新化学实体是解决这种未满足需求的重要策略，并且对新的或未经探索的目标的抑制剂进行筛查是一种有用的方法。这项研究的总体目的是发现一类新的药物，该药物靶向细菌类生物合成途径，用于治疗GR（ - ）非发酵剂感染。类种子对于细菌中的电子传输和细胞壁生物合成至关重要。许多细菌种类，包括GR（ - ）非摄入剂，使用替代的类异胞素合成途径，即2-C-甲基-D-雄性4-磷酸（MEP）途径，这与人类在人类，甲龙（MVA）途径中发现的途径截然不同，并提供了较高的选择性的可构症。尽管已经报道了MEP抑制剂的筛查，但这仍然是一种不流失的途径，部分原因是研究底物的可用性非常有限，并且因为大多数以前的策略都依赖生化酶筛选，因此产生了细胞活性较差的抑制剂。这项研究的新方法是用敏感的细胞生物发光记者测定法筛选铜绿假单胞菌MEP途径抑制剂，并导致轮廓 使用生化测定的命中，以验证化合物作为特定的MEP抑制剂并确定精确靶标。这是可行的，因为团队在构建和利用这些类型的屏幕方面的经验以及团队独特的访问来综合途径中七个酶促反应中的每一个。在第一阶段，我们将开发并将铜绿假单胞菌MEP途径蜂窝记者屏幕应用于> 300,000个离散小分子的多元化库。在筛选测定中将确认命中，并通过对携带MEP+MEP+MVA途径酶的菌株的差异生长抑制来评估MEP途径的特异性。对于使用细菌胞质溶胶和放射性标记的底物，针对每个酶促步骤的途径验证的非环毒性打击，将确定特异性的MEP反应。将评估抑制剂的生化和MIC分析中的效力，抑制模式和光谱，并通过其抗菌光谱进行优先次序。在第二阶段，我们将检查最高优先级命中的类似物，并化学优化这些结构的最有希望的类似物，以开发铅化合物，以实现动物模型中的疗效和毒性测试。