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: 8522430
• 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 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8522430
• 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/抑制患者（例如艾滋病毒和癌症）、囊性纤维化患者以及机械通气患者或烧伤患者严重感染的常见且毒性极强的原因。临床上重要的相关革兰氏阴性[Gr(-)]非发酵菌包括伯克霍尔德氏菌属、不动杆菌属和寡养单胞菌属，它们在严重的医院感染中普遍存在，例如肺炎、菌血症和尿路感染。 Gr(-)非发酵菌的多重耐药性正在增加，作为最后的手段，粘菌素等多粘菌素已重新用于对抗这些感染。发现不受现有基于靶点的耐药机制影响的新化学实体是解决这一未满足需求的重要策略，而筛选新的或未充分利用的靶点的抑制剂是一种有用的方法。本研究的总体目标是发现一类针对细菌类异戊二烯生物合成途径的新型药物，用于治疗 Gr(-) 非发酵菌感染。类异戊二烯对于细菌中的电子传输和细胞壁生物合成至关重要。许多细菌物种，包括 Gr(-) 非发酵菌，利用替代类异戊二烯合成途径，即 2-C-甲基-D-赤藓糖醇 4-磷酸 (MEP) 途径，该途径与人类中发现的甲羟戊酸 (MEP) 途径截然不同。 MVA) 途径，并为抑制剂提供了很高的选择性。虽然已经报道了 MEP 抑制剂的筛选，但这仍然是一个未充分利用的途径，部分原因是用于研究的底物的可用性相当有限，而且因为大多数先前的策略依赖于生化酶筛选，产生细胞活性较差的抑制剂。本研究的新方法是通过敏感的细胞生物发光报告分析来筛选铜绿假单胞菌 MEP 途径抑制剂，并分析所得结果 通过生化测定来验证化合物作为特定的 MEP 抑制剂并确定精确的靶标。这是可行的，因为该团队在构建和利用这些类型的屏幕方面拥有丰富的经验，并且因为该团队对途径中七个酶反应中每一个的合成底物具有独特的获取途径。在第一阶段，我们将开发铜绿假单胞菌 MEP 途径细胞报告基因筛选，并将其应用于超过 300,000 个离散小分子的多样化文库。将在筛选测定中确认命中，并通过携带 MEP 与 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。在每个酶促步骤中利用细菌胞质和放射性标记底物的生化测定中，将确定特定的靶向 MEP 反应，以用于经过途径验证的非细胞毒性命中。将在生化和 MIC 测定中评估抑制剂的效力、抑制模式和谱，并根据其抗菌谱进行优先排序。在第二阶段，我们将检查最优先命中的类似物，并化学优化这些结构中最有前途的结构，以开发用于动物模型功效和毒性测试的先导化合物。