Inhibitors of isoprenoid synthesis for antibacterial therapy

描述：铜绿假单胞菌是免疫功能低下/抑制患者（如HIV和癌症）、囊性纤维化患者、机械通气患者或烧伤创面患者严重感染的常见且毒性极大的原因。相关临床重要的革兰氏阴性[Gr(-)]非发酵菌包括伯克霍尔德菌属、不动杆菌属和窄养单胞菌属，它们普遍存在于严重的医院感染中，如肺炎、菌血症和尿路感染。Gr（-）非发酵菌的多药耐药性正在增加，作为最后的手段，多粘菌素（如粘菌素）已被重新用于对抗这些感染。发现不受现有基于靶标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，筛选新的或未开发的靶标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治疗Gr（-）非发酵菌感染的新型药物。类异戊二烯是细菌中电子传递和细胞壁生物合成所必需的。许多细菌，包括Gr（-）非发酵菌，利用另一种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，这与人类的甲羟戊酸（MVA）途径截然不同，并为抑制剂提供了高选择性的可能性。虽然MEP抑制剂的筛选已经有报道，但这仍然是一个未被充分利用的途径，部分原因是研究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，产生的抑制剂细胞活性差。本研究的新方法是用敏感的细胞生物发光报告试验筛选铜绿假单胞菌MEP途径抑制剂，并对结果进行分析

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.