Inhibitors of isoprenoid synthesis for antibacterial therapy

描述：铜绿假单胞菌是免疫受损/抑制患者严重感染的常见和极致命的原因（例如，艾滋病毒和癌症），囊性纤维化患者，以及机械通气或烧伤患者。相关的临床上重要的革兰氏阴性[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的菌种，这些菌种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。在Gr（-）非发酵菌中，多重耐药性正在增加，作为最后的手段，多粘菌素（如粘菌素）已被重新用于治疗这些感染。发现不受现有基于靶标的抗性机制影响的新化学实体是解决这种未满足需求的重要策略，并且筛选新的或未充分开发的靶标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靶向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。类异戊二烯是细菌电子传递和细胞壁生物合成所必需的。许多细菌物种，包括Gr（-）非发酵菌，利用替代的类异戊二烯合成途径，2-C-甲基-D-β 4-磷酸（MEP）途径，其与在人类中发现的甲羟戊酸（MVA）途径非常不同，并且提供了对抑制剂的高选择性可能性。虽然已经报道了MEP抑制剂的筛选，但这仍然是一种未充分开发的途径，部分原因是用于研究的底物的可用性非常有限，并且因为大多数先前的策略依赖于生物化学酶筛选，产生细胞活性差的抑制剂。本研究的新方法是用敏感的细胞生物发光报告基因检测筛选铜绿假单胞菌MEP途径抑制剂，并对结果进行分析。 通过生物化学测定来验证化合物作为特异性MEP抑制剂并鉴定精确的靶点。这是可行的，因为该团队在构建和利用这些类型的筛选方面的经验，以及该团队对途径中七个酶促反应中每一个的合成底物的独特访问。在第一阶段，我们将开发并应用铜绿假单胞菌MEP途径细胞报告筛选到> 300，000个离散小分子的多样化文库。将在筛选试验中确认命中，并通过携带MEP与MEP+MVA途径酶的菌株的差异生长抑制来评价MEP途径的特异性。在生物化学试验中，将利用细菌胞质溶胶和放射性标记底物对每个酶促步骤的经路径验证的非细胞毒性命中物鉴定特异性靶向MEP反应。将在生化和MIC试验中评价抑制剂的效价、抑制模式和谱，并根据其抗菌谱进行优先排序。在第二阶段，我们将研究最高优先级的类似物，并对这些结构中最有前途的结构进行化学优化，以开发用于动物模型中的功效和毒性测试的先导化合物。

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.