Therapeutics targeting fatty acid synthesis in P. aeruginosa

描述（由申请人提供）：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，以及机械通气或烧伤患者。频繁的抗生素耐药性和铜绿假单胞菌的高毒性使其比大多数其他细菌物种更致命。作用于新靶点的新型化学抗生素对于持续有效治疗铜绿假单胞菌至关重要，因为此类药物不会受到现有耐药机制的影响。最近在几个物种中鉴定的脂肪酸生物合成（FAB）抑制剂靶点FabI（烯酰-ACP还原酶）、FabF（缩合酶）和AccD（乙酰辅酶A羧化酶）已验证了这种大分子合成途径是发现新抗菌剂的未充分开发的潜在有用途径。然而，这些抑制剂不能阻断铜绿假单胞菌的生长或生存力，这主要是由于两个原因-（a）细胞渗透性和流出障碍和（B）第二种烯酰-ACP还原酶FabK的存在，该还原酶与FabI非同源，但复制了FabI的功能。该项目的策略是通过使用细胞报告筛选来检测FAB途径中任何几个步骤的抑制剂，开发对铜绿假单胞菌FAB有活性的新抗菌药物。这些将基于发光杆菌luxCDABE荧光素酶操纵子与启动子的融合，所述启动子响应于三种不同的必需FAB靶标AccD、FabF和循环中的第二还原酶FabG的耗尽而上调，FabG在结构上与FabI相关，但在铜绿假单胞菌中是必需的。这样的筛选将检测能够进入铜绿假单胞菌细胞并且能够充分逃逸外排以产生报告的抑制剂。将用FAB途径特异性细胞报告菌株筛选合成和天然产物化合物的多样化集合。将通过与报告菌株的重复试验、浓度依赖性活性的证明以及由整个重建脂肪酸生物合成循环组成的体外生化试验的抑制来确认命中。每种抑制剂的精确靶标将通过测定在FAB循环的受抑制生物化学测定中积累的FAB前体来鉴定，并且将在化合物存在下在推定靶标的直接测定中完成确认。通过证明MIC、对哺乳动物细胞活力的最小抑制、对细胞膜的最小破坏以及通过确定抗菌活性谱，对经验证的命中进行优先排序。将在第II阶段优化经优先验证的命中，以生成用于药物开发的先导化合物。公共卫生相关性：这项研究旨在发现新的抗生素有效地对抗一种常见的危险的临床细菌病原体，铜绿假单胞菌。细胞发光测定将用于鉴定必需脂肪酸生物合成途径的抑制剂。将通过在整个脂肪酸合成途径的生化测定中测试抑制剂来验证其对该途径的作用。

DESCRIPTION (provided by applicant): 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. Frequent antibiotic resistance and the highly virulent nature of P. aeruginosa make it deadlier than most other bacterial species. New chemical classes of antibiotics acting on novel targets are crucial for continued effective therapy against P. aeruginosa because such drugs will not be subject to existing resistance mechanisms. Recent identification of inhibitors of fatty acid biosynthesis (FAB) targets FabI (enoyl-ACP reductase), FabF (condensing enzyme), and AccD (acetyl-CoA carboxylase) in several species has validated this macromolecular synthesis pathway as an under-exploited potentially useful route for discovery of new antibacterials. However, these inhibitors fail to block growth or viability of P. aeruginosa for two primary reasons -- (a) cell permeability and efflux obstacles and (b) the existence of a second enoyl-ACP reductase FabK, which is non-homologous to, but duplicates the function of FabI. The strategy of this project is to develop new antibacterials, which are active against P. aeruginosa FAB, by using cellular reporter screens to detect inhibitors of any of several steps in the FAB pathway. These will be based on fusion of the Photorhabdus luminescens luxCDABE luciferase operon to promoters which are up-regulated in response to depletion of three different essential FAB targets -- AccD, FabF, and the second reductase in the cycle, FabG, which is structurally related to FabI but is essential in P. aeruginosa. Such screens will detect inhibitors capable of entering P. aeruginosa cells and capable of escaping efflux sufficiently to generate a report. A diverse collection of synthetic and natural product compounds will be screened with the FAB pathway-specific cellular reporter strain. Hits will be confirmed by repeat assay vs. the reporter strain, demonstration of concentration dependent activity, and inhibition of an in vitro biochemical assay consisting of the entire re-constituted fatty acid biosynthesis cycle. The precise target of each inhibitor will be identified by determination of FAB precursors that accumulate in inhibited biochemical assays of the FAB cycle, and confirmation will be accomplished in direct assays of the putative target in the presence of the compound. Validated hits will be prioritized by demonstrating MICs, minimal inhibition of mammalian cell viability, minimal disruption of cell membranes, and by determining the spectrum of anti-bacterial activity. Prioritized validated hits will be optimized in Phase II to generate lead compounds for drug development. PUBLIC HEALTH RELEVANCE: This research is aimed at discovering new antibiotics effective against a common dangerous clinical bacterial pathogen, Pseudomonas aeruginosa. Cellular luminescent assays will be used to identify inhibitors of the essential fatty acid biosynthesis pathway. Inhibitors will be verified to act on this pathway by testing them in a biochemical assay of the entire fatty acid synthesis pathway.