Therapeutics targeting fatty acid synthesis in P. aeruginosa

描述(申请人提供)：铜绿假单胞菌是一种常见且极其致命的原因，可导致免疫功能低下/抑制的患者(如艾滋病毒和癌症)、囊性纤维化患者、机械通气者或有烧伤创面的患者发生严重感染。频繁的抗生素耐药性和铜绿假单胞菌的高度毒力使其比大多数其他细菌物种更致命。作用于新靶点的新的抗生素化学类别对于持续有效地治疗铜绿假单胞菌至关重要，因为此类药物将不受现有耐药机制的影响。最近在几个物种中发现了针对脂肪酸生物合成(FAB)的靶标Fabi(环氧乙酰-ACP还原酶)、FabF(缩合酶)和ACCD(乙酰辅酶A羧基酶)的化合物，证实了这种大分子合成途径是发现新的抗菌药物的一条未被充分开发的潜在有用途径。然而，这些抑制剂不能阻止铜绿假单胞菌的生长或活力，主要有两个原因--(A)细胞通透性和外排障碍，(B)第二个Enoyl-ACP还原酶FabK的存在，它与Fabi不同源，但功能重复。该项目的战略是开发新的抗菌药，通过使用细胞报告屏幕来检测Fab途径中几个步骤中的任何一个步骤的抑制剂，从而对铜绿假单胞菌Fab具有活性。这将基于Photorhabdus Lighescens LuxCDABE荧光素酶操纵子与启动子的融合，启动子在三个不同的基本Fab靶标-ACCD、FabF和循环中的第二个还原酶FabG的耗尽时上调，FabG在结构上与Fabi相关，但在铜绿假单胞菌中是必不可少的。这样的筛查将检测能够进入铜绿假单胞菌细胞并能够充分逃脱外排的抑制剂，从而生成报告。将用FAB途径特异性细胞报告菌株筛选不同的合成和天然产物化合物。HITS将通过与报告菌株的重复检测、浓度依赖活性的证明以及由整个重组脂肪酸生物合成循环组成的体外生化检测的抑制来确认。每种抑制剂的精确靶标将通过测定FAB循环中被抑制的生化分析中积累的FAB前体来确定，并将在化合物存在的情况下直接对假定的靶标进行确认。验证的HITS将通过展示MICS、对哺乳动物细胞活性的最小抑制、对细胞膜的最小破坏以及通过确定抗菌活性的光谱来优先考虑。优先验证的HITS将在第二阶段进行优化，以产生用于药物开发的先导化合物。公共卫生相关性：这项研究旨在发现对一种常见的危险临床细菌病原体铜绿假单胞菌有效的新抗生素。细胞发光分析将被用来识别必需脂肪酸生物合成途径的抑制剂。通过对整个脂肪酸合成途径进行生化测试，将证实抑制剂对这一途径起作用。

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.