Investigation of the FAS-II enzyme, FabK, as a druggable target for C. difficile

项目摘要/摘要 艰难梭菌的超强毒力和抗药性菌株现在极大地导致了总体发病率。 艰难梭菌感染(CDI)导致的复发率高达35%，估计有29,000人死亡 每年，国家负担超过45亿美元。现在迫切需要的是 用于CDI治疗的新型抗菌靶点的表征。一种新的和潜在的选择性 目标是细菌酶FabK，烯醇酰载体蛋白(ACP)还原酶II，它是 细菌脂肪酸合成途径(Fas II)。抑制Fas II途径中的酶已被证明是 导致很强的抗菌作用，如与Fabi(环氧乙酰-ACP还原酶I)抑制剂三氯生，异烟肼， 以及目前正在进行临床试验的其他Fabi抑制剂。艰难梭菌将FabK表达为其唯一的enoyl还原酶， 在结构和机械上与Fabi完全不同的同工酶。我们假设，这些抑制因子 Cd FabK酶对艰难梭菌表现出选择性活性，同时对正常的 肠道下部的菌群。这一假说是基于现有的文献提出的，该文献证明了 Enoyl-Reductase同工酶在肠道菌群中的物种特异性分布，已知具有选择性活性的抑制剂 针对FabK酶，以及我们自己的初步数据证明了靶标的重要性。在这里，我们的 目的是验证艰难梭菌中的FabK酶作为窄谱CDI的可用药靶点 心理治疗。为了实现这些目标，我们组织了一支技术娴熟的科学家团队，他们在C。 艰难杆菌微生物学、蛋白质生物化学、结构生物学、合成和计算化学，谁将 追求以下具体目标：1.将FabK作为一种狭义的- 艰难梭菌的光谱靶标。我们对这一目标的工作假设是艰难梭菌无法克服 外源脂肪酸通过旁路途径抑制FabK/Fas-II的表达。我们将利用一系列 可诱导FabK高表达和低表达艰难梭菌的体内外研究及Key 肠道细菌来解决这一假说。2.FabK作为药物的结构和化学验证 艰难梭菌的目标。这一目的的工作假设是，FabK酶可以被小分子- 具有抗艰难梭菌活性的分子化合物。我们将通过使用组合的 一种虚拟与实验相结合的已知FabK抑制剂综合优化方法 筛选、开发和优化对CdFabK具有高亲和力和选择性的抑制剂。这些研究 这里提出的建议将促进我们对肠道菌群Fas-II要求的生物学理解，验证 诱人的抗菌药物靶点，为艰难梭菌脂肪酸调控提供了新的线索，并提供了强有力的证据 为进一步开发CdFabK抑制剂作为治疗药物的新策略提供了原则。 抗药性CDI。

PROJECT SUMMARY/ABSTRACT Hypervirulent, drug-resistant strains of Clostridium difficile now greatly contribute to the overall morbidity and mortality of C. difficile infection (CDI), resulting in relapse rates of up to 35%, an estimated 29,000 deaths per year, and a national burden in excess of $4.5 billion annually. There is an urgent need for the characterization of novel antibacterial targets for the treatment of CDI. One novel and potentially selective target is the bacterial enzyme FabK, enoyl-acyl carrier protein (ACP) reductase II, a key enzyme in the bacterial fatty acid synthesis pathway (FAS II). Inhibition of enzymes in the FAS II pathway has been shown to result in a strong antibacterial effect, as with the FabI (enoyl-ACP reductase I) inhibitors triclosan, isoniazid, and other FabI inhibitors currently in clinical trials. C. difficile expresses FabK as its sole enoyl reductase, an isozyme that is structurally and mechanistically distinct from FabI. We hypothesize that inhibitors of the CdFabK enzyme will show selective activity against C. difficile, while causing minimal disruption to the normal flora of the lower bowel. This hypothesis was formulated based upon existing literature demonstrating a species-specific distribution of enoyl-reductase isozymes in gut flora, known inhibitors with selective activity against the FabK enzyme, and our own preliminary data demonstrating target essentiality. Here, our objectives are to validate the FabK enzyme in C. difficile as a druggable target for narrow-spectrum CDI therapy. To achieve these objectives, we have organized a skilled team of scientists with expertise in C. difficile microbiology, protein biochemistry, structural biology, synthetic and computational chemistry, who will pursue the following specific aims: 1. Microbiological and in vivo validation of FabK as a narrow- spectrum target in C. difficile. Our working hypothesis for this aim is that C. difficile cannot overcome FabK/FAS-II inhibition by pathway bypass in the presence of exogenous fatty acids. We will utilize a series of in vitro and in vivo studies using inducible FabK over- and under-expressing C. difficile strains and tests on key gut bacteria to address this hypothesis. 2. Structural and chemical validation of FabK as a druggable target in C. difficile. The working hypothesis for this aim is that the FabK enzyme can be inhibited by small- molecule compounds resulting in anti-difficile activity. We will test this hypothesis by utilizing a combined approach of synthetic optimization of known FabK inhibitors, coupled with synergistic virtual and experimental screening, to develop and optimize inhibitors with both high affinity and selectivity for CdFabK. The studies proposed here will advance our biological understanding of the gut flora FAS-II requirements, validate an attractive antibacterial drug target, shed new light on fatty acid regulation in C. difficile and provide strong proof of principle for further development of CdFabK inhibitors as a new therapeutic strategy for treatment of drug- resistant CDI.