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

研究 FAS-II 酶 FabK 作为艰难梭菌的药物靶点

基本信息

批准号：
9440646
负责人：
Kirk Edward Hevener
金额：
$ 23.22万
依托单位：
UNIVERSITY OF TENNESSEE HEALTH SCI CTR
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-25 至 2019-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9440646
关键词：
Achievement Acyl Carrier Protein Address Adverse effects Affinity Animals Anti-Bacterial Agents Antibiotics Area Bacteria Biochemical Biological Bypass Cause of Death Cells Cessation of life Chemicals Chronic Disease Clinical Clinical Trials Clostridium difficile Coupled Data Development Drug Targeting Drug resistance Enzyme Inhibition Enzymes Evaluation Fatty Acids Funding Mechanisms Goals In Vitro Infection Intestines Investigation Isoenzymes Light Literature Medical Research Membrane Metronidazole Microbiology Morbidity - disease rate Nosocomial Infections Obesity Organism Oxidoreductase Pathway interactions Pharmaceutical Preparations Protein Biochemistry Public Health Recombinants Recurrence Regulation Relapse Reproduction spores Research Risk Factors Scientist Series Synthesis Chemistry Testing Triclosan Validation Vancomycin base computational chemistry enoyl reductase essential phospholipids evidence base expectation gastrointestinal infection gut microbiota in vivo inhibitor/antagonist insight isoniazid microbiota mortality neonate new therapeutic target novel novel therapeutic intervention novel therapeutics prevent resistant strain screening small molecule structural biology treatment strategy virtual

项目摘要

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.

项目总结/摘要高毒力、耐药的艰难梭菌菌株现在大大有助于整体发病率和死亡率。艰难梭菌感染（CDI），导致复发率高达35%，估计有29，000人死亡每年，国家负担超过45亿美元。迫切需要用于治疗CDI的新型抗菌靶标的表征。一种新颖的、潜在的选择性的目标是细菌酶FabK，烯酰-酰基载体蛋白（ACP）还原酶II，其是在细菌脂肪酸合成途径（FAS II）。FAS II途径中的酶的抑制已被证明导致强的抗菌作用，如FabI（烯酰-ACP还原酶I）抑制剂三氯生，异烟肼，和其他FabI抑制剂目前在临床试验中。C.艰难梭表达FabK作为其唯一的烯酰还原酶，在结构和机制上与FabI不同同工酶。我们假设， CdFabK酶对C.困难，同时对正常的下肠道的植物群。这一假设是根据现有文献提出的，肠道植物群中烯酰还原酶同工酶种特异性分布，已知具有选择活性的抑制剂和我们自己的初步数据证明了目标的重要性。这里我们的目的是在C.作为窄谱CDI的药物靶点疗法为了实现这些目标，我们组织了一个熟练的科学家团队，他们在C。艰难微生物学，蛋白质生物化学，结构生物学，合成和计算化学，谁将具体目标如下：1. FabK作为一种窄- C.光谱目标很难我们的工作假设是C。艰难不能克服在外源性脂肪酸存在下通过旁路途径抑制FabK/FAS-II。我们将利用一系列在体外和体内研究中使用诱导型FabK过表达和低表达C.艰难菌株和关键测试肠道细菌来解决这个假设。2. FabK作为药物制剂的结构和化学验证目标在C。很难这一目标的工作假设是FabK酶可以被小分子抑制。产生抗艰难梭菌活性的分子化合物。我们将通过使用一个组合来测试这个假设。已知FabK抑制剂的合成优化方法，结合协同虚拟和实验筛选，以开发和优化对CdFabK具有高亲和力和选择性的抑制剂。研究本文提出的建议将促进我们对肠道植物群FAS-II要求的生物学理解，验证有吸引力的抗菌药物靶点，揭示了C.难辩有力证明进一步开发CdFabK抑制剂作为药物治疗的新治疗策略的原则- 抗CDI。