STRUCTURE-BASED TUBERCULOSIS DRUG DESIGN TARGETED AT ACYL-COA CARBOXYLASE

针对酰基辅酶A羧化酶的基于结构的结核病药物设计

• 批准号: 7353357
• 负责人: Shiou-Chuan Tsai
• 金额: $ 33.37万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-17 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7353357
• 关键词: Acetyl Coenzyme A; Acids; Acquired Immunodeficiency Syndrome; Actinobacteria class; Actinomyces; Active Sites; Acyl Coenzyme A; Affinity; Anabolism; Animals; Antimycobacterial Agents; Antitubercular Agents; Bacteria; Binding; Biochemical Reaction; Biochemistry; Biological; Biological Assay; Biological Factors; Biological Process; Bioterrorism; Biotin; California; Categories; Cell Wall; Cessation of life; Cities; Commit; Communicable Diseases; Complex; Computer Assisted; Computer Simulation; Confidential Information; Data; Development; Docking; Drug Delivery Systems; Drug Design; Emerging Communicable Diseases; Environment; Enzyme Inhibition; Enzymes; Fatty Acids; Five-Year Plans; Funding; Future; Genes; Genetic; Genome; Goals; Health; Housing; In Vitro; Inhibitory Concentration 50; Inorganic Sulfates; Instruction; Investigation; Kinetics; Language; Lead; Libraries; Lipids; Methods; Mission; Molecular; Multi-Drug Resistance; Mutate; Mutation; Mycobacterium tuberculosis; Mycolic Acid; Occupations; Organism; Outcome; Pharmaceutical Preparations; Plants; Play; Positioning Attribute; Principal Investigator; Proteins; Public Health; Regulation; Research; Research Design; Research Personnel; Resources; Role; Screening procedure; Sequence Analysis; Side; Site-Directed Mutagenesis; Specificity; Structure; Structure-Activity Relationship; Substrate Specificity; Techniques; Therapeutic; Time; TimeLine; Training; Tuberculosis; Universities; Unspecified or Sulfate Ion Sulfates; Validation; Virulence; Work; X-Ray Crystallography; abstracting; analog; andrimid; antimicrobial; assay development; base; biodefense; cell envelope; chemotherapy; design; enzyme mechanism; fatty acid biosynthesis; graduate student; improved; in vitro Assay; in vivo; inhibitor/antagonist; isoniazid; lipid biosynthesis; mutant; novel; pathogen; performance site; programs; propionyl-coenzyme A; resistant strain; therapeutic target; tuberculosis drugs; tuberculosis treatment

Principal Investigator/Program Director (Last, First, Middle): Tsai, Shiou-Chuan DESCRIPTION: See instructions. State the application's broad, long-term objectives and specific aims, making reference to the health relatedness of the project (i.e., relevance to the mission of the agency). Describe concisely the research design and methods for achieving these goals. Describe the rationale and techniques you will use to pursue these goals. In addition, in two or three sentences, describe in plain, lay language the relevance of this research to public health. If the application is funded, this description, as is, will become public information. Therefore, do not include proprietary/confidential information. DO NOT EXCEED THE SPACE PROVIDED. Mycobacterium tuberculosis, the pathogen of tuberculosis (TB), has a cell envelope with chemically complex lipids that are closely related with its virulence and multi-drug resistance. Acyl-CoA carboxylase (ACCase) provides the building blocks for these complex lipids, and the importance and validity of ACCase as a drug target is well recognized. The M. tuberculosis ACCase include six ACCase subunits (accD1-6), and that AccD4, AccD5 and AccD6 play major roles in providing the building-blocks to cell wall lipid biosyntheses. However, very little is known about the substrate specificity or biological functions of these pathogen ACCases. Our long-term goal is to discover a library of novel anti-TB therapeutics against new M. tuberculosis protein targets. The objective of this particular application is to elucidate the substrate specificities, sequence-structure-function relationship, and biological roles of AccD4, AccD5 and AccD6, using X-ray crystallography, enzyme inhibition assays, and computer-assisted inhibitor design. The rationale is that, once we identified inhibitors of AccD4-6, we will be able to inhibit cell wall lipid biosynthesis, leading to pathogen death. This rationale has been validated by past genetic data, which indicate that mutations of AccD4 and AccD6 lead to pathogen death. In the next two years, we will persue three aims: AIM 1. Determine the molecular basis of substrate specificities in AccD4-6: (1.1) Refine the co-crystal structures of AccD5 bound with propionyl-CoA and biotin analogs. (1.2) Refine the co-crystal structures of AccD6 bound with acetyl-CoA and biotin analogs. (1.3) Solve the crystal structure of apo AccD4, and cocrystal structures of AccD4 bound with long chain acyl-CoA and biotin analogs. AIM 2. Determine the inhibitor-binding specificities of AccD5-6: (2.1). Screen in silico Sulfa, Propeller and andrimid (three identified inhibitors) analogs against AccD5-6 using UC Irvine's ChemDB and cross-validation with two docking softwares. (2.2) Screen in vitro the inhibitors predicted from 2.1 and elucidate the AccD5-6 enzyme mechanisms by inhibition kinetics. (2.3) Refine co-crystal structures of AccD5-6 bound with Sulfa, Propeller or andrimid. AIM 3. Compare the active site geometries and substrate binding pockets of AccD4, AccD5 and AccD6, and define the substrate/inhibitor binding residues by site-directed mutagenesis: (3.1) Systematically mutate AccD5 residue 437 to evaluate its importance for substrate specificity. (3.2) Mutate residues in the Acyl-CoA binding pocket to probe for AccD5 specificities for acyl-CoA and Sulfa analogs. The feasibility of the proposed studies are strongly supported by strong preliminary data, including diffracting crystals of all proposed structural studies (AIM 1 and AIM 2.3), as well as established enzyme assays, identification of more than 50 potent inhibitors in AIM 2, and complete construction of half mutants proposed in AIM 3. The proposed research is scientifically significant because, for the first time, the substrate/inhibitor specificities of these unique M. tuberculosis ACCases will be critically evaluated and dissected. Such findings are original, because no ACCase from any other organisms has such a uniquely diverse, yet precisely controlled substrate specificity. The outcome from this proposal will identify potent ACCase inhibitors. Therefore, the completion of this project will also have health significance on the development of new TB therapeutics. The proposed research will retain and increase job opportunities for two graduate students and two postdoctoral researcher, and the outcome will enable us to provide new building blocks for downstream polyketide biosynthesis in an one-pot, environmentally friendly fashion that completes multi-step total syntheses by turning the bacteria into drug-manufacturing factory. PERFORMANCE SITE(S) (organization, city, state) University of California, Irvine, CA 92697, USA REVISED ABSTRACT SECTION

首席调查员/计划主管（最后，第一，中间）： Tsai，Shiou-Chuan 描述：请参阅说明。陈述该应用程序的广泛，长期目标和具体目标，以参考项目的健康相关性（即与代理机构的任务相关）。简单地描述实现这些目标的研究设计和方法。描述您将用来追求这些目标的理由和技术。此外，用两个或三个句子描述，以简单的语言描述这项研究与公共卫生的相关性。如果申请是资助的，那么此描述将成为公共信息。因此，请勿包含专有/机密信息。不要超过提供的空间。 结核分枝杆菌（结核病的病原体（TB））具有一个细胞包膜，其化学复杂的脂质与其毒力和多药耐药性密切相关。酰基-COA羧化酶（ACACASE）为这些复杂的脂质提供了基础，并且可以很好地认识到ACCase作为药物靶标的重要性和有效性。结核分枝杆菌的ACCASE包括六个ACCASE亚基（ACCD1-6），ACCD4，ACCD5和ACCD6在为细胞壁脂质生物合成的建筑块提供方面起着重要作用。然而，关于这些病原体的底物特异性或生物学功能知之甚少。我们的长期目标是发现针对新的结核分枝杆菌蛋白靶标的新型抗TB疗法库。该特定应用的目的是使用X射线晶体学，酶抑制分析和计算机辅助抑制剂设计，阐明ACCD4，ACCD5和ACCD6的底物特异性，序列结构功能以及ACCD4，ACCD5和ACCD6的生物学作用。理由是，一旦我们确定了ACCD4-6的抑制剂，我们将能够抑制细胞壁脂质生物合成，从而导致病原体死亡。该基本原理已通过过去的遗传数据来验证，这表明ACCD4和ACCD6的突变导致病原体死亡。在接下来的两年中，我们将说明三个目标：目标1。确定ACCD4-6中底物特异性的分子基础：（1.1）完善与丙酰基-COA和生物素类似物结合的ACCD5的共结晶结构。 （1.2）完善与乙酰-COA和生物素类似物结合的ACCD6的共结晶结构。 （1.3）求解APO ACCD4的晶体结构，以及与长链酰基-COA和生物素类似物结合的ACCD4的共晶结构。目标2。确定ACCD5-6的抑制剂结合特异性：（2.1）。使用UC Irvine的ChemDB和两个docking ofking软件的筛选，螺旋，螺旋桨和雄激素（三个鉴定的抑制剂）类似物对ACCD5-6进行了类似物。 （2.2）在体外筛选抑制剂从2.1预测，并通过抑制动力学阐明ACCD5-6酶机制。 （2.3）与Sulfa，Propeller或Andrimid结合的ACCD5-6的精制共晶结构。 AIM 3。比较ACCD4，ACCD5和ACCD6的活性位点几何形状和底物结合袋，并通过定点诱变来定义底物/抑制剂结合残基：（3.1）系统地突变ACCD5残基437评估其对底物特异性的重要性。 （3.2）酰基辅酶A结合口袋中的突变残基，以探测酰基辅酶A和硫酸类似物的ACCD5特异性。强大的初步数据（包括所有提出的结构研究的衍射晶体（AIM 1和AIM 2.3）以及已建立的酶测定，在AIM 2中识别50多个有效的抑制剂，以及在AIM的一半构造中，对这些唯一的特定构建，因为在AIM 2中识别了50多个唯一的MEM，因为在AIM 2中识别了50多个有效的抑制剂。结核病将进行严格评估和解剖。这样的发现是原始的，因为任何其他生物的ACTAS都没有具有如此独特但精确控制的底物特异性的独特性。该提案的结果将确定有效的ACCASE抑制剂。因此，该项目的完成也将对新的结核病治疗剂的发展具有健康意义。拟议的研究将保留并增加两名研究生和两名博士后研究人员的工作机会，结果将使我们能够以一种单锅，环保的方式为下游聚酮化合物生物合成提供新的基础，从而完成多步骤的总合成，通过将细菌变成药物制造工厂。 绩效网站（组织，城市，州） 加利福尼亚大学，欧文，美国加利福尼亚州92697，美国 修订的摘要部分