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/项目主任(最后、第一、中间)： 蔡秀川 说明：请参阅说明。说明申请的广泛、长期目标和具体目标，并提及项目与健康的相关性(即与机构使命的相关性)。简明扼要地描述实现这些目标的研究设计和方法。描述你将用来实现这些目标的基本原理和技术。此外，用两三句简单明了的话描述这项研究与公共卫生的相关性。如果申请得到资助，这一描述将成为公开信息。因此，不包括专有/机密信息。不要超过所提供的空间。 结核分枝杆菌是结核病的病原体，其细胞膜含有复杂的化学脂质，与其毒力和多药耐药密切相关。酰辅酶A羧基酶(ACCase)为这些复杂的脂质提供了基础，ACCase作为药物靶点的重要性和有效性得到了很好的认识。结核分枝杆菌ACCase包括6个ACCase亚基(acc1-6)，AccD4、AccD5和AccD6在细胞壁脂生物合成中起主要作用。然而，人们对这些病原菌ACCase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点的新型抗结核疗法的库。这一特殊应用的目的是利用X射线结晶学、酶抑制试验和计算机辅助抑制剂设计来阐明AccD4、AccD5和AccD6的底物特异性、序列-结构-功能关系和生物学作用。其基本原理是，一旦我们确定了AccD4-6的抑制剂，我们将能够抑制细胞壁脂的生物合成，导致病原体死亡。这一理论已被过去的遗传数据证实，这些数据表明AccD4和AccD6的突变会导致病原体死亡。在接下来的两年里，我们将致力于三个目标：目的1.确定AccD4-6底物特异性的分子基础：(1.1)提纯与丙酰辅酶A和生物素类似物结合的AccD5的共晶结构。(1.2)提纯乙酰辅酶A和生物素类似物结合的AccD6的共晶结构。(1.3)解出载脂蛋白AccD4的晶体结构，以及与长链酰辅酶A和生物素类似物结合的AccD4的共晶结构。目的2.确定AccD5-6：(2.1)与抑制物结合的特异性。使用加州大学欧文分校的ChemDB和两个对接软件进行交叉验证，针对AccD5-6筛选硅磺胺、推进剂和雄胺(三种已确定的抑制剂)类似物。(2.2)体外筛选从2.1预测的抑制物，并通过抑制动力学阐明AccD5-6酶的作用机制。(2.3)细化与磺胺、螺旋桨或雄胺结合的AccD5-6的共晶结构。目的3.比较AccD4、AccD5和AccD6的活性部位几何构型和底物结合口袋，并通过定点突变确定底物/抑制物结合残基：(3.1)系统突变AccD5残基437，以评价其对底物特异性的重要性。(3.2)在酰基-辅酶A结合口袋中突变残基，以探测酰基-辅酶A和磺胺类似物的AccD5特异性。这项研究的可行性得到了强大的初步数据的有力支持，包括所有拟议的结构研究(AIM 1和AIM 2.3)的衍射晶体，以及建立的酶分析，鉴定AIM 2中50多个有效的抑制剂，以及AIM 3中建议的一半突变体的完整构建。拟议的研究具有重要的科学意义，因为将首次对这些独特的结核分枝杆菌ACCase的底物/抑制剂的特异性进行严格的评估和解剖。这样的发现是原创的，因为没有任何其他生物的ACCase具有如此独特的多样性，但精确控制底物专一性。这项提案的结果将确定有效的ACCase抑制剂。因此，该项目的完成对结核病新疗法的开发也将具有健康意义。这项拟议的研究将保留和增加两名研究生和两名博士后研究员的就业机会，其结果将使我们能够以一锅环保的方式为下游聚酮生物合成提供新的构件，通过将细菌转化为药物制造工厂来完成多步骤全合成。 演出现场(S)(组织、市、州) 加州大学欧文分校，邮编：92697，美国 修订摘要部分