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: 7895564
• 负责人: Shiou-Chuan Tsai
• 金额: $ 30.6万
• 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 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7895564
• 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; Biotin; California; Cell Wall; Cells; Cessation of life; Cities; Commit; Communicable Diseases; Complex; Computer Assisted; Computer Simulation; Confidential Information; Data; Development; Docking; Drug Delivery Systems; Drug Design; Drug resistance; Environment; Enzyme Inhibition; Enzymes; Fatty Acids; Funding; Genes; Genetic; Genome; Genus Mycobacterium; Goals; Health; 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; Role; Sequence Analysis; Site-Directed Mutagenesis; Specificity; Structure; Structure-Activity Relationship; Substrate Specificity; Techniques; Therapeutic; Time; Toxic effect; Training; Tuberculosis; Universities; Unspecified or Sulfate Ion Sulfates; Validation; Virulence; Work; X-Ray Crystallography; abstracting; analog; andrimid; antimicrobial; assay development; base; cell envelope; chemotherapy; design; enzyme mechanism; fatty acid biosynthesis; graduate student; improved; 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

首席研究员/项目总监（最后、第一、中间）： 蔡修全 描述：参见说明。说明申请的广泛、长期目标和具体目标，并参考项目的健康相关性（即与机构使命的相关性）。简明地描述实现这些目标的研究设计和方法。描述您将用于实现这些目标的基本原理和技术。此外，用两到三个句子，用通俗易懂的语言描述这项研究与公共卫生的相关性。如果申请获得资助，该描述将按原样成为公共信息。因此，请勿包含专有/机密信息。请勿超出所提供的空间。 结核分枝杆菌是结核病（TB）的病原体，其细胞包膜含有化学复杂的脂质，这与其毒力和多重耐药性密切相关。酰基辅酶A羧化酶 (ACCase) 为这些复杂脂质提供了构建模块，并且 ACCase 作为药物靶点的重要性和有效性已得到广泛认可。结核分枝杆菌 ACCase 包括六个 ACCase 亚基 (accD1-6)，并且 AccD4、AccD5 和 AccD6 在为细胞壁脂质生物合成提供构建模块方面发挥着重要作用。然而，人们对这些病原体 ACCas 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结核分枝杆菌蛋白靶标的新型抗结核疗法库。这一特定应用的目的是利用 X 射线晶体学、酶抑制测定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-功能关系以及生物学作用。其基本原理是，一旦我们鉴定出 AccD4-6 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。这一基本原理已被过去的遗传数据所验证，这些数据表明 AccD4 和 AccD6 的突变会导致病原体死亡。未来两年，我们将追求三个目标： 目的 1.确定AccD4-6底物特异性的分子基础：（1.1）精炼AccD5与丙酰辅酶A和生物素类似物结合的共晶结构。 (1.2)精制AccD6与乙酰辅酶A和生物素类似物结合的共晶结构。 (1.3)解析apo AccD4的晶体结构以及AccD4与长链酰基辅酶A和生物素类似物结合的共晶结构。目的 2. 确定 AccD5-6 的抑制剂结合特异性：(2.1)。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，在计算机上筛选针对 AccD5-6 的 Sulfa、Propeller 和 andrimid（三种已鉴定的抑制剂）类似物。 (2.2)体外筛选2.1预测的抑制剂，并通过抑制动力学阐明AccD5-6酶机制。 (2.3)精制AccD5-6与Sulfa、Propeller或andrimid结合的共晶结构。目的 3. 比较 AccD4、AccD5 和 AccD6 的活性位点几何结构和底物结合口袋，并通过定点诱变定义底物/抑制剂结合残基： (3.1) 系统突变 AccD5 残基 437，以评估其对底物特异性的重要性。 (3.2) 突变酰基辅酶A结合袋中的残基以探测酰基辅酶A和磺基类似物的AccD5特异性。拟议研究的可行性得到了强有力的初步数据的有力支持，包括所有拟议结构研究（AIM 1 和 AIM 2.3）的衍射晶体，以及已建立的酶测定、AIM 2 中 50 多种有效抑制剂的鉴定，以及完整的AIM 3 中提出的半突变体的构建。拟议的研究具有科学意义，因为这些独特的结核分枝杆菌 ACCase 的底物/抑制剂特异性将首次受到至关重要的影响。评估和剖析。这些发现是原创的，因为任何其他生物体的 ACCase 都没有如此独特的多样性，但精确控制的底物特异性。该提案的结果将确定有效的 ACCase 抑制剂。因此，该项目的完成对于结核病新疗法的开发也具有健康意义。拟议的研究将为两名研究生和两名博士后研究员保留和增加就业机会，其成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建模块，通过以下方式完成多步全合成：将细菌变成制药工厂。 绩效站点（组织、城市、州） 加州大学欧文分校, CA 92697, 美国 修订后的摘要部分

项目成果

ACCase 6 is the essential acetyl-CoA carboxylase involved in fatty acid and mycolic acid biosynthesis in mycobacteria.

• DOI: 10.1099/mic.0.027714-0
• 发表时间: 2009-08
• 期刊: Microbiology (Reading, England)
• 作者: Kurth DG;Gago GM;de la Iglesia A;Bazet Lyonnet B;Lin TW;Morbidoni HR;Tsai SC;Gramajo H
• 通讯作者: Gramajo H