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

主要研究者/项目负责人（最后一名、第一名、中间名）： 蔡秀娟 描述：参见说明。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，与原子能机构的使命相关）。简要描述研究设计和实现这些目标的方法。描述你将用来实现这些目标的原理和技术。此外，用两三句话，用通俗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，此描述将成为公共信息。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（Mycobacterium tuberculosis，TB）是结核病的病原菌，其细胞膜上含有化学复杂的脂质，这些脂质与结核分枝杆菌的毒力和耐药性密切相关。酰基辅酶A羧化酶（ACCase）为这些复杂的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得到了充分的认可。分枝结核杆菌ACCase包括六个ACCase亚基（AccD 1 -6），AccD 4、AccD 5和AccD 6在提供细胞壁脂质生物合成结构单元中起主要作用。然而，对这些病原体ACCases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一个新的抗结核药物库，以对抗新的结核分枝杆菌。结核蛋白靶点。本申请的目的是利用X射线晶体学、酶抑制试验和计算机辅助抑制剂设计阐明AccD 4、AccD 5和AccD 6的底物特异性、序列-结构-功能关系和生物学作用。基本原理是，一旦我们确定了AccD 4 -6的抑制剂，我们将能够抑制细胞壁脂质生物合成，导致病原体死亡。这一基本原理已被过去的遗传数据所证实，这些数据表明AccD 4和AccD 6的突变导致病原体死亡。在未来两年，我们将追求三个目标：目标1。确定AccD 4 -6中底物特异性的分子基础：（1.1）优化与丙酰辅酶A和生物素类似物结合的AccD 5的共晶结构。(1.2)优化与乙酰辅酶A和生物素类似物结合的AccD 6的共晶结构。(1.3)解析载脂蛋白AccD 4的晶体结构，以及AccD 4与长链酰基辅酶A和生物素类似物结合的共晶体结构。AIM 2.测定AccD 5 -6的底物结合特异性：（2.1）。使用UC Irvine的ChemDB和两个对接软件的交叉验证，在计算机上筛选针对AccD 5 -6的Sulfa、Propeller和CyclomidTM（三种已鉴定的抑制剂）类似物。(2.2)体外筛选2.1中预测的抑制剂，并通过抑制动力学研究阐明AccD 5 -6酶的作用机制。(2.3)优化AccD 5 -6与Sulfa、Propeller或Alcohol结合的共晶结构。AIM 3.比较AccD 4、AccD 5和AccD 6的活性位点几何形状和底物结合口袋，并通过定点诱变确定底物/抑制剂结合残基：（3.1）系统地突变AccD 5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结合口袋中的残基以探测酰基辅酶A和Sulfa类似物的AccD 5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，以及建立的酶测定，AIM 2中50多种强效抑制剂的鉴定，以及AIM 3中提出的半突变体的完整构建。拟议的研究具有科学意义，因为第一次，这些独特的M.结核病病例将被严格评估和解剖。这些发现是原创的，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，该项目的完成也将对结核病新疗法的开发具有健康意义。该研究将为两名研究生和两名博士后研究人员保留并增加就业机会，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建模块，通过将细菌转化为药物制造工厂来完成多步全合成。 履约地点（组织、城市、州） 加州大学欧文分校，加利福尼亚州92697，美国 修订摘要部分

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