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

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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