This is a new initiative within LICP to begin a program in mycobacterial pathogenesis. To date the only effort on this project has centered on equipping a P-3 facility in which to grow the virulent organism and designing potential projects to begin work. A description of the proposed course of work follows; (1) Elucidation of the immediate enzyme target of isoniazid action. Isoniazid (INH) represents the front-line defense against tuberculosis yet its mode of action and the reason for its mycobacterial specificity is not understood. INH has a quantitative effect on mycolic acid biosynthesis and as a consequence an effect on cell-wall integrity. This proposal will attempt to identify the enzyme target of INH sensitivity by photoaffinity-labelling of the enzyme involved with radiolabelled INH. INH has also been proposed to act thru interference with nicotinamide cofactor activity. This will be explored through differential competitive photolabelling with 2-azido-NAD+/H. The availability of a specific enzyme target will allow determination of the biosynthetic step affected and will allow a direct assay to guide the development of anti-mycobacterial chemotherapeutics. (2) Regulation of cell-wall biosynthesis in Mycobactrium tuberculosis H37Rv. In E. coli fatty acid metabolism/biosynthesis is coordinately regulated by FadR which has a dual role as both repressor of transcription of catabolic enzymes and activator of transcription of anabolic pathways. FadR binds to long- chain fatty acyl coenzyme A esters within the cytoplasm and undergoes a conformational change which relieves repression of the catabolic enzymes necessary for fatty acid degradation. The regulation of the biosynthesis of complex lipid structures in mycobacteria is essentially unknown but perhaps operates using similar mechanisms. This research will investigate the effect of various lipids and lipid precursors on total incorporation of acetyl-CoA into lipid fractions specific for the cell-wall. Cytoplasmic proteins which bind to labelled target lipids will be identified and their role in regulation of lipid metabolism will be explored. (3) Determination of the mechanism of formation and function of cyclopropanated mycolic acids in the cell-wall core of Mycobacterium tuberculosis. Cyclopropanated mycolic acids occur at the interface of hydrophobic domains in the mycobacterial cell-wall. The function of these modifications may be structural or they may serve to deactivate potential targets for oxidative attack by host organisms. The proposed experiments will attempt to demonstrate cyclopropanation of precursors to mycolic acids in purified cell-free extracts and utilize this activity to purify, clone and sequence the enzyme from M. tuberculosis. Targeted gene disruption of this activity will allow a direct assessment of the role of this structure in mycobacterial virulence. (4) Distribution and function of fatty acid synthase-like complexes in Mycobactrium tuberculosis H37Rv. Many of the wide variety of complex lipids encountered in the mycobacterial cell-wall appear to be biosynthetically related to fatty acids. The biosynthetic mechanism of fatty acid formation is well understood for most organisms and involves common enzymatic modules. Using motifs common to these sytems which catalyze biological """"""""Claisen- condensations"""""""" we will attempt to identify and analyze the genetic loci responsible for the biosynthesis of different families of mycobacterial lipids.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Intramural Research (Z01)
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Murugesan, Dinakaran; Ray, Peter C; Bayliss, Tracy et al. (2018) 2-Mercapto-Quinazolinones as Inhibitors of Type II NADH Dehydrogenase and Mycobacterium tuberculosis: Structure-Activity Relationships, Mechanism of Action and Absorption, Distribution, Metabolism, and Excretion Characterization. ACS Infect Dis 4:954-969
Kastrinsky, David B; Barry, Clifton E (2010) Synthesis of labeled meropenem for the analysis of M. tuberculosis transpeptidases. Tetrahedron Lett 51:197-200
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Goodwin, Michael B; Boshoff, Helena I; Barry 3rd, Clifton E et al. (2006) Quantification of small molecule organic acids from Mycobacterium tuberculosis culture supernatant using ion exclusion liquid chromatography/mass spectrometry. Rapid Commun Mass Spectrom 20:3345-50
Vannada, Jagadeshwar; Bennett, Eric M; Wilson, Daniel J et al. (2006) Design, synthesis, and biological evaluation of beta-ketosulfonamide adenylation inhibitors as potential antitubercular agents. Org Lett 8:4707-10
Kim, Pilho; Zhang, Yong-Mei; Shenoy, Gautham et al. (2006) Structure-activity relationships at the 5-position of thiolactomycin: an intact (5R)-isoprene unit is required for activity against the condensing enzymes from Mycobacterium tuberculosis and Escherichia coli. J Med Chem 49:159-71
Kim, Pilho; Barry, Clifton E; Dowd, Cynthia S (2006) Novel route to 5-position vinyl derivatives of thiolactomycin: Olefination vs. deformylation. Tetrahedron Lett 47:3447-3451
Barry 3rd, Clifton E; Boshoff, Helena I M; Dowd, Cynthia S (2004) Prospects for clinical introduction of nitroimidazole antibiotics for the treatment of tuberculosis. Curr Pharm Des 10:3239-62

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