CELL WALL BIOGENESIS--TARGET FOR NEW ANTI-TB DRUGS

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The lack of fundamental knowledge of the biochemistry and genetics of Mycobacterium tuberculosis and the lack of a variety of drugs against tuberculosis have become serious problems in the face of increased prevalence and multiple drug resistance. Basic studies on the cell wall of mycobacteria emerging from this laboratory have revealed the presence of a unique tetramycolated penta-D-arabinoside (McNeil et al. , J. Biol, Chem, 266, 13217-13223, 1991). This unit is essential for the structural integrity and permeability characteristics of the cell wall of M. tuberculosis and provides a germane target for a new generation of anti-tuberculosis drugs. Neither mycolic acids nor D-arabinose are present in the host, and two effective antimycobacterials, isoniazid (INH) and ethambutol, are known to inhibit the biogenesis of mycolylarabinoside. In this proposal, we present a synergistic balance of fundamental biochemical and genetic research coupled with the design, chemical synthesis, and antimicrobial testing of new enzyme antagonists. Specifically, we will elucidate the pathway for arabinan biosynthesis, develop assays for relevant enzymes, and clone the genes for particularly promising drug targets. To accomplish this, we have developed an active enzyme system capable of converting ribulose-5-phosphate to arabinose-5-phosphate and beyond, to cell wall arabinan. In parallel studies, we will chemically synthesize transition state analogs and substrate analogs of key enzymes. The effect of these inhibitors on enzymatic conversions will be determined separately from their effect on whole bacteria, allowing compounds which inhibit enzymes but are unable to penetrate the bacteria to be recognized and then chemically modified to allow for permeation. As warranted, the effects of the new compounds on bacterial growth in mice will be determined. As well as the new antagonists, INH and ethambutol will be studied. Preliminary results have identified the general site of action of ethambutol on arabinan synthesis; the specific enzymes involved will be identified. In this regard, genes encoding for resistance to ethambutol have been cloned; recent evidence suggests that one of the ethambutol-resistance determinants is involved in arabinan biosynthesis. The cloning of genes encoding for INH resistance is proposed. In addition, enzymes susceptible to INH will be identified using an enzyme system capable of synthesizing mycolic acids from 14C labeled medium chain fatty acids. Finally, the ability of novel, tailored inhibitors such as cyclopropene-containing analogs to inhibit mycolic acid synthesis will be explored.