The goal is to rapidly identify effective agents for treatment of tuberculosis. The driving force behind this urgent need is increasing drug resistance of Mycobacterium tuberculosis. Limited data suggest that quinolones, which inhibit DNA gyrase, and beta-lactam antibiotics, which inhibit penicillin binding proteins (PBPs) (cell wall synthetic enzymes), may be effective. The clinical fate of these agents ultimately depends upon the ease with which resistance develops. Therefore, this research will focus on understanding resistance mechanisms for these two classes of drugs. A rapid method for developing drugs for clinical use is presented. There are four aims: 1) To characterize cell wall permeability of M. tuberculosis to quinolones and beta-lactam antibiotics. The barrier to penetration and partitioning of drugs in cell wall will be measured quantitatively for susceptible and resistant cells and the contribution of penetration to resistance determined. 2) Molecular cloning and sequence analysis of gyrase genes and identification of the DNA site cleaved by gyrase. A M. tuberculosis genomic DNA library will be screened with PCR-amplified DNA probes to obtain recombinant DNA for sequence analysis of gyrase genes. The effect of antibiotic selection on gyrase mutation will be determined by comparing gyrase gene sequence and DNA cleavage site of susceptible and resistant strains. 3) Identification of penicillin binding proteins of M. tuberculosis. PBPs will be identified and binding constants determined by fluorography. Drug inactivation by beta-lactamase will be assayed. Knowledge of binding, drug inactivation, and penetration will define the contribution of each to resistance. 4) To determine activity of quinolones and beta-lactam antibiotics against M. tuberculosis in vivo. Human subjects with pulmonary tuberculosis will be treated with single agents for 7 days to determine by quantitative culture the rate of elimination of organisms from sputum. Rate of elimination is predictive of efficacy. These data combined with knowledge of resistance mechanisms, will speed development of quinolones and beta-lactam antibiotics for treatment of tuberculosis.
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