New drugs are desperately needed for tuberculosis both to reduce the long duration of treatment and to cure and stop transmission of multiple-drug-resistant Mycobacterium tuberculosis. Recent studies, including the completion of the TB genome sequence, suggest that efflux pump inhibitors may play a significant role in the intrinsic resistance of M. tuberculosis to most common antibiotics. Influx has identified several low molecular weight inhibitors of the NorA efflux pump in Staphylococcus aureus which demonstrated synergistic activity with ciprofloxacin and which dramatically reduced the frequency of emergence of ciprofloxacin-resistant mutants. In preliminary in vitro experiments with M. tuberculosis, two of these compounds demonstrated marked synergy when combined with two tetracyclines and a macrolide. We propose to a) test additional congeners of these inhibitors to determine structure-activity relationships with respect to M. tuberculosis, b) determine the substrate specificity of the effected efflux pump by testing for synergy with a wide variety of antimicrobials, c) determine the effect of the inhibitors on the frequency of emergence of drug-resistant mutants and d) with a variety of TB drug-susceptible and resistant clinical isolates, and e) assess synergistic activity of inhibitors with antimicrobial agents in a macrophage model of TB infection. It is anticipated that these studies will result in the identification of one or more lead compounds that would be assessed for activity in animal models of acute and chronic TB in a phase II project. In addition these compounds would serve as starting points for synthesis of additional congeners. PROPOSED COMMERCIAL APPLICATION: The envisioned commercial product is a combination of tetracycline, a macrolide, or possibly another antibiotic with an efflux pump inhibitor. This combination can form a powerful anti-TB agent which can be effective against resistant strains and prevent further emergence of resistance.
