Abstract
A mutant of Mycobacterium smegmatis is a potential class I model substitute for Mycobacterium tuberculosis. Because not all of the rifamycins have been tested in this organism, we determined bactericidal profiles for the 6 major rifamycin derivatives. The profiles closely mirrored those established for M. tuberculosis. Rifalazil was confirmed to be the most potent rifamycin. Because the tuberculous granuloma presents a harshly oxidizing environment we explored the effects of oxidation on rifamycins. Mass spectrometry confirmed that three of the six major rifamycins showed autoxidation in the presence of trace metals. Oxidation could be monitored by distinctive changes including isosbestic points in the ultraviolet-visible spectrum. Oxidation of rifamycins abrogated anti-mycobacterial activity in M. smegmatis. Protection from autoxidation was conferred by binding susceptible rifamycins to tear lipocalin, a promiscuous lipophilic protein. Rifalazil was not susceptible to autoxidation but was insoluble in aqueous solution. Solubility was enhanced when complexed to tear lipocalin and was accompanied by a spectral red shift. The positive solvatochromism was consistent with robust molecular interaction and binding. Other rifamycins also formed a complex with lipocalin, albeit to a lesser extent. Protection from oxidation and enhancement of solubility with protein binding may have implications for delivery of select rifamycin derivatives.