Antibacterial activity of rifamycins for M. smegmatis with comparison of oxidation and binding to tear lipocalin

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 that established for Mycobacterium 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 antimycobacterial activity in Mycobacterium 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. 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.

Repositioning rifamycins for Mycobacterium abscessus lung disease

“…The hydroquinone of this ring (i.e., hydroxyl groups at C1 and C4 positions) is susceptible to autoxidation in the presence of oxygen and divalent metal cations as a catalyst, generating rifampicin quinone ( Figure 1) [77]. Consistent with this, autoxidation of rifampicin results in a loss of anti-mycobacterial activity [78]. Similar results are observed with rifapentine, a rifamycin with the same hydroquinone Rifabutin (gold circles) accumulates to higher levels in M. abscessus than rifampicin (purple circles) (our unpublished data).…”

“…Rifabutin, however, does not have a susceptible hydroquinone ( Figure 1). As a result, rifabutin is resistant to autoxidation and does not lose potency under oxidizing conditions [78]. As oxidizing conditions in the cytosol of M. tuberculosis and M. abscessus are likely to be similar, autoxidation cannot explain the differential potency of rifampicin in these species.…”

“…structure and no activity against M. abscessus [64,78]. Rifabutin, however, does not have a susceptible hydroquinone ( Figure 1).…”

“…Scaffolds in which the napthoquinone has a stable oxidation state may thus exhibit good potency against M. abscessus. For example, rifalazil also lacks a hydroquinone and is resistant to autoxidation [78]; its activity against M. abscessus, however, has yet to be determined. A better understanding of how rifamycin oxidation in M. abscessus (both via autoxidation and enzyme-mediated mechanisms) impacts potency is needed.…”

Restoration of structural stability and ligand binding after removal of the conserved disulfide bond in tear lipocalin

“…In this study tear lipocalin (TL) was selected for experiments because the ligand binding of TL has been well characterized. The demonstrated ligands include fatty acids of varying lengths, phospholipids, alkyl alcohols, glycolipids, as well as various synthetic ligands and drugs [17-21]. Comprehensive information about structure, dynamics and function is also available for TL.…”