Reduction of clofazimine by mycobacterial type 2 NADH:quinone oxidoreductase: a pathway for the generation of bactericidal levels of reactive oxygen species |
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Authors: | Yano Takahiro Kassovska-Bratinova Sacha Teh J Shin Winkler Jeffrey Sullivan Kevin Isaacs Andre Schechter Norman M Rubin Harvey |
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Institution: | Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. |
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Abstract: | The mechanism of action of clofazimine (CFZ), an antimycobacterial drug with a long history, is not well understood. The present study describes a redox cycling pathway that involves the enzymatic reduction of CFZ by NDH-2, the primary respiratory chain NADH:quinone oxidoreductase of mycobacteria and nonenzymatic oxidation of reduced CFZ by O(2) yielding CFZ and reactive oxygen species (ROS). This pathway was demonstrated using isolated membranes and purified recombinant NDH-2. The reduction and oxidation of CFZ was measured spectrally, and the production of ROS was measured using a coupled assay system with Amplex Red. Supporting the ROS-based killing mechanism, bacteria grown in the presence of antioxidants are more resistant to CFZ. CFZ-mediated increase in NADH oxidation and ROS production were not observed in membranes from three different Gram-negative bacteria but was observed in Staphylococcus aureus and Saccharomyces cerevisiae, which is consistent with the known antimicrobial specificity of CFZ. A more soluble analog of CFZ, KS6, was synthesized and was shown to have the same activities as CFZ. These studies describe a pathway for a continuous and high rate of reactive oxygen species production in Mycobacterium smegmatis treated with CFZ and a CFZ analog as well as evidence that cell death produced by these agents are related to the production of these radical species. |
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Keywords: | Amyloid Blood Brain RNA Binding Protein RNA Folding Translation Control |
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