title: Reduction of clofazimine by mycobacterial type 2 NADH:quinone oxidoreductase: a pathway for the generation of bactericidal levels of reactive oxygen species.
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.
authors.affiliation: Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Yano
authors.forename: Sacha
authors.initials: S
authors.lastname: Kassovska-Bratinova
authors.forename: J Shin
authors.initials: JS
authors.lastname: Teh
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Winkler
authors.forename: Kevin
authors.initials: K
authors.lastname: Sullivan
authors.forename: Andre
authors.initials: A
authors.lastname: Isaacs
authors.forename: Norman M
authors.initials: NM
authors.lastname: Schechter
authors.forename: Harvey
authors.initials: H
authors.lastname: Rubin
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D002417:Cattle; D002991:Clofazimine / Q000494:pharmacology*; D007917:Leprostatic Agents / Q000494:pharmacology*; D020102:Mycobacterium smegmatis / Q000201:enzymology*; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013211:Staphylococcus aureus / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D007917:Leprostatic Agents; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D002991:Clofazimine; D016660:NAD(P)H Dehydrogenase (Quinone)