Mitochondrial production of reactive oxygen species is often considered an unavoidable consequence of aerobic metabolism and currently cannot be manipulated without perturbing oxidative phosphorylation. Antioxidants are widely used to suppress effects of reactive oxygen species after formation, but they can never fully prevent immediate effects at the sites of production. To identify site-selective inhibitors of mitochondrial superoxide/H2O2 production that do not interfere with mitochondrial energy metabolism, we developed a robust small-molecule screen and secondary profiling strategy. We describe the discovery and characterization of a compound (N-cyclohexyl-4-(4-nitrophenoxy)benzenesulfonamide; CN-POBS) that selectively inhibits superoxide/H2O2 production from the ubiquinone-binding site of complex I (site I(Q)) with no effects on superoxide/H2O2 production from other sites or on oxidative phosphorylation. Structure/activity studies identified a core structure that is important for potency and selectivity for site I(Q). By employing CN-POBS in mitochondria respiring on NADH-generating substrates, we show that site I(Q) does not produce significant amounts of superoxide/H2O2 during forward electron transport on glutamate plus malate. Our screening platform promises to facilitate further discovery of direct modulators of mitochondrially derived oxidative damage and advance our ability to understand and manipulate mitochondrial reactive oxygen species production under both normal and pathological conditions.
Keywords: Antioxidant; CN-POBS; Complex II; Complex III; DMSO; Energy metabolism; FCCP; Free radicals; Glycerol-3-phosphate dehydrogenase; Hydrogen peroxide; N-cyclohexyl-4-(4-nitrophenoxy)benzenesulfonamide; NADH:Q oxidoreductase; ROS; Respiratory complexes; Superoxide; TMRM; TPMP; carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; dimethyl sulfoxide; flavin mononucleotide site of complex I; flavin site of complex II; mGPDH; methyltriphenylphosphonium; mitochondrial sn-glycerol-3-phosphate dehydrogenase; outer ubiquinone-binding site of complex III; pH difference across the mitochondrial inner membrane; potential difference across the mitochondrial inner membrane; reactive oxygen species; site I(F); site I(F) plus matrix NAD-linked dehydrogenases; site I(F)/DH; site I(Q); site II(F); site III(Qo); tetramethylrhodamine methyl ester; ubiquinone-binding site of complex I; ΔpH; ΔΨm.
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