Inhibitors of ROS production by the ubiquinone-binding site of mitochondrial complex I identified by chemical screening

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• 作者：Adam L. Orr ; Deepthi Ashok ; Melissa R. Sarantos ; Tong Shi ; Robert E. Hughes ; Martin D. Br
• 关键词：mGPDH ; mitochondrial sn-glycerol-3-phosphate dehydrogenase ; TPMP ; methyltriphenylphosphonium ; TMRM ; tetramethylrhodamine methyl ester ; 螖唯m ; potential difference across the mitochondrial inner membrane ; 螖pH ; pH difference across the mitochondrial inner membrane ; site IF ; flavin mononucleotide site of complex I ; site IF/DH ; site IF plus matrix NAD-linked dehydrogenases ; site IQ ; ubiquinone-binding site of complex I ; site IIF ; flavin site of complex II ; site IIIQo ; outer ubiquinone-binding site
• 刊名：Free Radical Biology and Medicine
• 出版年：December, 2013
• 年：2013
• 卷：65
• 期：Complete
• 页码：1047-1059
• 全文大小：2338 K

文摘

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.