Redox and structural determinants of mitochondrial complex III inhibition by triphenylphosphonium-conjugated...

Growing evidence indicates that cancer cell mitochondria remain functional and represent attractive therapeutic targets. Mitochondria-targeted drug delivery commonly uses triphenylphosphonium (TPP⁺)-conjugated compounds, which preferentially accumulate in cancer cell mitochondria because of their highly negative membrane potential. Many TPP⁺-conjugated agents inhibit mitochondrial electron transport chain complexes I and II, suppressing mitochondrial respiration and cancer cell proliferation. Recent studies suggest that electron-withdrawing substituents, such as trifluoromethyl groups, on the TPP⁺ phenyl rings alter electron density around the phosphorus center, enhancing mitochondrial uncoupling activity and antiproliferative effects. To determine how TPP⁺ electronic substituents influence redox properties, mitochondrial complex III interactions, and biological activity, we used mitochondria-targeted atovaquone (Mito-ATO) as a model system. Atovaquone (ATO), a hydroxy-1,4-naphthoquinone and the only FDA-approved mitochondrial complex III inhibitor, is currently undergoing clinical evaluation for cancer therapy. We synthesized a series of mitochondria-targeted ATO derivatives (MitoR-ATOs) containing electron-donating or electron-withdrawing substituents on the TPP⁺ moiety. Their effects on enzymatic superoxide generation were assessed by EPR spin trapping, mitochondrial oxygen consumption by Seahorse XF96 analysis, complex III Qi-site binding by computational modeling, and cancer cell proliferation using IncuCyte live-cell imaging. Unexpectedly, several MitoR-ATO derivatives emerged as potent complex III inhibitors through enhanced binding at the Qi site. Structure-activity relationship analysis revealed that the intrinsic redox properties of TPP⁺-conjugated MitoR-ATO analogs alone do not predict biological activity. Instead, accurate prediction of the therapeutic efficacy of mitochondria-targeted complex III inhibitors requires integrating redox properties with computational analyses of ligand-protein interactions at the mitochondrial complex III Qi site.