The antiseizure medication stiripentol inhibits mitochondrial complex I by blocking early-stage electron transfer

The oxidation of NADH is essential for maintaining cellular redox balance and supporting cell metabolism. Mitochondrial complex I (NADH:ubiquinone oxidoreductase) plays a central role in this process by coupling NADH oxidation to electron transfer and proton translocation across the inner mitochondrial membrane. We previously reported that the antiseizure medication stiripentol decreases lactate production and mitochondrial respiration, suggesting an impact on NADH turnover beyond its known inhibition of lactate dehydrogenase. In this study, we identify complex I as a target of stiripentol across multiple species and cell types. Biochemical and spectroscopic analyses demonstrate that stiripentol inhibits NADH oxidation and electron transfer through a mechanism distinct from that of classical ubiquinone pocket inhibitors such as rotenone or piericidin A. Remarkably, stiripentol acts upstream of the ubiquinone reduction site, representing the first example of a complex I inhibitor with a binding site within the N-module. These findings uncover a previously unrecognized mode of complex I inhibition and link stiripentol's metabolic effects to direct modulation of mitochondrial NADH oxidation. This work broadens the understanding of stiripentol's mechanism of action and highlights its potential to modulate redox metabolism in cancer cells.