The Thienopyrimidinone Gamhépathiopine Targets the Q_O Site of <i>Plasmodium falciparum</i> Cytochrome <i>b</i>

Chemotherapy remains a key component of the arsenal of tools to fight malaria. Specifically, new drugs with diverse mechanism(s) of action are required to combat existing drug resistance. Here, we describe comprehensive studies to determine the molecular target(s) of gamhépathiopine, a thienopyrimidinone showing promise for the treatment of malaria. <i>In vitro</i> evolution of gamhépathiopine resistance and whole genome analyses identified mutations within the Q_O site of <i>Plasmodium falciparum</i> cytochrome <i>b</i>, part of complex III of the electron transport chain. Subsequent biochemical assays demonstrated that gamhépathiopine directly inhibits complex III activity. Furthermore, exogenous expression of <i>Saccharomyces cerevisiae</i> dihydroorotate dehydrogenase, known to render the electron transport chain dispensable in <i>Plasmodium</i>, results in complete abrogation of gamhépathiopine activity. Cross-resistance profiling and docking studies indicate that gamhépathiopine occupies a similar, but not identical, binding pose to the established Q_O-targeting antimalarial atovaquone. The implications of these findings for the future development of gamhépathiopine are discussed.