Natalie Wiedemar, Rachel Milne, Sandra Carvalho, Stephen Patterson, Mike Bodkin, Nicolas Masurier, Vincent Lisowski, Nicolas Primas, Pierre Verhaegh, Graeme M. Sloane, Susan Wyllie
ACS Infect. Dis. 2025, XXXX, XXX, XXX-XXX
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. In vitro evolution of gamhépathiopine resistance and whole genome analyses identified mutations within the QO site of Plasmodium falciparum cytochrome b, part of complex III of the electron transport chain. Subsequent biochemical assays demonstrated that gamhépathiopine directly inhibits complex III activity. Furthermore, exogenous expression of Saccharomyces cerevisiae dihydroorotate dehydrogenase, known to render the electron transport chain dispensable in Plasmodium, 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 QO-targeting antimalarial atovaquone. The implications of these findings for the future development of gamhépathiopine are discussed.