Subtle changes in endochin-like quinolone structure alter the site of inhibition within the cytochrome bc₁ complex of plasmodium falciparum

The cytochrome bc₁ complex (cyt bc₁) is the third component of the mitochondrial electron transport chain and is the target of several potent antimalarial compounds, including the naphthoquinone atovaquone (ATV) and the 4(1H)-quinolone ELQ-300. Mechanistically, cyt bc₁ facilitates the transfer of electrons from ubiquinol to cytochrome c and contains both oxidative (Q_o) and reductive (Q_i) catalytic sites that are amenable to small-molecule inhibition. Although many antimalarial compounds, including ATV, effectively target the Q_o site, it has been challenging to design selective Q_i site inhibitors with the ability to circumvent clinical ATV resistance, and little is known about how chemical structure contributes to site selectivity within cyt bc₁. Here, we used the proposed Q_i site inhibitor ELQ-300 to generate a drug-resistant Plasmodium falciparum clone containing an I22L mutation at the Q_i region of cyt b. Using this D1 clone and the Y268S Q_o mutant strain, P. falciparum Tm90-C2B, we created a structureactivity map of Q_i versus Q_o site selectivity for a series of endochin-like 4(1H)-quinolones (ELQs). We found that Q_i site inhibition was associated with compounds containing 6-position halogens or aryl 3-position side chains, while Q_o site inhibition was favored by 5,7-dihalogen groups or 7-position substituents. In addition to identifying ELQ-300 as a preferential Q_i site inhibitor, our data suggest that the 4(1H)-quinolone scaffold is compatible with binding to either site of cyt bc₁ and that minor chemical changes can influence Q_o or Q_i site inhibition by the ELQs.