Subtle Changes in Endochin-Like Quinolone Structure Alter the Site of Inhibition within the Cytochrome bc1 Complex of Plasmodium falciparum

The cytochrome bc(1) complex (cyt bc(1)) 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 bc(1) 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 bc(1). 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 structure-activity 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 bc(1) and that minor chemical changes can influence Q(o) or Q(i) site inhibition by the ELQs.