4-nitrobenzoate inhibits 4-hydroxybenzoate polyprenyltransferase in malaria parasites and enhances atovaquone efficacy.

Ubiquinone (UQ) is essential for the electron transport chain in Plasmodium falciparum, the causative agent of severe malaria. Its biosynthesis begins with the condensation of 4-hydroxybenzoate (4-HB) and an isoprenoid chain, catalyzed by 4-HB polyprenyltransferase (4-HPT; COQ2 gene). Atovaquone (AV) inhibits the mitochondrial bc1 complex by competing with ubiquinol (UQH2), but resistance to the synergic combination AV/proguanil therapy has emerged. Here, we show that 4-nitrobenzoate (4-NB) inhibits Pf4-HPT, enhances AV efficacy and selectivity, while preserving proguanil synergy. In Saccharomyces cerevisiae expressing PfCOQ2, 4-NB inhibited UQ biosynthesis. In vivo, 4-NB improved AV efficacy in Plasmodium berghei-infected mice. Structure-activity studies with 4-HB analogs further defined chemical features for potentiation. These findings support PfCOQ2 as a target to boost AV-based antimalarial therapy. Impact statement This study identifies a molecular rationale for enhancing atovaquone efficacy through targeted inhibition of ubiquinone biosynthesis. By validating PfCOQ2 as a druggable target and demonstrating in vivo potentiation, our findings offer strategic advance toward rational antimalarial combination therapies, moving beyond empirical approaches and addressing current resistance.