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 (UQH 2 ), but resistance to the synergic combination AV/proguanil therapy has emerged. Here, we show that 4‐nitrobenzoate (4‐NB) inhibits Pf 4‐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.