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

Ubiquinone (UQ) is a critical component of the electron transport chain in Plasmodium falciparum, the etiological agent of human malaria. The first step in UQ biosynthesis is the condensation of 4-hydroxybenzoate (4-HB) and an isoprenic chain by the enzyme 4-hydroxybenzoate polyprenyltransferase (4-HPT; COQ2 gene). Atovaquone (AV), an antimalarial drug, competes with ubiquinol (UQH2) for binding to the mitochondrial bc1 complex, preventing the redox recycling of UQ. In clinical practice, AV is combined with proguanil, a dehydrofolate reductase inhibitor, in a single formulation. However, parasitic resistance to this combination has been demonstrated, indicating the need for new pharmacological combinations to potentiate AV. Previously, 4-nitrobenzoate (4-NB) demonstrated the ability to inhibit UQ biosynthesis in P. falciparum parasites as well as potentiate AV efficacy in vitro. However, both its pharmacodynamics and whether this potentiation could be useful in vivo remained obscure. Here we show that 4-NB enhances AV antiplasmodial efficacy to kill parasites, increases its selectivity compared with animal cells, and preserves proguanil efficacy. 4-NB specifically inhibited the 4-HPT enzymatic activity in mutant strains of Saccharomyces cerevisiae complemented with PfCOQ2. Finally, 4-NB also improved AV antimalarial efficacy in mice infected with Plasmodium berghei parasites. Finally, work with various 4-HB analogs delineated the chemical requirements to potentiate AV activity. These findings clarify the importance of UQ biosynthesis for malaria parasites and suggest that PfCOQ2 could be a therapeutic target to enhance the efficacy of AV.