# 4‐nitrobenzoate inhibits 4‐hydroxybenzoate polyprenyltransferase in malaria parasites and enhances atovaquone efficacy

## TL;DR

4-nitrobenzoate inhibits a key enzyme in malaria parasite ubiquinone biosynthesis, enhancing the effectiveness of the antimalarial drug atovaquone.

## Contribution

4-nitrobenzoate is shown to inhibit Pf4-HPT, offering a new strategy to enhance atovaquone efficacy and combat resistance.

## Key findings

- 4-nitrobenzoate inhibits Pf4-HPT and reduces ubiquinone biosynthesis in malaria parasites.
- 4-nitrobenzoate enhances atovaquone efficacy in vitro and in Plasmodium berghei-infected mice.
- Structure–activity studies identified chemical features for potentiation of atovaquone.

## Abstract

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 statementThis 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.

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.

Atovaquone is an antimalarial requiring potentiation for sufficient efficacy. We pursued strategies to enhance its activity, showing that 4‐nitrobenzoate inhibits 4‐hydroxybenzoate polyprenyltransferase, decreasing ubiquinone biosynthesis. Since atovaquone competes with ubiquinol in mitochondria, 4‐nitrobenzoate facilitates its action, potentiating activity in vitro and in vivo. The figure illustrates drug potentiation (crossed rings), ubiquinone role (green), atovaquone (blue), and 4‐nitrobenzoate blocking 4‐hydroxybenzoate polyprenyltransferase (yellow).