Structural insights and mechanistic inhibition of Plasmodium falciparum dihydroorotate dehydrogenase by newly identified chemotypes

Resistance to frontline antimalarial drugs continues to threaten malaria control, highlighting the need for new chemotypes. Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) catalyzes a key step in de novo pyrimidine biosynthesis and remains a validated antimalarial target. Although the PfDHODH inhibitor DSM265 advanced to clinical development, resistance-associated mutations highlight the need for structurally distinct inhibitors. Here, we report the discovery and structural characterization of PfDHODH inhibitors identified through target-based high-throughput screening of 432,592 compounds. Primary hits were evaluated by dose-response assays against asexual blood-stage parasites, mammalian mitochondrial toxicity assays, and HepG2 cytotoxicity profiling, yielding 348 hits. Seven representative compounds were prioritized based on efficacy against PfDHODH and orthologs from P. vivax and P. berghei, and their X-ray co-crystal structures revealed distinct binding modes within the active site. Ferulenol exhibited sub-micromolar inhibition of recombinant PfDHODH (rPfDHODH) and rPvDHODH, together with potent blood-stage activity, with its prenylated tail contributing additional hydrophobic interactions. Compound 2 showed the highest potency across the tested DHODH enzymes but reduced selectivity for PfDHODH, potentially due to its distinct binding orientation and furylmethyl substituent. Comparative structural analyses with DSM265 identified binding-site features that may guide the design of next-generation PfDHODH inhibitors with improved potency and selectivity.