High-throughput phenotypic screening identifies novel antimalarial scaffolds and target-associated chemotypes

The emergence and spread of artemisinin-resistant Plasmodium falciparum threaten recent progress in malaria control and highlight the urgent need for antimalarial agents with new mechanisms of action. Here, we performed a high-throughput phenotypic screening of the Nagoya Chemical Library, comprising 36,160 compounds. This yielded 1391 (3.8%) primary hits, further reduced by cytotoxicity profiling. Differential screening against wild-type 3D7 and transgenic 3D7-yDHODH parasites expressing yeast dihydroorotate dehydrogenase (DHODH) was done to identify mitochondrial electron transport chain inhibitors. This approach identified a compound with modest activity against parasite DHODH, confirmed by enzymatic assays with recombinant PfDHODH. A subset of 441 potent compounds with half-maximal concentration (EC₅₀) < 6.5 μM was selected and profiled against the Dd2-derived multidrug-resistant panel. This analysis identified compounds likely targeting key parasite pathways, including phosphatidylinositol 4-kinase (PfPI4K) and P-type ATPase 4 (PfATP4). Chemical clustering of a selected subset of 350 compounds (<10 μM) revealed novel scaffolds with antiplasmodial potency that bear no structural resemblance to known antimalarial compounds. Collectively, these findings highlight novel chemotypes associated with validated antiplasmodial targets (PfDHODH, PfPI4K, and PfATP4) and uncover previously uncharacterized scaffolds with unknown targets. These compounds provide a foundation for further investigation of their mechanisms of action and optimization as potential antimalarial agents.