Functional Phenotyping of MMV Pandemic Response Box Identifies Stage and Mechanism-Specific Inhibitors against Blood Stage Plasmodium falciparum

Widespread drug resistance necessitates the prioritization of novel scaffolds with alternate mechanisms as possible partner drugs to artemisinin to combat malaria. We utilized the Pandemic Response Box chemical library of the Medicines for Malaria Venture, launched in 2019, to identify inhibitors with stage-specific potency and phenotypic signatures against the blood stage development of Plasmodium falciparum (P. falciparum) toward exploring drug repurposing. From this screening, we initially identified 60 molecules active at 10 μM against both drug-sensitive (3D7) and chloroquine-resistant (Dd2) strains of P. falciparum. Furthermore, 28 compounds active below 3 μM were prioritized, several of which specifically impaired stage transitions of ring (MMV001014), trophozoite (MMV1593540 and MMV1634402), and schizonts (MMV1580844, MMV1580496, MMV1580173, and MMV1580483), confirmed through microscopic phenotypes and flow cytometry. The ring stage inhibitor, MMV001014, was irreversible, led to no recrudescence, and showed antagonistic effects with artemisinin, indicative of overlapping mechanisms. Both the trophozoite inhibitors, MMV1593540 and MMV1634402, exhibited nanomolar EC₅₀, among which MMV1593540 was additive with artemisinin while antagonistic with chloroquine. Two of the schizont stage inhibitors (MMV1580844 and MMV1580173) appeared to operate through a mechanism driven by the generation of reactive oxygen species, and all of them with molecule-specific effects on infected red blood cell (iRBC) membrane integrity, confirmed through confocal microscopy. Taken together, these results highlight interesting starting points with likely unique modes of action from MMV’s pandemic response box for drug repurposing to combat human malaria that continues to impact the developing world.