Functional Phenotyping of MMV Pandemic Response Box Identifies Stage-specific inhibitors Against Blood stage Plasmodium

Wide-spread resistance to clinically used antimalarials necessitates the prioritization of novel scaffolds with alternate mechanisms, as possible partner drugs to artemisinin. 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 P. falciparum towards exploring the possibility of drug repurposing. From this screening, we initially identified 60 molecules active against both drug sensitive (3D7) and chloroquine resistant (Dd2) strains of P. falciparum. Further, 28 active compounds active below 3{micro}M 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 mechanism. Both the trophozoite inhibitors exhibited nanomolar IC50 with non-compromised digestive vacuole. MMV1593540 was partially additive with artemisinin while antagonistic with chloroquine. Two among the schizont stage inhibitors (MMV1580844 and MMV1580496) appeared to operate through a mechanism driven by the generation of reactive oxygen species and all of them with molecule-specific effect on infected red blood cell (RBC) membrane integrity confirmed through confocal microscopy. Taken together, these results highlight interesting starting points derived from MMVs Pandemic Response Box for repurposing to combat Malaria that continues to morbidly affect the developing world. ImportanceMalaria caused by infectious parasites belonging to the Plasmodium family continues to morbidly affect the marginalized populations. The situation is further complicated by lack of mass vaccination, drug resistance, and emergence of new parasitic forms. To alleviate the threat of drug resistance, it is important to identify new drugs acting through mechanisms distinct from the existing ones such as artemisinin. This work describes the screening of a chemical compound library against blood stage development of malaria parasites and prioritization of molecules that can inhibit parasite development in a stage-specific manner. Several of these compounds demonstrate nanomolar potency against sensitive and resistant forms of the parasites acting through distinctive mechanisms. Exploring the modes of action of these molecules will facilitate their optimization and possible clinical applications against the deadly diseases, Malaria.