Whole-Cell Phenotypic Screening of Medicines for Malaria Venture Pathogen Box Identifies Specific Inhibitors of Plasmodium falciparum Late-Stage Development and Egress.

We report a systematic, cellular phenotype-based antimalarial screening of the Medicines for Malaria Venture Pathogen Box collection, which facilitated the identification of specific blockers of late-stage intraerythrocytic development of Plasmodium falciparum First, from standard growth inhibition assays, we identified 173 molecules with antimalarial activity (50% effective concentration [EC50] ≤ 10 µM), which included 62 additional molecules over previously known antimalarial candidates from the Pathogen Box. We identified 90 molecules with EC50 of ≤1 µM, which had significant effect on the ring-trophozoite transition, while 9 molecules inhibited the trophozoite-schizont transition and 21 molecules inhibited the schizont-ring transition (with ≥50% parasites failing to proceed to the next stage) at 1 µM. We therefore rescreened all 173 molecules and validated hits in microscopy to prioritize 12 hits as selective blockers of the schizont-ring transition. Seven of these molecules inhibited the calcium ionophore-induced egress of Toxoplasma gondii, a related apicomplexan parasite, suggesting that the inhibitors may be acting via a conserved mechanism which could be further exploited for target identification studies. We demonstrate that two molecules, MMV020670 and MMV026356, identified as schizont inhibitors in our screens, induce the fragmentation of DNA in merozoites, thereby impairing their ability to egress and invade. Further mechanistic studies would facilitate the therapeutic exploitation of these molecules as broadly active inhibitors targeting late-stage development and egress of apicomplexan parasites relevant to human health.

A reference document on Permissible Limits for solvents and buffers during in vitro antimalarial screening.

Antimalarial drug discovery expands on targeted and phenotype-based screening of potential inhibitory molecules to ascertain overall efficacy, phenotypic characteristics and toxicity, prior to exploring pharmacological optimizations. Candidate inhibitors may have varying chemical properties, thereby requiring specific reconstitution conditions to ensure solubility, stability or bioavailability. Hence, a variety of solvents, buffers, detergents and stabilizers become part of antimalarial efficacy assays, all of which, above certain threshold could interfere with parasite viability, invasion or red blood cell properties leading to misinterpretation of the results. Despite their routine use across malaria research laboratories, there is no documentation on non-toxic range for common constituents including DMSO, glycerol, ethanol and methanol. We herein constructed a compatibility reference guide for 14 such chemicals and estimated their Permissible Limit against P. falciparum asexual stages at which viability and replication of parasites are not compromised. We also demonstrate that at the estimated Permissible Limit, red blood cells remain healthy and viable for infection by merozoites. Taken together, this dataset provides a valuable reference tool for the acceptable concentration range for common chemicals during in vitro antimalarial tests.