Repurpose antimalarials to target Toxoplasma gondii dihydrofolate reductase thymidylate synthase

Toxoplasma gondii is an obligate intracellular blood and tissue protozoan parasite that infects up to a third of the population worldwide. Several antimalarial drugs, in particular pyrimethamine (PYR), have been used for decades to treat toxoplasmosis. Here, the clinical candidate P218, a potent inhibitor of Plasmodium falciparum dihydrofolate reductase (PfDHFR), and a series of flexible diaminopyrimidine butyrolactone analogues were identified as potent T. gondii dihydrofolate reductase (TgDHFR) inhibitors. The most promising butyrolactone analogue, LA4, displayed an improved TgDHFR inhibition (K_i 1.71 nM), increased antiparasitic properties in vitro (IC₅₀ 0.44 nM), and a higher cell selectivity compared to PYR (K_i 13.0 nM, IC₅₀ 410 nM) while P218 (K_i 2.19 nM, IC₅₀ 370 nM) presented an improved activity with comparable cell selectivity to PYR. The in vivo results against T. gondii RH strain-infected mice showed that P218 reduced parasitic burden in blood whereas LA4 decreased parasite load in peritoneal fluid and blood with an extended mice survival. These findings position butyrolactone LA4 as a new potential for the treatment of acute toxoplasmosis.