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Apicomplexan infections cause substantial morbidity and mortality, worldwide. New, improved therapies are needed. Herein, we create a next generation anti-apicomplexan lead compound, JAG21, a tetrahydroquinolone, with increased sp3-character to improve parasite selectivity. Relative to other cytochrome b inhibitors, JAG21 has improved solubility and ADMET properties, without need for pro-drug. JAG21 significantly reduces Toxoplasma gondii tachyzoites and encysted bradyzoites in vitro, and in primary and established chronic murine infections. Moreover, JAG21 treatment leads to 100% survival. Further, JAG21 is efficacious against drug-resistant Plasmodium falciparum in vitro. Causal prophylaxis and radical cure are achieved after P. berghei sporozoite infection with oral administration of a single dose (2.5 mg/kg) or 3 days treatment at reduced dose (0.625 mg/kg/day), eliminating parasitemia, and leading to 100% survival. Enzymatic, binding, and co-crystallography/pharmacophore studies demonstrate selectivity for apicomplexan relative to mammalian enzymes. JAG21 has significant promise as a pre-clinical candidate for prevention, treatment, and cure of toxoplasmosis and malaria.

Twenty-two compounds belonging to several classes of polyamine analogs have been examined for their ability to inhibit the growth of the human malaria parasite Plasmodium falciparum in vitro and in vivo. Four lead compounds from the thiourea sub-series and one compound from the urea-based analogs were found to be potent inhibitors of both chloroquine-resistant (Dd2) and chloroquine-sensitive (3D7) strains of Plasmodium with IC50 values ranging from 150 to 460 nM. In addition, the compound RHW, N1,N7-bis (3-(cyclohexylmethylamino) propyl) heptane-1,7-diamine tetrabromide was found to inhibit Dd2 with an IC50 of 200 nM. When RHW was administered to P. yoelii-infected mice at 35 mg/kg for 4 days, it significantly reduced parasitemia. RHW was also assayed in combination with the ornithine decarboxylase inhibitor difluoromethylornithine, and the two drugs were found not to have synergistic antimalarial activity. Furthermore, these inhibitors led to decreased cellular spermidine and spermine levels in P. falciparum, suggesting that they exert their antimalarial activities by inhibition of spermidine synthase.

Toxoplasma gondii, an Apicomplexan parasite, causes significant morbidity and mortality, including severe disease in immunocompromised hosts and devastating congenital disease, with no effective treatment for the bradyzoite stage. To address this, we used the Tropical Disease Research database, crystallography, molecular modeling, and antisense to identify and characterize a range of potential therapeutic targets for toxoplasmosis. Phosphoglycerate mutase II (PGMII), nucleoside diphosphate kinase (NDK), ribulose phosphate 3-epimerase (RPE), ribose-5-phosphate isomerase (RPI), and ornithine aminotransferase (OAT) were structurally characterized. Crystallography revealed insights into the overall structure, protein oligomeric states and molecular details of active sites important for ligand recognition. Literature and molecular modeling suggested potential inhibitors and druggability. The targets were further studied with vivoPMO to interrupt enzyme synthesis, identifying the targets as potentially important to parasitic replication and, therefore, of therapeutic interest. Targeted vivoPMO resulted in statistically significant perturbation of parasite replication without concomitant host cell toxicity, consistent with a previous CRISPR/Cas9 screen showing PGM, RPE, and RPI contribute to parasite fitness. PGM, RPE, and RPI have the greatest promise for affecting replication in tachyzoites. These targets are shared between other medically important parasites and may have wider therapeutic potential.