Marvin J. Meyers

Cryptosporidium parasites are a leading cause of waterborne disease worldwide, causing life-threatening diarrhea and malnutrition in young children and people with HIV. Currently, nitazoxanide is the only FDA-approved treatment, but it is largely ineffective in severely immunocompromised patients, highlighting the need for new therapies. Here, we use a ligand-based virtual screening workflow that integrates Thompson Sampling, a pharmacophore model, and a machine learning classifier to screen a subset of Enamine’s REAL database comprising 51 million virtual compounds. Across two screening campaigns, 20 novel micromolar hits were identified among 69 tested compounds (29%), eight of which exhibited confirmed activity at or below 21 μM. These findings expand the chemical diversity of the original series by introducing new tail and head chemotypes and demonstrate the effectiveness of active learning strategies for improving ligand-based virtual screening performance.

Cryptosporidiosis is a major cause of diarrheal disease worldwide, particularly in young children and immunocompromised individuals, yet effective therapeutic options remain limited. Previously, the triazolopyridazine derivative SLU-10482 was identified as a potent anti-Cryptosporidium lead, but it suffers from modest affinity for the hERG ion channel. In the present study, structure–activity relationships (SAR) were investigated through systematic modification of the 3-position of the aryl ring and by designing gutrestricted analogues intended to reduce systemic exposure while maintaining antiparasitic activity. A diverse library of analogues was synthesized using amide coupling, Sonogashira, Suzuki, and Buchwald–Hartwig cross-coupling reactions, followed by hydrogenation and late-stage functional group transformations to access alkynyl, alkyl, tertiary amine, quaternary ammonium, and substituted aryl derivatives. Compounds were evaluated for anti-Cryptosporidium parvum activity in infected HCT-8 cells. SAR studies revealed that smaller, less polar substituents at the 3-position were generally associated with improved potency, whereas increasing substituent size and polarity resulted in diminished activity, although several larger polar analogues retained measurable potency. To develop gut-restricted agents, molecular weight, polarity, and conformational flexibility were intentionally increased to reduce intestinal permeability and systemic exposure. While these modifications were expected to preserve local intestinal activity, the resulting analogues exhibited substantially reduced antiparasitic potency. Because the molecular target of this chemotype remains unknown, the observed loss of activity may arise from reduced target engagement, impaired penetration across the parasite membrane barriers, or a combination of both.

Cryptosporidiosis is a major cause of life-threatening diarrhea in children and chronic diarrhea in immunocompromised individuals. Our previous work identified pyrazolo[3,4-d]pyrimidine benzoxaborole (2) as an orally efficacious inhibitor of Cryptosporidium with a downside of relapse after 7 days post drug treatment. Here, we report a structure–activity relationship study around the benzoxaborole ring of compound 2, resulting in rac- 11 with improved in vitro potency (EC50 = 0.025 μM). Remarkably, rac- 11 is noncytotoxic and displayed curative in vivo efficacy in Cryptosporidium-infected immunocompromised NSG mice. Furthermore, resolution of rac-11 into its enantiomers demonstrated that one enantiomer is 2-fold more potent (ent2-11; EC50 = 0.011 μM), whereas the other is 400-fold less potent, highlighting the importance of the methyl group at the C3 position of benzoxaborole. The ent2-11 also had reduced adverse binding to hPDE5 compared to 2. Collectively, these findings advance new and effective candidates for the treatment of cryptosporidiosis.

Toxoplasmosis, caused by the protozoan Toxoplasma gondii , affects nearly one-third of the global population and may result in severe congenital, ocular, and neurological manifestations. Current therapies are limited by toxicity, poor efficacy against chronic infection, and lack of activity against tissue cysts, highlighting the need for new therapeutic strategies. Aspartic proteases represent promising but underexplored drug targets in T. gondii . In this study, we evaluated the anti- T . gondii potential of a panel of aspartic protease inhibitors initially developed for inhibition of Plasmodium . Eleven compounds were screened in vitro against intracellular tachyzoites (RH strain) in human foreskin fibroblasts (HFF), and their cytotoxicity was assessed to determine EC ₅₀ , CC ₅₀ , and selectivity indices. Most compounds displayed micromolar activity (EC ₅₀ range: 1.06–75.86 µM), with CWHM-032 (=TCMDC-134675), CWHM-033 (=TCMDC-136879), and CWHM-117 emerging as the most potent inhibitors. Based on its in vitro selective activity, predicted pharmacokinetic and safety profile, and previously reported efficacy against experimental malaria, CWHM-117 was selected for in vivo evaluation. In an acute murine model of toxoplasmosis, treatment with CWHM-117 delayed mortality compared to the vehicle-treated group. In a chronic infection model, CWHM-117 significantly reduced cerebral cyst burden ( P < 0.05), demonstrating activity against the bradyzoite stage, which remains a major therapeutic challenge. Overall, these findings indicate that aspartic protease inhibitors, particularly CWHM-117, represent promising lead compounds for the treatment of toxoplasmosis. This study supports T. gondii aspartic proteases as druggable targets and encourages further optimization and mechanistic studies to advance this class of compounds toward preclinical development.

Egress from host cells is fundamental for the spread of infection by apicomplexan parasites, including Babesia species. These tick-borne pathogens represent emerging zoonoses, but treatment options are limited. Here, using microscopy, transcriptomics and chemical genetics, we identified signalling, proteases and gliding motility as key drivers of egress by Babesia divergens . We developed reverse genetic tools in B. divergens to perform a knockdown screen of putative mediators of egress, identifying kinases and proteases involved in distinct steps of egress (aspartyl protease (ASP) 3 and kinases cGMP-dependent protein kinase (PKG) and calcium-dependent protein kinase (CDPK4)) and invasion (ASP2, ASP3 and PKG) of red blood cells. Inhibition of egress stimulates additional rounds of intracellular replication, indicating that exit from the replication cycle is uncoupled from egress. Chemical genetics validated PKG, CDPK4, ASP2 and ASP3 as druggable targets in Babesia spp. and identified promising compounds for babesiosis treatment. Taken together, egress in B. divergens more closely resembles egress in Toxoplasma gondii than in the more evolutionarily related Plasmodium spp.

Malaria is a parasitic disease caused by the parasite Plasmodium which results in hundreds of thousands of deaths per year. The need for new drugs for the treatment of this disease is imperative as new resistant strains of Plasmodium emerge. Previously, our group reported two related series of pyrrolidine‐based antimalarial compounds. While both series were found to be orally efficacious in a mouse infection model, some analogues also showed modest affinities for the hERG ion channel. Herein, we describe the attempted synthesis of β‐fluoro substituted pyrrolidine analogues of both chemotypes. In this work, we observed position‐dependent chemical instability of fluorine on the pyrrolidine ring. β‐fluoro substituted pyrrolidine (±)‐ 3 was tested in the P. falciparum 3D7 assay and showed a 40‐fold loss of anti‐ Plasmodium potency with little change in weak hERG affinity at 10 µM. In an effort to expand the utility of this work, pyrrolidine (±)‐ 1 and fluoropyrrolidine (±)‐ 3 were also tested against the related apicomplexan parasite Cryptosporidium parvum which causes cryptosporidiosis. Both compounds showed modest potency (EC ₅₀ = 10 µM and 12 µM, respectively), implying this series could be further optimized for treatment for cryptosporidiosis.

Cryptosporidiosis is a diarrheal disease caused by the parasite Cryptosporidium resulting in over 100,000 deaths annually. Here, we present a structure-activity relationship study of the benzoic acid position (R⁶) of pyrazolo[3,4-d]pyrimidine lead SLU-2815 (1), an inhibitor of parasite phosphodiesterase CpPDE1, resulting in the discovery of benzoxaborole SLU-10906 (63) as a benzoic acid bioisostere. Benzoxaborole 63 is 10-fold more potent than 1 against the parasite in a cell-based infection model (EC₅₀ = 0.19 μM) and non-cytotoxic. Furthermore, 63 has a fast rate of parasite-killing and is orally efficacious in a Cryptosporidium mouse infection model (50 mg/kg BID), although relapse was observed 7 days post-drug treatment. The partial selectivity profile versus human phosphodiesterases is preserved with the benzoxaborole motif and represents an important feature to improve in future optimization. Benzoxaborole 63 represents an important advance toward the optimization of the pyrazolo[3,4-d]pyrimidine series and the identification of a drug to treat cryptosporidiosis.