Figure S3 shows competitive proteomic profiling of 1553-N-Me.

Figure S8 shows effects of BKIDC-1553 and 1553-N-Me on intracellular ATP levels in LNCaP, PC3, and DU145 cells.

Cryptosporidium parvum, an apicomplexan parasite and the causative agent of cryptosporidiosis, contributes to a high burden in mortality and morbidity for humans and livestock. Currently, there are no reliably successful parasite-specific treatments for the debilitating diarrhea associated with the infection. Bumped kinase inhibitors (BKIs), which selectively target parasite calcium-dependent protein kinases (CDPKs), have been shown to decrease infections caused by several medical and veterinary-relevant parasites, including Toxoplasma gondii, Plasmodium falciparum, and Cryptosporidium parvum. In the present study, various dosing regimens of BKI-1708 were evaluated for safety and clinical efficacy in the calf model for cryptosporidiosis, specifically with the aim of finding a minimum effective dose. The majority of the different BKI dosages produced notable improvements across nearly all clinical and parasitological measures, including diarrhea severity, oocyst shedding, and overall health status. These results provide strong evidence for advancing BKI-1708 as a preclinical candidate for treatment of cryptosporidiosis.

Table S7 shows pharmacokinetic parameters after a single dose in mouse, rat, dog, and cynomolgus monkey.

Figure S15 shows measured and simulated plasma levels in SCID mice.

Figure S2 shows crystal violet-based cell proliferation assay.

Figure S5 shows cell cycle profiles and histograms of DAPI-stained cells with BKIDCs.

Figure S12 shows antiproliferative activity of BKIDCs on HK1 and HK2 KO LNCaP cell lines.

Figure S7 shows Western blot analysis confirming the findings with RPPA.

Table S1 shows BKIDC-1553 intratumoral concentrations after at least 4 weeks of thrice weekly oral dosing at 20 mg/kg.

Background Calcium-dependent protein kinase 1 (CDPK1) has emerged as a protozoan-specific target for the treatment of cryptosporidiosis. A previous study identified pyridopyrimidinones as new Cryptosporidium parvum CDPK1 (CpCDPK1) inhibitors with potent growth inhibition against C. parvum and Cryptosporidium hominis. Docking analyses suggested the unique positioning of the kinase's alpha C-helix could present refinement opportunities.Methods Compounds designed to optimize the pyridopyrimidinones focused on the back-pocket region predicted to be proximal to the alpha C-helix, the solvent-exposed region, and the ATP ribose-binding site. Designed derivatives were synthesized and assessed for CpCDPK1 and Src kinase inhibition and for Cryptosporidium spp growth inhibition in mammalian cells. AMP/Mg+2 and 3 inhibitors were cocrystalized with CpCDPK1, and 2 inhibitors were profiled for kinase selectivity.Results WIN 4-88 was identified with CpCDPK1 (half maximum inhibitory concentration [IC50] = 0.056 mu M), and growth inhibition of zoonotic C. parvum NLuc (half maximum effective concentration [EC50] = 0.042 mu M), anthroponotic C. parvum Tu114 (EC50 = 0.030 mu M), and C. hominis Tu502 (EC50 = 0.062 mu M), as well as enhanced kinome selectivity. The crystal structures confirmed the predicted binding mode, indicating key interactions with hinge residue Y155, similar orientations of the solvent-expose moieties, occupancy of the back pocket near the alpha C-helix, and for 1 inhibitor containing a solubilizing hydroxyethyl attached to the central heterocycle extension into the ATP ribose-binding site.Conclusions The expanded structure-activity relationship and structural insights will potentially be applicable to other chemotypes with similar binding modes and will enhance development of CpCDPK1 inhibitors for the treatment of cryptosporidiosis. Expanded structure-activity relationship and structural analyses provide new insights into the design of potent and selective CpCDPK1 inhibitors that effectively block growth of zoonotic C. parvum and anthroponotic C. parvum and C. hominis for the treatment of cryptosporidiosis.

BACKGROUND:Calcium-dependent protein kinase 1 (CDPK1) has emerged as a protozoan specific target for the treatment of cryptosporidiosis. A previous study identified pyridopyrimidinones as new Cryptosporidium parvum (Cp) CDPK1 inhibitors with potent growth inhibition against C. parvum and C. hominis. Docking analyses suggested the unique positioning of the kinase's αC-helix could present refinement opportunities. METHODS:Compounds designed to optimize the pyridopyrimidinones focused on the back-pocket region predicted to be proximal to the αC-helix, the solvent exposed region and the ATP ribose-binding site. Designed derivatives were synthesized and assessed for CpCDPK1 and Src kinase inhibition and for Cryptosporidium spp., growth inhibition in mammalian cells. AMP/Mg+2 and three inhibitors were co-crystalized with CpCDPK1, and two inhibitors were profiled for kinase selectivity. RESULTS:WIN 4-88 was identified with CpCDPK1 (IC50 = 0.056 μM), and growth inhibition of zoonotic C. parvum (NLuc EC50 = 0.042 μM), anthroponotic C. parvum (Tu114 EC50 = 0.030 μM), and C. hominis Tu502 (EC50 = 0.062 μM), as well as enhanced kinome selectivity. The crystal structures confirmed the predicted binding mode, indicating key interactions with hinge residue Y155, similar orientations of the solvent expose moieties, occupancy of the back-pocket near the αC-helix and for one inhibitor containing a solubilizing hydroxyethyl attached to the central heterocycle extension into the ATP ribose-binding site. CONCLUSIONS:The expanded structure-activity relationship and structural insights will potentially be applicable to other chemotypes with similar binding modes and will enhance development of CpCDPK1 inhibitors for the treatment of cryptosporidiosis.

Table S5 shows antibodies and corresponding RPPA signal intensities relative to β-actin signals.

Supplementary Image shows uncropped Western blot images.

BackgroundUnlike mouse models of congenital toxoplasmosis, pregnant sheep models provide the opportunity to evaluate treatment strategies that more closely resemble clinical practice in pregnant women, including chemotherapeutic interventions initiated after specific IgM seroconversion. BKI-1748, which targets Toxoplasma gondii CDPK1 and MAPKL-1 protein kinases, has demonstrated an excellent safety profile and efficacy when administered repeatedly to pregnant sheep, starting at 2 and 7 days after challenge.MethodsIn this study, treatment was initiated at day 14 post-infection (p.i.), following T. gondii IgM seroconversion. Twenty-three sheep were orally inoculated with 10 TgShSp1 oocysts at 90 days of gestation, while three sheep remained uninfected. On day 14 p.i., infected sheep carrying live fetuses (n = 10) received 10 doses of BKI-1748 orally at 15 mg/kg every 2 days, whereas 10 infected sheep were left untreated.ResultsAll infected sheep, both treated and untreated, seroconverted to serum IgG by day 21 p.i., with treated sheep showing a marked reduction in IgG levels from day 28 p.i. onward. Administration of the compound significantly enhanced lamb viability in infected sheep, resulting in 91% viable lambs in treated animals compared to 52% in untreated sheep. Whereas all lambs born to untreated sheep were congenitally infected, only 17% of lambs in the treated group were infected. Nevertheless, congenitally infected lambs in the treated group had lower birth weights than T. gondii-free lambs.ConclusionThis study highlights the potential utility of BKI-1748 for prenatal treatment of human congenital toxoplasmosis, in which IgM seroconversion prompts the need for intervention.

Figure S9 shows cooperation of BKIDCs with oligomycin to induce apoptotic cell death.

Table S2 shows rat cardiovascular assessment (nonGLP) after IV administration of increasing dosages of BKIDC-1553.

Figure S4 shows dose response of multiple cancer cell lines to BKIDC-1553-N-Me and 1817.

Figure S16 shows metabolites of BKIDC-1553 produced by liver microsomes from rats, dogs, and humans.

Figure S10 shows measurement of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the Seahorse assay.