Novel Bumped Kinase Inhibitors Are Safe and Effective Therapeutics in the Calf Clinical Model for Cryptosporidiosis

Abstract

Cryptosporidiosis, caused by the apicomplexan parasite Cryptosporidium parvum, is a diarrheal disease that has produced a large global burden in mortality and morbidity in humans and livestock. There are currently no consistently effective parasite-specific pharmaceuticals available for this disease. Bumped kinase inhibitors (BKIs) specific for parasite calcium-dependent protein kinases (CDPKs) have been shown to reduce infection in several parasites having medical and veterinary importance, including Toxoplasma gondii, Plasmodium falciparum, and C. parvum. In the present study, BKIs were screened for efficacy against C. parvum infection in the neonatal mouse model. Three BKIs were then selected for safety and clinical efficacy evaluation in the calf model for cryptosporidiosis. Significant BKI treatment effects were observed for virtually all clinical and parasitological scoring parameters, including diarrhea severity, oocyst shedding, and overall health. These results provide proof of concept for BKIs as therapeutic drug leads in an animal model for human cryptosporidiosis.

Advances in bumped kinase inhibitors for human and animal therapy for cryptosporidiosis

“…Methods for the propagation of C. parvum (IOWA-II strain) oocysts in calves (Riggs and Perryman, 1987; Riggs et al, 1989), propagation of transgenic Nanoluciferase (Nluc) expressing C. parvum (UGA1 strain) oocysts in mice (Vinayak et al, 2015; Hulverson et al, 2017), in vitro microsomal stability (Tatipaka et al, 2014), pharmacokinetic analysis of mouse plasma, faecal, and urine BKI concentrations by LC-MS/MS analysis (Ojo et al, 2012; Schaefer et al, 2016; Hulverson et al, 2017), in vitro protein binding using dialysis membranes (Tatipaka et al, 2014), Nluc expressing C. parvum in vitro growth inhibition in infected HCT-8 cells (Hulverson et al, 2017), in vitro cytotoxicity in CRL-8155 and HepG2 cells (Tatipaka et al, 2014), and in vivo mouse gastrointestinal (GI) tract tissue exposure by LC-MS/MS analysis (Arnold et al, 2017) have all been previously described. Detailed descriptions of these methods are included in Supplementary Data S1.…”

“…Detailed descriptions of these methods are included in Supplementary Data S1. Methods for high-content imaging of Cryptosporidium proliferation in HCT-8 cells (Love et al, 2017), QPatch hERG (Danker and Moller, 2014), rat cardiovascular screening (Banfor et al, 2016), in vitro micronucleus assay (Nicolette et al, 2011), the 24-well Ames screening assay, kinome profiling (Goedken et al, 2015), Nluc C. parvum in infected adult IFN-γ KO mouse efficacy (Hulverson et al, 2017), Gastroplus (Simulations Plus, Inc., Lancaster, CA, USA) modelling of efficacy and GI tissue and lumen exposures (Arnold et al, 2017), and IOWA-II strain C. parvum in infected neonatal calf efficacy (Schaefer et al, 2016) have all been previously described. All LC-MS/MS analytes were measured with an Acquity ultra performance liquid chro6 matography (UPLC) system in tandem with a Xevo TQ-S mass spectrometer (Waters, Milford, MA, USA).…”

5-Aminopyrazole-4-Carboxamide-Based Compounds Prevent the Growth of Cryptosporidium parvum

“…Several studies explored the activity of bumped kinase inhibitors (BKIs) on C. parvum calcium-dependent protein kinase 1 (CpCDPK1). Pyrazolopyrimidine (PP-BKIs) and 5-aminopyrazole-4-carboxamide (AC-BKIs) have been shown to block the activity of CpCDPK1 (7,8), prevent the growth and proliferation of C. parvum in vitro (9), and reduce oocyst shedding in infected SCID beige mice (10,11). Furthermore, BKIs 1 (compound 1294, PP), 2 (compound 1517, AC), and 3 (compound 1553, PP) were effective for treatment of clinical diarrhea and reduction of oocyst excretion in the neonatal calf C. parvum challenge model ( Fig.…”

“…Furthermore, BKIs 1 (compound 1294, PP), 2 (compound 1517, AC), and 3 (compound 1553, PP) were effective for treatment of clinical diarrhea and reduction of oocyst excretion in the neonatal calf C. parvum challenge model ( Fig. 1) (9). Based on the cited studies, additional AC analogues were screened for activity against CpCDPK1 and inhibition of C. parvum growth.…”

“…Methods for solubility testing, inhibition of CpCDPK1 and C. parvum cellular growth, drug pharmacokinetic and fecal concentration analysis, plasma protein binding, and determination of efficacy in neonatal mice were performed as previously described (9 Fig. 1).…”