Pyridopyrimidinones as Cryptosporidium parvum Calcium Dependent Protein Kinase 1 (CpCDPK1) Inhibitors

Cryptosporidium parvum, Cp, is a protozoan parasite that can cause life-threatening diarrhea. Given that only one drug is currently FDA approved for the treatment of cryptosporidiosis but with only limited efficacy, new medications are needed. In this dissertation compounds directed towards tRNA synthetase and calcium dependent protein kinase 1 (CDPK1), two promising molecular targets in the field, were assessed. In the case of tRNA synthetase, a collection of previously identified eukaryotic and prokaryotic tRNA synthetase inhibitors were screened for Cp growth inhibition in HCT-8 host cells. Three compounds, halofuginone, borrelidin and AN3661, were found to potently block Cp growth. A limited structure-activity relationship (SAR) analysis of AN3361 found that the propionic acid moiety present in the compound is critical for potent anti-Cryptosporidium activity. In the case of CpCDPK1, a diversified set of known protein kinases inhibitors were similarly assessed for Cp growth inhibition. This exercise identified pyridopyrimidones as a new chemotype of potent Cp growth inhibitors that were confirmed to block CpCDPK1 kinase activity. SAR analysis provided further insights into two structural features for achieving potent CpCDPK1 kinase and Cp growth inhibition, namely appropriate occupancy of the ATP binding pocket near the C-helix and the presence of a hydrogen bond acceptor in the solvent exposed portion of the inhibitor. A promising derivative, UH15_16, potently inhibited Cp growth (EC50 = 14 nM) and CpCDPK1 kinase (IC50 = 5.4 nM) activities, as well as displayed moderate selectivity based on kinase profiling. UH15_16 formulated as a 2:1 mixture with Captisol® in water demonstrated limited systemic exposure following oral administration, was well tolerated at 50 mg/kg once per day dosing for 5 days in mice and resulted in a 75% reduced burden of the gut pathogen at 50 mg/kg once per day dosing for 7 days in an acute mouse model of cryptosporidiosis. The third formulation (5x Captisol with 2% Tween 80 at ...