Structure-Guided Design of Calcium-Dependent Protein Kinase 1 Inhibitors for 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 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.