Abstract
Despite the enormous economic importance of Neospora caninum related veterinary diseases, the number of effective therapeutic agents is relatively small. Development of new therapeutic strategies to combat the economic impact of neosporosis remains an important scientific endeavor. This study demonstrates molecular, structural and phenotypic evidence that N. caninum calcium-dependent protein kinase 1 (NcCDPK1) is a promising molecular target for neosporosis drug development. Recombinant NcCDPK1 was expressed, purified and screened against a select group of bumped kinase inhibitors (BKIs) previously shown to have low IC50s against Toxoplasma gondii CDPK1 and T. gondii tachyzoites. NcCDPK1 was inhibited by low concentrations of BKIs. The three-dimensional structure of NcCDPK1 in complex with BKIs was studied crystallographically. The BKI-NcCDPK1 structures demonstrated the structural basis for potency and selectivity. Calcium-dependent conformational changes in solution as characterized by small-angle X-ray scattering are consistent with previous structures in low Calcium-state but different in the Calcium-bound active state than predicted by X-ray crystallography. BKIs effectively inhibited N. caninum tachyzoite proliferation in vitro. Electron microscopic analysis of N. caninum cells revealed ultra-structural changes in the presence of BKI compound 1294. BKI compound 1294 interfered with an early step in Neospora tachyzoite host cell invasion and egress. Prolonged incubation in the presence of 1294 interfered produced observable interference with viability and replication. Oral dosing of BKI compound 1294 at 50 mg/kg for 5 days in established murine neosporosis resulted in a 10-fold reduced cerebral parasite burden compared to untreated control. Further experiments are needed to determine the PK, optimal dosage, and duration for effective treatment in cattle and dogs, but these data demonstrate proof-of-concept for BKIs, and 1294 specifically, for therapy of bovine and canine neosporosis.
Highlights
The apicomplexan parasite, Neospora caninum, is the leading cause of epidemic abortion in cattle and is a frequent cause of neuromuscular diseases in dogs
The seven bumped kinase inhibitors (BKIs) with 1 to 3 nM IC50s for inhibiting NcCDPK1 enzyme activity inhibit the growth of N. caninum tachyzoites in vitro with EC50 values in the range of 49 to 140 nM
Proliferation occurs after successful invasion of a host cell by a complex process of coordinated secretion of proteins regulated in part by changes in intracellular calcium concentrations
Summary
The apicomplexan parasite, Neospora caninum, is the leading cause of epidemic abortion in cattle and is a frequent cause of neuromuscular diseases in dogs. More recent trials revealed fewer efficacies in the field, and even suggested that vaccination may increase the risk of early embryonic death [7]. Chemotherapy could provide a viable alternative if appropriate compounds are identified Both in vivo and in vitro studies have been performed to determine efficacy of treatments with lasalocid, monensin, pirithrexim, pyrimethamine, clindamycin, robenidine and trimethoprim [8], artemisinin and artemisone [9], [10], depudecin [11], toltrazuril, and ponazuril [12] none of these studies showed that treatments were effective in cattle. To date there is no approved vaccine and no approved treatment for cattle that are infected with N. caninum
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