title: Neospora caninum calcium-dependent protein kinase 1 is an effective drug target for neosporosis therapy.
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.
authors.affiliation: Center for Emerging and Re-emerging Infectious Diseases (CERID), Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, Washington, United States of America.
authors.forename: Kayode K
authors.initials: KK
authors.lastname: Ojo
authors.affiliation: Center for Emerging and Re-emerging Infectious Diseases (CERID), Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, Washington, United States of America.
authors.forename: Molly C
authors.initials: MC
authors.lastname: Reid
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.
authors.forename: Latha
authors.initials: L
authors.lastname: Kallur Siddaramaiah
authors.affiliation: Institute of Parasitology, Vetsuisse Faculty, University of Berne, Berne, Switzerland.
authors.forename: Joachim
authors.initials: J
authors.lastname: Müller
authors.affiliation: Institute of Parasitology, Vetsuisse Faculty, University of Berne, Berne, Switzerland.
authors.forename: Pablo
authors.initials: P
authors.lastname: Winzer
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.
authors.forename: Zhongsheng
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Center for Emerging and Re-emerging Infectious Diseases (CERID), Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, Washington, United States of America.
authors.forename: Katelyn R
authors.initials: KR
authors.lastname: Keyloun
authors.affiliation: Department of Chemistry, University of Washington, Seattle, Washington, United States of America.
authors.forename: Rama Subba Rao
authors.initials: RS
authors.lastname: Vidadala
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.
authors.forename: Ethan A
authors.initials: EA
authors.lastname: Merritt
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.
authors.forename: Wim G J
authors.initials: WG
authors.lastname: Hol
authors.affiliation: Department of Chemistry, University of Washington, Seattle, Washington, United States of America.
authors.forename: Dustin J
authors.initials: DJ
authors.lastname: Maly
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.
authors.forename: Erkang
authors.initials: E
authors.lastname: Fan
authors.affiliation: Center for Emerging and Re-emerging Infectious Diseases (CERID), Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, Washington, United States of America.
authors.forename: Wesley C
authors.initials: WC
authors.lastname: Van Voorhis
authors.affiliation: Institute of Parasitology, Vetsuisse Faculty, University of Berne, Berne, Switzerland.
authors.forename: Andrew
authors.initials: A
authors.lastname: Hemphill
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray / Q000379:methods; D016503:Drug Delivery Systems / Q000379:methods; D055808:Drug Discovery / Q000379:methods; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D018688:Neospora / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D014122:Toxoplasma / Q000187:drug effects / Q000378:metabolism*; D014123:Toxoplasmosis / Q000188:drug therapy; D017871:Calcium-Calmodulin-Dependent Protein Kinases
chemical_list: D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases