title: Selective inhibition of Sarcocystis neurona calcium-dependent protein kinase 1 for equine protozoal myeloencephalitis therapy.
abstract: Sarcocystis neurona is the most frequent cause of equine protozoal myeloencephalitis, a debilitating neurological disease of horses that can be difficult to treat. We identified SnCDPK1, the S. neurona homologue of calcium-dependent protein kinase 1 (CDPK1), a validated drug target in Toxoplasma gondii. SnCDPK1 shares the glycine "gatekeeper" residue of the well-characterized T. gondii enzyme, which allows the latter to be targeted by bumped kinase inhibitors. This study presents detailed molecular and phenotypic evidence that SnCDPK1 can be targeted for rational drug development. Recombinant SnCDPK1 was tested against four bumped kinase inhibitors shown to potently inhibit both T. gondii (Tg) CDPK1 and T. gondii tachyzoite growth. SnCDPK1 was inhibited by low nanomolar concentrations of these BKIs and S. neurona growth was inhibited at 40-120nM concentrations. Thermal shift assays confirmed these bumped kinase inhibitors bind CDPK1 in S. neurona cell lysates. Treatment with bumped kinase inhibitors before or after invasion suggests that bumped kinase inhibitors interfere with S. neurona mammalian host cell invasion in the 0.5-2.5μM range but interfere with intracellular division at 2.5μM. In vivo proof-of-concept experiments were performed in a murine model of S. neurona infection. The experimental infected groups treated for 30days with compound BKI-1553 (n=10 mice) had no signs of disease, while the infected control group had severe signs and symptoms of infection. Elevated antibody responses were found in 100% of control infected animals, but only 20% of BKI-1553 treated infected animals. Parasites were found in brain tissues of 100% of the control infected animals, but only in 10% of the BKI-1553 treated animals. The bumped kinase inhibitors used in these assays have been chemically optimized for potency, selectivity and pharmacokinetic properties, and hence are good candidates for treatment of equine protozoal myeloencephalitis.
authors.affiliation: Department of Medicine, Division of Allergy and Infectious Disease, Center for Emerging and Reemerging Infectious Disease (CERID), University of Washington, Seattle, WA 98109, USA. Electronic address: ojo67kk@u.washington.edu.
authors.forename: Kayode K
authors.initials: KK
authors.lastname: Ojo
authors.affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA.
authors.forename: Sriveny
authors.initials: S
authors.lastname: Dangoudoubiyam
authors.affiliation: United States Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Parasitic Diseases Laboratory, Beltsville, MD 20705, USA.
authors.forename: Shiv K
authors.initials: SK
authors.lastname: Verma
authors.affiliation: Center for Infectious Disease Research, Seattle, WA 98109, USA.
authors.forename: Suzanne
authors.initials: S
authors.lastname: Scheele
authors.affiliation: Center for Infectious Disease Research, Seattle, WA 98109, USA.
authors.forename: Amy E
authors.initials: AE
authors.lastname: DeRocher
authors.affiliation: Department of Veterinary Science, University of Kentucky, Lexington, KY 40546, USA.
authors.forename: Michelle
authors.initials: M
authors.lastname: Yeargan
authors.affiliation: Department of Medicine, Division of Allergy and Infectious Disease, Center for Emerging and Reemerging Infectious Disease (CERID), University of Washington, Seattle, WA 98109, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Choi
authors.affiliation: Department of Medicine, Division of Allergy and Infectious Disease, Center for Emerging and Reemerging Infectious Disease (CERID), University of Washington, Seattle, WA 98109, USA.
authors.forename: Tess R
authors.initials: TR
authors.lastname: Smith
authors.affiliation: Department of Medicine, Division of Allergy and Infectious Disease, Center for Emerging and Reemerging Infectious Disease (CERID), University of Washington, Seattle, WA 98109, USA.
authors.forename: Kasey L
authors.initials: KL
authors.lastname: Rivas
authors.affiliation: Department of Medicine, Division of Allergy and Infectious Disease, Center for Emerging and Reemerging Infectious Disease (CERID), University of Washington, Seattle, WA 98109, USA.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Hulverson
authors.affiliation: Department of Medicine, Division of Allergy and Infectious Disease, Center for Emerging and Reemerging Infectious Disease (CERID), University of Washington, Seattle, WA 98109, USA.
authors.forename: Lynn K
authors.initials: LK
authors.lastname: Barrett
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
authors.forename: Erkang
authors.initials: E
authors.lastname: Fan
authors.affiliation: Department of Chemistry, University of Washington, Seattle, WA 98195, USA.
authors.forename: Dustin J
authors.initials: DJ
authors.lastname: Maly
authors.affiliation: Center for Infectious Disease Research, Seattle, WA 98109, USA; Department of Global Health, University of Washington, Seattle, WA 98195, USA.
authors.forename: Marilyn
authors.initials: M
authors.lastname: Parsons
authors.affiliation: United States Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Parasitic Diseases Laboratory, Beltsville, MD 20705, USA.
authors.forename: Jitender P
authors.initials: JP
authors.lastname: Dubey
authors.affiliation: Department of Veterinary Science, University of Kentucky, Lexington, KY 40546, USA. Electronic address: daniel.howe@uky.edu.
authors.forename: Daniel K
authors.initials: DK
authors.lastname: Howe
authors.affiliation: Department of Medicine, Division of Allergy and Infectious Disease, Center for Emerging and Reemerging Infectious Disease (CERID), University of Washington, Seattle, WA 98109, USA; Department of Global Health, University of Washington, Seattle, WA 98195, USA. Electronic address: wesley@uw.edu.
authors.forename: Wesley C
authors.initials: WC
authors.lastname: Van Voorhis
mesh_terms: D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D004679:Encephalomyelitis / Q000188:drug therapy* / Q000469:parasitology; D005260:Female; D006734:Horse Diseases / Q000188:drug therapy / Q000469:parasitology; D006736:Horses; D007371:Interferon-gamma / Q000235:genetics; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use*; D011494:Protein Kinases / Q000187:drug effects*; D011817:Rabbits; D012522:Sarcocystis / Q000187:drug effects / Q000201:enzymology*; D012523:Sarcocystosis / Q000188:drug therapy*; D013696:Temperature; D014122:Toxoplasma / Q000187:drug effects / Q000201:enzymology; D017871:Calcium-Calmodulin-Dependent Protein Kinases
chemical_list: D007371:Interferon-gamma; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases
keywords: Bumped kinase inhibitors; Calcium-dependent protein kinase 1; Drug target; Equine protozoal myeloencephalitis; Gatekeeper residue; Sarcocystis neurona