Selective inhibition of Sarcocystis neurona calcium-dependent protein kinase 1 for equine protozoal myeloencephalitis therapy

Kayode K Ojo; Sriveny Dangoudoubiyam; Shiv K Verma; Suzanne Scheele; Amy E DeRocher; Michelle Yeargan; Ryan Choi; Tess R Smith; Kasey L Rivas; Matthew A Hulverson; Lynn K Barrett; Erkang Fan; Dustin J Maly; Marilyn Parsons; Jitender P Dubey; Daniel K Howe; Wesley C Van Voorhis

doi:10.1016/j.ijpara.2016.08.003

Selective inhibition of Sarcocystis neurona calcium-dependent protein kinase 1 for equine protozoal myeloencephalitis therapy

Int J Parasitol. 2016 Dec;46(13-14):871-880. doi: 10.1016/j.ijpara.2016.08.003. Epub 2016 Oct 8.

Authors

Kayode K Ojo¹, Sriveny Dangoudoubiyam², Shiv K Verma³, Suzanne Scheele⁴, Amy E DeRocher⁴, Michelle Yeargan⁵, Ryan Choi⁶, Tess R Smith⁶, Kasey L Rivas⁶, Matthew A Hulverson⁶, Lynn K Barrett⁶, Erkang Fan⁷, Dustin J Maly⁸, Marilyn Parsons⁹, Jitender P Dubey³, Daniel K Howe¹⁰, Wesley C Van Voorhis¹¹

Affiliations

¹ 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: [email protected].
² Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA.
³ United States Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Parasitic Diseases Laboratory, Beltsville, MD 20705, USA.
⁴ Center for Infectious Disease Research, Seattle, WA 98109, USA.
⁵ Department of Veterinary Science, University of Kentucky, Lexington, KY 40546, USA.
⁶ Department of Medicine, Division of Allergy and Infectious Disease, Center for Emerging and Reemerging Infectious Disease (CERID), University of Washington, Seattle, WA 98109, USA.
⁷ Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
⁸ Department of Chemistry, University of Washington, Seattle, WA 98195, USA.
⁹ Center for Infectious Disease Research, Seattle, WA 98109, USA; Department of Global Health, University of Washington, Seattle, WA 98195, USA.
¹⁰ Department of Veterinary Science, University of Kentucky, Lexington, KY 40546, USA. Electronic address: [email protected].
¹¹ 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: [email protected].

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.

Keywords: Bumped kinase inhibitors; Calcium-dependent protein kinase 1; Drug target; Equine protozoal myeloencephalitis; Gatekeeper residue; Sarcocystis neurona.

MeSH terms

Animals
Cell Line
Chlorocebus aethiops
Encephalomyelitis / drug therapy*
Encephalomyelitis / parasitology
Female
Horse Diseases / drug therapy
Horse Diseases / parasitology
Horses
Interferon-gamma / genetics
Male
Mice
Mice, Knockout
Protein Kinase Inhibitors / chemistry
Protein Kinase Inhibitors / pharmacology
Protein Kinase Inhibitors / therapeutic use*
Protein Kinases / drug effects*
Rabbits
Sarcocystis / drug effects
Sarcocystis / enzymology*
Sarcocystosis / drug therapy*
Temperature
Toxoplasma / drug effects
Toxoplasma / enzymology

Substances

Protein Kinase Inhibitors
Interferon-gamma
Protein Kinases
calcium-dependent protein kinase

Abstract

MeSH terms

Substances

Grants and funding