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

Authors: OJO, KAYODE - University Of Washington; DANGOUDOUBIYAM, SRIVENY - Texas A&M University; VERMA, SHIV - Non ARS Employee; SCHEELE, SUZANNE - Center For Infectious Disease Research; DEROCHER, AMY - Center For Infectious Disease Research; YEARGAN, MICHELLE - University Of Kentucky; CHOI, RYAN - University Of Washington; SMITH, TESS - University Of Washington; RIVAS, KASEY - University Of Washington; HULVERSON, MATTHEW - University Of Washington; BARRETT, LYNN - University Of Washington; FAN, ERKANG - University Of Washington; MALY, DUSTIN - University Of Washington; PARSONS, MARILYN - Center For Infectious Disease Research; Dubey, Jitender; HOWE, DANIEL - University Of Kentucky; VAN VOORHIS, WESLEY - University Of Washington

Submitted to: International Journal for Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/5/2016
Publication Date: 10/8/2016
Citation: Ojo, K., Dangoudoubiyam, S., Verma, S.K., Scheele, S., Derocher, A.D., Yeargan, M., Choi, R., Smith, T.R., Rivas, K.L., Hulverson, M.A., Barrett, L.K., Fan, E., Maly, D., Parsons, M., Dubey, J.P., Howe, D., Van Voorhis, W.C. 2016. Selective inhibition of Sarcocystis neurona calcium-dependent protein kinase 1 for equine protozoal myeloencephalitis therapy. International Journal for Parasitology. 46:871-880.

Interpretive Summary: Toxoplasmosis, caused by the single celled parasite, Toxoplasma gondii, continues to be a public health problem. Sarcocystis, is very similar to Toxoplasma, and some species of Sarcocystis are zoonotic, while others cause severe disease in livestock. Sarcocystis neurona causes a fatal neurologic disease (called EPM) in horses and many other species of animals. There is no satisfactory treatment for EPM. In the present study authors found that a new group of compounds, BK1-1533, is effective in killing S. neurona in a mouse model of EPM. S.neurona infected mice dosed orally with BK1-1533 remained healthy whereas untreated mice died of S. neurona infection. These results will be of interest to veterinarians, biologists, and parasitologists.

Technical Abstract: Sarcocystis neurona is the most frequent cause of Equine Protozoal Myeloencephalitis (EPM), a debilitating neurologic 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 (BKIs). This study presents detailed molecular and phenotypic evidence that SnCDPK1 can be targeted for rational drug development. Recombinant SnCDPK1 was tested against four BKIs shown to potently inhibit both TgCDPK1 and T. gondii tachyzoite growth. SnCDPK1 was inhibited by low nanomolar concentrations of these BKIs and S. neurona growth was inhibited at 40 to 120 nM concentrations. Thermal shift assays confirmed these BKIs bind CDPK1 in S. neurona cell lysates. Temporal dose-response analysis suggests that BKIs interfere with S. neurona mammalian host cell invasion in the 0.5-2.5 µM range but at 2.5 µM BKIs interfere with intracellular cell division. In vivo proof of concept experiments were performed in a murine model of S. neurona infection. The experimental infected groups treated for 30 days with compound BKI-1553 (N=10) 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 BKIs used in these assays have been chemically optimized for potency, selectivity, and pharmacokinetic properties and hence are good candidates for treatment of EPM.