Protein Kinase Inhibitors Arrested the In-Vitro Growth of Theileria equi.

Abstract

Theileria equi is an intra-erythrocytic apicomplexean protozoa that infect equines. Protein kinases (PK), key molecules of the apicomplexean life cycle, have been implicated as significant drug targets. The growth inhibitory efficacy of PK inhibitors against Theileria/Babesia animal parasites have not been documented so far. The present study aimed to carry out in-vitro growth inhibitory efficacy studies of four novel drug molecules-SB239063, PD0332991 isethionate, FR180204 and apigenin, targeting different protein kinases of T. equi. A continuous microaerophilic stationary-phase culture (MASP) system was established for propagation of T. equi parasites. This in-vitro culture technique was used to assess the growth inhibitory effect of proteinkinase targeted drug molecules, whereas diminazene aceturate was taken as control drug against T. equi. The inhibitory concentration (IC50) was determined for comparative analysis. The potential cytotoxicity of the drug molecule was also assessed on horse's peripheral blood mononuclear cells (PBMCs) cell line. SB239063 and diminazene aceturate drugs significantly inhibited (p < 0.05) the in-vitro growth of T. equi parasite at 0.1µM, 1µM, 10µM, 50µM and 100µM concentration at ≥ 48h of incubation period and respective IC50 values were 4.25µM and 1.23µM. Furthermore, SB239063 was not cytotoxic to the horse PBMCs and found safer than diminazine aceturate drug. PD0332991 isethionate and FR180204 are extracellular signal-regulated kinase (ERK) inhibitors and significantly (p < 0.05) inhibited T. equi in-vitro growth at higher concentrations (≥ 48h of incubation period) with respective IC50 value of 10.41µM and 21.0µM. Lower concentrations of these two drugs were not effective (p > 0.05) even after 96h of treatment period. Apigenin (protein kinase-C inhibitor) drug molecule was unsuccessful in inhibiting the T. equi parasite growth completely. After 96h of in-vitro treatment period, a parasite viability study was performed on drug-treated T. equi parasitized RBCs. These drugs-treated parasitized RBCs were collected and transferred to wells containing fresh culture media (without drug) and naïve host RBCs. Drug-treated RBCs collected from SB239063, PD0332991, diminazene aceturate treatment (1µM to 100µM concentration) were unsuccessful in growing/multiplying further. Apigenin drug-treated T. equi parasites were live after 96h of treatment. It may be concluded that SB239063 was the most effective drug molecule (being lowest in IC50 value) out of the four different protein kinase inhibitors tested in this study. This drug molecule has insignificant cytotoxic activity against horse's PBMCs.