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

• Published in: Acta parasitologica Vol. 65; no. 3; pp. 644 - 651
• Main Authors: Yadav, Jyotika, Goel, Praveen, Mandal, Kruti Debnath, Yadav, Rahul, Kumar, Naveen, Kumar, Rajender, Tripathi, B. N., Kumar, Sanjay
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 01-09-2020; Springer Nature B.V
• Subjects: Animal parasites; Animal Systematics/Taxonomy/Biogeography; Biomedical and Life Sciences; Biomedicine; Cell culture; Comparative analysis; Culture media; Culture techniques; Cytotoxicity; Drugs; Ecology; Enzyme inhibitors; Extracellular signal-regulated kinase; Horses; Incubation; Inhibitors; Kinases; Leukocytes (mononuclear); Life cycles; Medical Microbiology; Microbiology; Original Paper; Parasites; Parasitology; Peripheral blood mononuclear cells; Propagation; Protein kinase inhibitors; Proteins; Protozoa; Theileria equi; Therapeutic targets; Toxicity; Drug molecule; MASP; Protein kinase inhibitor; Protein kinase; Theileria equi
• ISSN: 1230-2821; 1896-1851
• DOI: 10.2478/s11686-020-00202-5

Introduction
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.
Methods
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 protein kinase targeted drug molecules, whereas diminazene aceturate was taken as control drug against
T. equi
. The inhibitory concentration (IC
50
) 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.
Results
SB239063 and diminazene aceturate drugs significantly inhibited (
p
 < 0.05) the in-vitro growth of
T. equi
parasite at 0.1 µM, 1 µM, 10 µM, 50 µM and 100 µM concentration at ≥ 48 h of incubation period and respective IC
50
values were 4.25 µM and 1.23 µM. 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 (≥ 48 h of incubation period) with respective IC
50
value of 10.41 µM and 21.0 µM. Lower concentrations of these two drugs were not effective (
p
 > 0.05) even after 96 h of treatment period. Apigenin (protein kinase-C inhibitor) drug molecule was unsuccessful in inhibiting the
T. equi
parasite growth completely. After 96 h 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 (1 µM to 100 µM concentration) were unsuccessful in growing/multiplying further. Apigenin drug-treated
T. equi
parasites were live after 96 h of treatment.
Conclusion
It may be concluded that SB239063 was the most effective drug molecule (being lowest in IC
50
value) out of the four different protein kinase inhibitors tested in this study. This drug molecule has insignificant cytotoxic activity against horse’s PBMCs.
Graphic Abstract