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

Abstract

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 (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.

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 IC50 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 IC50 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 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.

Graphic Abstract

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References

  1. 1.

    Wise LN, Kappmeyer LS, Mealey RH, Knowles DP (2013) Review of equine piroplasmosis. J Vet Intern Med 27(6):1334–1346

    CAS  PubMed  Article  Google Scholar 

  2. 2.

    Hines SA, Ramsay JD, Kappmeyer LS, Lau AO, Ojo KK, Van Voorhis WC, Knowles DP, Mealey RH (2015) Theileria equi isolates vary in susceptibility to imidocarb dipropionate but demonstrate uniform in-vitro susceptibility to a bumped kinase inhibitor. Parasit Vectors 8:33

    PubMed  PubMed Central  Article  Google Scholar 

  3. 3.

    Haubrich BA, Swinney DC (2016) Enzyme activity assay for protein kinases: strategies to identify active substrate. Curr Drug Discov Techno 13(1):2–15

    CAS  Article  Google Scholar 

  4. 4.

    Schuster FL (2002) Cultivation of Babesia and Babesia-like blood parasites: agents of an emerging zoonotic disease. Clin Microbiol Rev 15:365–373

    PubMed  PubMed Central  Article  Google Scholar 

  5. 5.

    Gopalakrishnan A, Maji C, Dahiya RK, Suthar A (2015) Oxidative damage inflicted by Theileria equi on horse erythrocytes when cultured in-vitro by microaerophilous stationary phase technique. J Equine Vet Sci 35:763–767

    Article  Google Scholar 

  6. 6.

    Kumar S, Gupta AK, Pal Y, Dwivedi SK (2003) In-vivo therapeutic efficacy trial with artemisinin derivative, buparvaquone and imidocarb dipropionate against Babesia equi infection in donkeys. J Vet Med Sci 65(11):1171–1177

    CAS  PubMed  Article  Google Scholar 

  7. 7.

    Kumar S, Kumar R, Sugimoto C (2009) A perspective on Theileria equi infections in donkeys. J Vet Res 56:171–180

    Google Scholar 

  8. 8.

    Gopalakrishnan A, Maji C, Dahiya RK, Suthar A, Kumar R, Gupta AK (2016) In-vitro growth inhibitory efficacy of some target specific novel drug molecules against Theileria equi. Vet Parasitol 217:1–6

    CAS  PubMed  Article  Google Scholar 

  9. 9.

    Holbrook AA (1969) Biology of equine piroplasmosis. J Am Vet Med Assoc 155:453–454

    CAS  PubMed  Google Scholar 

  10. 10.

    Kumar S, Malhotra DV, Sangwan AK, Goel P, Kumar A, Kumar S (2007) Infectivity rate and transmission potential of Hyalomma anatolicum ticks for Babesia equi infection. Vet Parasitol 144:338–343

    PubMed  Article  Google Scholar 

  11. 11.

    Kumar S, Kumar R, Gupta AK, Yadav SC, Goyal SK, Khurana SK, Singh RK (2013) Development of EMA-2 recombinant antigen based enzyme-linked immunosorbent assay for seroprevalence studies of Theileria equi infection in Indian equine population. Vet Parasitol 198(1–2):10–17

    CAS  PubMed  Article  Google Scholar 

  12. 12.

    Dahiya R, Salar RK, Mandal KD, Kumar R, Tripathi BN, Pal Y, Kumar S (2018) Risk factor analysis associated with Theileria equi infected equines in semi-arid and sub-humid ecological enzootic zones of India. Vet Parasitol Reg Stud Reports 12:17–21

    PubMed  Google Scholar 

  13. 13.

    Kumar S, Malhotra DV, Dhar S (1997) Serodiagnosis of Babesia equi infection-a comparison of Dot-ELISA, complement fixation test and capillary tube agglutination test. Vet Parasitol 69:171–176

    CAS  PubMed  Article  Google Scholar 

  14. 14.

    Bork S, Yokoyama N, Igarashi I (2005) Recent advances in the chemotherapy of babesiosis by Asian scientist: toxoplasmosis and babesiosis in Asia. Asian Parasitol 4:233–242

    Google Scholar 

  15. 15.

    Vial HJ, Gorenflot A (2006) Chemotherapy against babesiosis. Vet Parasitol 138:147–160

    CAS  PubMed  Article  Google Scholar 

  16. 16.

    Kumar S, Malhotra DV, Dhar S, Nichani AK (2002) Vaccination of donkeys against Babesia equi using killed merozoite immunogen. Vet Parasitol 106(1):19–33

    CAS  PubMed  Article  Google Scholar 

  17. 17.

    Nakamura K, Yokoyama N, Igarashi I (2007) Cyclin-dependent kinase inhibitors block erythrocyte invasion and intraerythrocytic development of Babesia bovis in-vitro. Parasitol 134:1347–1353

    CAS  Article  Google Scholar 

  18. 18.

    Doerig C, Billker O, Haystead T, Sharma P, Tobin AB, Waters NC (2008) Protein kinases of malaria parasites: an update trends. Parasitol 24(12):570–577

    CAS  Google Scholar 

  19. 19.

    Jirage D, Keenan SM, Waters NC (2010) Exploring novel targets for antimalarial drug discovery: plasmodial protein kinases. Infect Disord Drug Targets 10(3):134–146

    CAS  PubMed  Article  Google Scholar 

  20. 20.

    Rothschild CM (2013) Equine piroplasmosis. J Equine Vet Sci 33:497–508

    Article  Google Scholar 

  21. 21.

    Igarashi I, Njonge FK, Kaneko Y, Nakamura Y (1998) Babesia bigemina: in-vitro and in vivo effects of curdlan sulfate on growth of parasites. Exp Parasitol 90:290–293

    CAS  PubMed  Article  Google Scholar 

  22. 22.

    Bork S, Yokoyama N, Ikehara Y, Kumar S, Sugimoto C, Igarashi I (2004) Growth-inhibitory effect of heparin on Babesia parasites. Antimicrob Agents Chemother 48:236–241

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23.

    AbouLaila M, Nakamura K, Govind Y, Yokoyama N, Igarashi I (2010) Evaluation of the in-vitro growth inhibitory effect of epoxomicin on Babesia parasites. Vet Parasitol 167:19–27

    CAS  PubMed  Article  Google Scholar 

  24. 24.

    O’Brien J, Wilson I, Orton T, Pognan F (2000) Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 267(17):5421–5426

    PubMed  Article  Google Scholar 

  25. 25.

    Ward P, Equinet L, Packer J, Doerig C (2004) Protein kinases of the human malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote. BMC Genom 5:79

    Article  Google Scholar 

  26. 26.

    Cohen A, Dumètre A, Azas N (2013) A decade of Plasmodium falciparum metabolic pathways of therapeutic interest to develop new selective antimalarial drugs. Mini Rev Med Chem 13(9):1340–1347

    CAS  PubMed  Article  Google Scholar 

  27. 27.

    Lacey MR, Brumlik MJ, Yenni RE, Burow ME, Curiel TJ (2007) Toxoplasma gondii expresses two mitogen-activated protein kinase genes that represent distinct protozoan subfamilies. J Mol Evol 64(1):4–14

    CAS  PubMed  Article  Google Scholar 

  28. 28.

    Dorin-Semblat D, Quashie N, Halbert J, Sicard A, Doerig C, Peat E, Ranford-Cartwright L, Doerig C (2017) Functional characterization of both MAP kinases of the human malaria parasite Plasmodium falciparum by reverse genetics. Mol Microbiol 65(5):1170–1180

    Article  Google Scholar 

  29. 29.

    Seitzer U, Schnittger L, Boguslawski K, Ahmed JS (2006) Investigation of MAP kinase activation in Theileria-infected cell lines. Ann N Y Acad Sci 1081:473–475

    PubMed  Article  Google Scholar 

  30. 30.

    Brumlik MJ, Nkhoma S, Kious MJ, Thompson GR III, Patterson TF, Siekierka JJ, Anderson TJC, Curiel TJ (2011) Human p38 mitogen-activated protein kinase inhibitor drugs inhibit Plasmodium falciparum replication. Exp Parasitol 128(2):170–175

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 31.

    Ray A, Quade J, Carson CA, Ray BK (1990) Calcium-dependent protein phosphorylation in Babesia bovis and its role in growth regulation. J Parasitol 76:153–161

    CAS  PubMed  Article  Google Scholar 

  32. 32.

    Kim A, Nam YJ, Lee MS, Shin YK, Sohn DS, Lee CS (2016) Apigenin reduces proteasome inhibition-induced neuronal apoptosis by suppressing the cell death process. Neurochem Res 41(11):2969–2980

    CAS  PubMed  Article  Google Scholar 

  33. 33.

    Shukla S, Gupta S (2010) Apigenin: a promising molecule for cancer prevention. Pharm Res 27(6):962–978

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. 34.

    Tasdemir D, Kaiser M, Brun R, Yardley V, Schmidt TJ, Tosun F, Rüedi P (2006) Antitrypanosomal and antileishmanial activities of flavonoids and their analogues: in-vitro, in vivo, structure-activity relationship, and quantitative structure-activity relationship studies. Antimicrob Agents Chemother 50(4):1352–1364

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. 35.

    Ohori M, Takeuchi M, Maruki R, Nakajima H, Miyake H (2006) FR180204, a novel and selective inhibitor of extracellular signal-regulated kinase, ameliorates collagen-induced arthritis in mice. Naunyn Schmiedebergs Arch Pharmacol 374(4):311–316

    PubMed  Article  Google Scholar 

  36. 36.

    Dobbelaere DA, Fernandez PC, Heussler VT (2000) Theileria parva: taking control of host cell proliferation and survival mechanisms. Cell Microbiol 2(2):91–99

    CAS  PubMed  Article  Google Scholar 

  37. 37.

    Galley Y, Hagens G, Glaser I, Davis W, Eichhorn M, Dobbelaere D (1997) Jun NH2-terminal kinase is constitutively activated in T cells transformed by the intracellular parasite Theileria parva. Proc Natl Acad Sci USA 94(10):5119–5124

    CAS  PubMed  Article  Google Scholar 

  38. 38.

    Chaussepied M, Lallemand D, Moreau MF, Adamson R, Hall R, Langsley G (1998) Upregulation of Jun and Fos family members and permanent JNK activity lead to constitutive AP-1 activation in Theileria-transformed leukocytes. Mol Biochem Parasitol 94(2):215–226

    CAS  PubMed  Article  Google Scholar 

  39. 39.

    Bison S, Razzoli M, Arban R, Michielin F, Bertani S, Carboni L (2011) Effect of the p38 MAPK inhibitor SB-239063 on lipopolysaccharide-induced psychomotor retardation and peripheral biomarker alterations in rats. Eur J Pharmacol 661(1–3):49–56

    CAS  PubMed  Article  Google Scholar 

Download references

Acknowledgements

Authors wish to acknowledge their gratitude to the Director, ICAR-National Research Centre on Equines, Hisar, Haryana, India for providing all the necessary facilities for conducting this study and Head, Department of Veterinary Medicine, Lala Lajpat Rai University of Veterinary and Animal Science, Hisar, Haryana, India for managing the administrative matters of the first author as a student of department.

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Correspondence to Sanjay Kumar.

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Yadav, J., Goel, P., Mandal, K.D. et al. Protein Kinase Inhibitors Arrested the In-Vitro Growth of Theileria equi. Acta Parasit. 65, 644–651 (2020). https://doi.org/10.2478/s11686-020-00202-5

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Keywords

  • Theileria equi
  • Protein kinase
  • Protein kinase inhibitor
  • MASP
  • Drug molecule