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

中文翻译：

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蛋白激酶抑制剂阻止了Theileria equi的体外生长。

介绍

Equiile equi是一种感染马的红细胞内蜂复合体原生动物。蛋白激酶（PK）是apicomplexean生命周期的关键分子，已被认为是重要的药物靶标。到目前为止，尚未发现PK抑制剂对Theileria / Babesia动物寄生虫的生长抑制作用。

方法

旨在本研究中进行的体外的四种新型药物分子-SB239063，PD0332991羟乙基磺酸盐，FR180204和芹菜素，靶向的不同蛋白激酶生长抑制效力研究T.球菌。建立了一个连续的微需氧固定相培养（MASP）系统来繁殖马鞭毛虫寄生虫。使用该体外培养技术来评估蛋白质的生长抑制效果激酶靶向药物分子，而diminazene aceturate被作为对控制药物T.球菌。测定抑制浓度（IC ₅₀）用于比较分析。还对马的外周血单核细胞（PBMC）细胞系评估了该药物分子的潜在细胞毒性。

结果

SB239063和diminazene aceturate药物显著抑制（p  <0.05）的体外生长T.球菌在0.1μM，1μM，10μM，50μM，并在温育期的≥48小时100μM浓度寄生虫和各自的IC _50个值分别为4.25 µM和1.23 µM。此外，SB239063对马PBMC无细胞毒性，并且发现它比醋酸二甲嗪更安全。PD0332991羟乙基磺酸盐和FR180204是细胞外信号调节激酶（ERK）抑制剂和显著（p  <0.05）抑制了T.球菌与各自的IC在体外在较高浓度（≥48潜伏期上的H）生长₅₀ 10.41μM和21.0的值微米 降低这两种药物的浓度无效（p  > 0.05），即使在96小时的治疗期后也是如此。芹菜素（蛋白激酶-C抑制剂）药物分子不能完全抑制马鞭毛虫寄生虫的生长。体外治疗96小时后，对药物处理过的马鞭毛虫寄生红细胞进行了寄生虫生存力研究。收集这些用药物处理过的寄生红细胞，并将其转移至含有新鲜培养基（不含药物）和幼稚宿主红细胞的孔中。从SB239063，PD0332991进行药物处理的RBC，醋酸二咪唑乙酸酯处理（浓度1 µM至100 µM）无法成功生长/繁殖。芹菜素药物治疗的马鞭毛虫寄生虫在治疗96小时后仍活着。

结论

可以得出结论，在这项研究中测试的四种不同的蛋白激酶抑制剂中，SB239063是最有效的药物分子（IC ₅₀值最低）。该药物分子对马的PBMC具有微不足道的细胞毒性活性。

图形摘要