两种不同选择性的BTK抑制剂胞内摄取和靶蛋白结合特点的比较

Comparison of intracellular uptake and target protein binding characteristics of two BTK inhibitors with different selectivities

目的通过研究两种不同选择性的布鲁顿酪氨酸激酶抑制剂(Bruton’s tyrosine kinase inhibitor,BTKi)的胞内摄取及靶蛋白结合特点,为进一步了解药物脱靶相关出血风险的机制提供参考。方法采用伊布替尼(非选择性BTKi)和泽布替尼(选择性BTKi)为研究药物。MEC-1细胞人血小板加药孵育后,细胞热转移实验(cellular thermal shift assay,CETSA)结合Western blot法得到熔解曲线和等温曲线,分析两种药物与靶蛋白BTK的结合特点。MEC-1细胞和人血小板加药孵育后,采用液相色谱-串联质谱法(LC-MS/MS)检测两种药物浓度,分析两种药物胞内摄取的情况。结果CETSA分析结果证实,相较于伊布替尼,泽布替尼对靶蛋白BTK的选择性更高。LC-MS/MS分析结果显示,两种药物均被MEC-1细胞与血小板胞内摄取,并呈浓度依赖性。结论BTKi靶向作用于B淋巴细胞的BTK发挥治疗作用的同时,由于其胞内摄取和靶向结合特点的差异,对血小板产生的脱靶作用可能是不同选择性BTKi出血风险存在差异的原因之一。

Aim To investigate the intracellular uptake and target protein binding characteristics of two Bruton’s tyrosine kinase inhibitors(BTKi)with different selectivities to provide further insights into the mechanisms of drug off-target-related bleeding risk.Methods Ibrutinib(non-selective BTKi)and zanubrutinib(selective BTKi)were used as study drugs.After incubation of MEC-1 cells and human platelets with drugs,the cellular thermal shift assay(CETSA)was combined with Western blot to obtain the melting curve and isothermal curve to analyze the binding characteristics of the two drugs with the target protein BTK.After incubation of MEC-1 cells and human platelets with drugs,the concentrations of the two drugs were detected by liquid chromatography-tandem mass spectrometry(LC-MS/MS)to analyze the intracellular uptake of the two drugs.Results CETSA analysis confirmed that zanubrutinib was more selective for the target protein BTK compared to ibrutinib.LC-MS/MS analysis showed that both drugs were uptaken intracellularly by MEC-1 cells and platelets in a concentration-dependent manner.Conclusions While BTKi targeting BTK to B lymphocytes exerts therapeutic effects,off-target effects on platelets due to differences in their intracellular uptake,and target-binding characteristics may be one of the reasons for the differences in bleeding risk across selective BTKi.