Futosi Krisztina, Bajza Boglárka, Deli Dorottya, Erdélyi András, Tusnády Simon, Mócsai Attila
Department of Physiology, Semmelweis University School of Medicine, Budapest, Hungary.
ELKH-SE Inflammation Physiology Research Group, Eötvös Loránd Research Network, Budapest, Hungary.
Front Pharmacol. 2023 Apr 6;14:1056154. doi: 10.3389/fphar.2023.1056154. eCollection 2023.
Tyrosine kinases are crucial signaling components of diverse biological processes and are major therapeutic targets in various malignancies and immune-mediated disorders. A critical step of development of novel tyrosine kinase inhibitors is the transition from the confirmation of the effects of drug candidates to the analysis of their efficacy. To facilitate this transition, we have developed a rapid assay for the analysis of the effect of oral tyrosine kinase inhibitors on basal tyrosine phosphorylation of circulating mouse neutrophils. The assay uses a single drop of peripheral blood without sacrificing the mice. Flow cytometry using intracellular staining by fluorescently labeled anti-phosphotyrosine antibodies revealed robust basal tyrosine phosphorylation in resting circulating neutrophils. This signal was abrogated by the use of isotype control antibodies or by pre-saturation of the anti-phosphotyrosine antibodies with soluble phosphotyrosine amino acids or tyrosine-phosphorylated peptides. Basal tyrosine phosphorylation was dramatically reduced in neutrophils of triple knockout mice lacking the Src-family tyrosine kinases Hck, Fgr, and Lyn. Neutrophil tyrosine phosphorylation was also abrogated by oral administration of the Abl/Src-family inhibitor dasatinib, a clinically used anti-leukemic agent. Detailed dose-response and kinetic studies revealed half-maximal reduction of neutrophil tyrosine phosphorylation by 2.9 mg/kg dasatinib, with maximal reduction observed 2 h after inhibitor administration. Taken together, our assay allows highly efficient analysis of the effect of orally administered tyrosine kinase inhibitors, and may be used as a suitable alternative to other existing approaches.
酪氨酸激酶是多种生物学过程的关键信号传导成分，也是各种恶性肿瘤和免疫介导疾病的主要治疗靶点。新型酪氨酸激酶抑制剂开发的关键步骤是从确认候选药物的作用过渡到分析其疗效。为促进这一过渡，我们开发了一种快速检测方法，用于分析口服酪氨酸激酶抑制剂对循环小鼠中性粒细胞基础酪氨酸磷酸化的影响。该检测方法只需一滴外周血，无需牺牲小鼠。使用荧光标记的抗磷酸酪氨酸抗体进行细胞内染色的流式细胞术显示，静息循环中性粒细胞中存在强大的基础酪氨酸磷酸化。使用同型对照抗体或用可溶性磷酸酪氨酸氨基酸或酪氨酸磷酸化肽预饱和抗磷酸酪氨酸抗体可消除该信号。在缺乏Src家族酪氨酸激酶Hck、Fgr和Lyn的三联敲除小鼠的中性粒细胞中，基础酪氨酸磷酸化显著降低。口服临床上使用的抗白血病药物Abl/Src家族抑制剂达沙替尼也可消除中性粒细胞酪氨酸磷酸化。详细的剂量反应和动力学研究表明，2.9 mg/kg达沙替尼可使中性粒细胞酪氨酸磷酸化降低一半，在给予抑制剂后2小时观察到最大降低。综上所述，我们的检测方法能够高效分析口服酪氨酸激酶抑制剂的作用，可作为其他现有方法的合适替代方法。
