For kinase inhibitors, intracellular target selectivity is fundamental to pharmacological mechanism. Although a number of acellular techniques have been developed to measure kinase binding or enzymatic inhibition, such approaches can fail to accurately predict engagement in cells. Here we report the application of an energy transfer technique that enabled the first broad-spectrum, equilibrium-based approach to quantitatively profile target occupancy and compound affinity in live cells. Using this method, we performed a selectivity profiling for clinically relevant kinase inhibitors against 178 full-length kinases, and a mechanistic interrogation of the potency offsets observed between cellular and biochemical analysis. For the multikinase inhibitor crizotinib, our approach accurately predicted cellular potency and revealed improved target selectivity compared with biochemical measurements. Due to cellular ATP, a number of putative crizotinib targets are unexpectedly disengaged in live cells at a clinically relevant drug dose.

中文翻译：

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活细胞中的定量、广谱激酶分析用于评估细胞 ATP 对靶点参与的影响

对于激酶抑制剂，细胞内靶点选择性是药理学机制的基础。尽管已经开发了许多非细胞技术来测量激酶结合或酶抑制，但此类方法可能无法准确预测细胞中的参与。在这里，我们报告了能量转移技术的应用，该技术实现了第一个广谱、基于平衡的方法来定量分析活细胞中的目标占有率和化合物亲和力。使用这种方法，我们对 178 种全长激酶进行了临床相关激酶抑制剂的选择性分析，并对细胞分析和生化分析之间观察到的效力偏移进行了机械询问。对于多激酶抑制剂克唑替尼，我们的方法准确预测了细胞效力，并显示与生化测量相比，靶点选择性有所提高。由于细胞 ATP，许多假定的克唑替尼靶标在临床相关药物剂量下意外地在活细胞中脱离。