BACKGROUND Bruton's tyrosine kinase (BTK) is a key component of the B-cell receptor (BCR) pathway and a clinically validated target for small molecule inhibitors such as ibrutinib in the treatment of B-cell malignancies. Tirabrutinib (GS-4059/ONO-4059) is a selective, once daily, oral BTK inhibitor with clinical activity against many relapsed/refractory B-cell malignancies. METHODS Covalent binding of tirabrutinib to BTK Cys-481 was assessed by LC-MSMS analysis of BTK using compound as a variable modification search parameter. Inhibition potency of tirabrutinib, ibrutinib, acalabrutinib, and spebrutinib against BTK and related kinases was studied in a dose-dependent manner either after a fixed incubation time (as used in conventional IC50 studies) or following a time course where inactivation kinetics were measured. RESULTS Tirabrutinib irreversibly and covalently binds to BTK Cys-481. The inactivation efficiency kinact/Ki was measured and used to calculate selectivity among different kinases for each of the four inhibitors studied. Tirabrutinib showed a kinact/Ki value of 2.4 ± 0.6 × 104 M-1 s-1 for BTK with selectivity against important off-targets. CONCLUSIONS For the BTK inhibitors tested in this study, analysis of the inactivation kinetics yielded a more accurate measurement of potency and selectivity than conventional single-time point inhibition measurements. Subtle but clear differences were identified between clinically tested BTK inhibitors which may translate into differentiated clinical efficacy and safety. GENERAL SIGNIFICANCE This is the first study that offers a detailed side-by-side comparison of four clinically-relevant BTK inhibitors with respect to their inactivation of BTK and related kinases.

中文翻译：

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替拉鲁替尼和其他不可逆的布鲁顿酪氨酸激酶抑制剂的生化特征揭示了靶标抑制作用的差异。

背景技术布鲁顿酪氨酸激酶（BTK）是B细胞受体（BCR）通路的关键组成部分，是小分子抑制剂（如依鲁替尼）在B细胞恶性肿瘤治疗中的临床验证靶标。替拉鲁替尼（GS-4059 / ONO-4059）是一种每日一次的选择性口服BTK抑制剂，对许多复发/难治性B细胞恶性肿瘤具有临床活性。方法采用化合物作为变量修饰搜索参数，通过LC-MSMS分析BTK，评估替拉鲁替尼与BTK Cys-481的共价结合。在固定的孵育时间后（如常规IC50研究中所用）或在测定失活动力学的时间过程后，以剂量依赖的方式研究了替拉鲁替尼，依鲁替尼，阿卡鲁替尼和斯培鲁替尼对BTK和相关激酶的抑制能力。结果Tirabrutinib与BTK Cys-481不可逆地共价结合。测量失活效率kinact / Ki，并将其用于计算所研究的四种抑制剂中每种激酶在不同激酶之间的选择性。替拉鲁替尼对BTK的运动/ Ki值显示为2.4±0.6×104 M-1 s-1，对重要的脱靶分子具有选择性。结论对于在这项研究中测试的BTK抑制剂，灭活动力学的分析比常规的单时间点抑制测量产生了更准确的效价和选择性测量。经临床测试的BTK抑制剂之间鉴定出细微但明显的差异，这可能会转化为差异化的临床疗效和安全性。