Introduction:Bruton9s tyrosine kinase (BTK) plays a crucial role in B-cell development, differentiation, and signaling in the B-cell receptor (BCR) signaling pathway, and is a validated target for aberrant B-cell activity in malignancy and autoimmune diseases. GS-4059 (ONO-4059) is a selective, once-daily, oral inhibitor of BTK with clinical activity against a number of relapsed/refractory B-cell malignancies. GS-4059 was designed to specifically form a covalent bond with a cysteine residue in the BTK active site. The studies presented here confirmed the biochemical mechanism of action of GS-4059, assessed the inactivation kinetics of BTK by GS-4059 and comparator compounds, and measured the selectivity of GS-4059 for BTK over other related kinases. These studies will help to establish pharmacokinetic-pharmacodynamic models to guide clinical studies, and the evaluation of potential for inhibition of other kinases will assist in understanding the safety of the molecule. Methods: The binding of GS-4059 to BTK was characterized using mass spectrometry to detect covalent adduct formation. Ibrutinib, a known irreversible covalent inhibitor, and staurosporine, a known reversible noncovalent inhibitor, were used as positive controls. The covalent nature of GS-4059 was further tested by chasing with excess amount of ibrutinib. GS-4059 and other covalent, irreversible inhibitors of BTK including CC-292, acalabrutinib, and ibrutinib were studied to determine the inactivation kinetics of BTK and related kinases, such as epidermal growth factor receptor (EGFR) and IL2-inducible T-cell kinase (ITK), for these compounds. The rate of enzyme inactivation was studied as a function of inhibitor concentration using a Sox-based fluorescence assay that allows real-time measurement of enzyme activity. Results: Mass spectrometry studies showed that GS-4059 is a covalent inhibitor of BTK, based on an observed mass shift of BTK following preincubation with GS-4059 (Figure 1). This mass shift was consistent with the molecular weight of GS-4059. The irreversibility of GS-4059 binding was further demonstrated by the observation that GS-4059 could not be displaced from BTK by chasing with ibrutinib, as measured by mass spectrometry. GS-4059 efficiently inactivated BTK with a rate constant kinact/Ki of 2.4 ± 0.6 ´ 104 M-1s-1, similar to that of the covalent BTK inhibitors CC-292 and acalabrutinib, but >10 times lower than that of ibrutinib (Figure 2, Figure 3, and Table 1). In addition, GS-4059 is a poor inhibitor of the related kinases EGFR and ITK, supporting the clinical observation of a favorable safety profile for GS-4059 (Table 1). Conclusions: In summary, GS-4059 is a potent and selective covalent irreversible inhibitor of BTK with a different selectivity profile than other BTK inhibitors, and warrants further studies against B-cell malignancies and autoimmune diseases. Disclosures Liclican:Gilead Sciences: Employment, Equity Ownership. Xing:Gilead Sciences: Employment, Equity Ownership. Serafini:Gilead Sciences: Employment, Equity Ownership. Wang:Gilead Sciences: Employment, Equity Ownership. Brendza:Gilead Sciences: Employment, Equity Ownership. Lutz:Gilead Sciences: Employment, Equity Ownership. Ray:Gilead Sciences: Employment, Equity Ownership. Schultz:Gilead Sciences: Employment, Equity Ownership. Sakowicz:Gilead Sciences: Employment, Equity Ownership. Feng:Gilead Sciences: Employment, Equity Ownership.

中文翻译：

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GS-4059 作为 Bruton 酪氨酸激酶的强效选择性共价不可逆抑制剂的生化表征

简介：Bruton9s 酪氨酸激酶 (BTK) 在 B 细胞受体 (BCR) 信号通路中的 B 细胞发育、分化和信号传导中起关键作用，并且是恶性肿瘤和自身免疫性疾病中异常 B 细胞活性的有效靶标. GS-4059 (ONO-4059) 是一种选择性的、每日一次的 BTK 口服抑制剂，具有针对多种复发/难治性 B 细胞恶性肿瘤的临床活性。GS-4059 旨在与 BTK 活性位点中的半胱氨酸残基特异性形成共价键。此处介绍的研究证实了 GS-4059 的生化作用机制，评估了 GS-4059 和比较化合物对 BTK 的失活动力学，并测量了 GS-4059 对 BTK 的选择性优于其他相关激酶。这些研究将有助于建立药代动力学-药效学模型来指导临床研究，对其他激酶抑制潜力的评估将有助于了解该分子的安全性。方法：GS-4059 与 BTK 的结合使用质谱法检测共价加合物的形成进行表征。已知的不可逆共价抑制剂依鲁替尼和已知的可逆非共价抑制剂星形孢菌素用作阳性对照。GS-4059 的共价性质进一步通过追踪过量的依鲁替尼进行测试。研究了 GS-4059 和其他 BTK 共价、不可逆抑制剂，包括 CC-292、acalabrutinib 和 ibrutinib，以确定 BTK 和相关激酶（如表皮生长因子受体 (EGFR) 和 IL2 诱导型 T 细胞激酶）的失活动力学(ITK), 对于这些化合物。使用允许实时测量酶活性的基于 Sox 的荧光测定，将酶失活率作为抑制剂浓度的函数进行研究。结果：质谱研究表明 GS-4059 是 BTK 的共价抑制剂，这是基于在与 GS-4059 预孵育后观察到的 BTK 质量转移（图 1）。这种质量转移与 GS-4059 的分子量一致。GS-4059 结合的不可逆性通过观察到 GS-4059 不能通过用 ibrutinib 进行追踪而从 BTK 中置换出来（如通过质谱法测量）得到进一步证明。GS-4059 以 2.4 ± 0.6 ´ 104 M-1s-1 的速率常数 kinact/Ki 有效灭活 BTK，类似于共价 BTK 抑制剂 CC-292 和 acalabrutinib，但比依鲁替尼低 10 倍以上（图2、图 3 和表 1)。此外，GS-4059 是相关激酶 EGFR 和 ITK 的不良抑制剂，支持 GS-4059 具有良好安全性的临床观察（表 1）。结论：总而言之，GS-4059 是一种有效的、选择性的 BTK 共价不可逆抑制剂，其选择性与其他 BTK 抑制剂不同，值得进一步研究对抗 B 细胞恶性肿瘤和自身免疫性疾病。披露 Liclican：Gilead Sciences：就业、股权。Xing：Gilead Sciences：就业，股权。Serafini：Gilead Sciences：就业、股权。王：吉利德科学：就业，股权。布伦扎：吉利德科学：就业、股权。Lutz：Gilead Sciences：就业、股权。Ray：Gilead Sciences：就业、股权。舒尔茨：吉利德科学：就业，股权。Sakowicz：Gilead Sciences：就业，股权。冯：吉利德科学：就业、股权。