Discovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca , Building 310, Cambridge Science Park, Milton Road, Cambridge CB4 0WG, United Kingdom.
Proteomic Resource Center, The Rockefeller University , New York, New York 10065, United States.
J Am Chem Soc. 2016 Aug 24;138(33):10554-60. doi: 10.1021/jacs.6b05483. Epub 2016 Aug 11.
Erythropoietin-producing human hepatocellular carcinoma (Eph) receptor tyrosine kinases (RTKs) regulate a variety of dynamic cellular events, including cell protrusion, migration, proliferation, and cell-fate determination. Small-molecule inhibitors of Eph kinases are valuable tools for dissecting the physiological and pathological roles of Eph. However, there is a lack of small-molecule inhibitors that are selective for individual Eph isoforms due to the high homology within the family. Herein, we report the development of the first potent and specific inhibitors of a single Eph isoform, EphB3. Through structural bioinformatic analysis, we identified a cysteine in the hinge region of the EphB3 kinase domain, a feature that is not shared with any other human kinases. We synthesized and characterized a series of electrophilic quinazolines to target this unique, reactive feature in EphB3. Some of the electrophilic quinazolines selectively and potently inhibited EphB3 both in vitro and in cells. Cocrystal structures of EphB3 in complex with two quinazolines confirmed the covalent linkage between the protein and the inhibitors. A "clickable" version of an optimized inhibitor was created and employed to verify specific target engagement in the whole proteome and to probe the extent and kinetics of target engagement of existing EphB3 inhibitors. Furthermore, we demonstrate that the autophosphorylation of EphB3 within the juxtamembrane region occurs in trans using a specific inhibitor. These exquisitely specific inhibitors will facilitate the dissection of EphB3's role in various biological processes and disease contribution.
促红细胞生成素产生的人肝细胞癌 (Eph) 受体酪氨酸激酶 (RTKs) 调节多种动态细胞事件，包括细胞突出、迁移、增殖和细胞命运决定。Eph 激酶的小分子抑制剂是解析 Eph 生理和病理作用的有价值工具。然而，由于家族内的高度同源性，缺乏对单个 Eph 同工型具有选择性的小分子抑制剂。在此，我们报告了第一个针对单个 Eph 同工型 EphB3 的有效和特异性抑制剂的开发。通过结构生物信息学分析，我们在 EphB3 激酶结构域的铰链区域鉴定出一个半胱氨酸，这是与任何其他人类激酶都不共享的特征。我们合成并表征了一系列亲电喹唑啉，以针对 EphB3 中的这种独特、反应性特征。一些亲电喹唑啉在体外和细胞中选择性地和有效地抑制 EphB3。EphB3 与两种喹唑啉复合物的晶体结构证实了蛋白质和抑制剂之间的共价结合。创建了一个“可点击”的优化抑制剂版本，并用于验证整个蛋白质组中的特定靶标结合，并探测现有 EphB3 抑制剂的靶标结合程度和动力学。此外，我们证明了在特定抑制剂的作用下，EphB3 跨膜区域内的自身磷酸化是发生在反式的。这些高度特异性的抑制剂将有助于解析 EphB3 在各种生物过程和疾病中的作用。
