Structure-guided development of affinity probes for tyrosine kinases using chemical genetics
Structure-guided development of affinity probes for tyrosine kinases using chemical genetics
复制标题
DOI：
10.1038/nchembio866
复制
发表时间：
2007-04-01
影响因子：
14.8
通讯作者：
Shokat, Kevan M.
中科院分区：
文献类型：
--
作者：
Blair, Jimmy A.;Rauh, Daniel;Shokat, Kevan M.
作为几乎所有信号转导途径的关键组分，蛋白激酶是小分子抑制剂调节细胞信号传导的有吸引力的靶点。我们报告了6-丙烯酰胺基-4-苯胺基喹唑啉不可逆激酶抑制剂的结构导向开发，该抑制剂有效地和选择性地靶向合理设计的激酶，该激酶具有两个在任何野生型激酶中都没有发现的选择性元素：亲电靶向的半胱氨酸残基和甘氨酸看门人残基。与表皮生长因子受体（EGFR）或工程化c-Src结合的两种不可逆喹唑啉抑制剂的共晶体结构显示出与靶向半胱氨酸（EGFR中的Cys 797和工程化c-Src中的Cys 345）的共价抑制剂结合。为了适应新的共价键，喹唑啉核心采用的位置与可逆喹唑啉抑制剂的激酶结构中所见的位置不同。基于这些结构，我们开发了一种荧光6-丙烯酰胺基-4-苯胺基喹唑啉亲和探针来报告细胞信号传导所需的激酶的分数，并使用这些试剂来定量EGF刺激EGFR与其下游产物Akt，Erk 1和Erk 2之间的关系。
As key components in nearly every signal transduction pathway, protein kinases are attractive targets for the regulation of cellular signaling by small-molecule inhibitors. We report the structure-guided development of 6-acrylamido-4-anilinoquinazoline irreversible kinase inhibitors that potently and selectively target rationally designed kinases bearing two selectivity elements that are not found together in any wild-type kinase: an electrophile-targeted cysteine residue and a glycine gatekeeper residue. Cocrystal structures of two irreversible quinazoline inhibitors bound to either epidermal growth factor receptor (EGFR) or engineered c-Src show covalent inhibitor binding to the targeted cysteine (Cys797 in EGFR and Cys345 in engineered c-Src). To accommodate the new covalent bond, the quinazoline core adopts positions that are different from those seen in kinase structures with reversible quinazoline inhibitors. Based on these structures, we developed a fluorescent 6-acrylamido-4-anilinoquinazoline affinity probe to report the fraction of kinase necessary for cellular signaling, and we used these reagents to quantitate the relationship between EGFR stimulation by EGF and its downstream outputs-Akt, Erk1 and Erk2.