Hill Zachary B, Perera B Gayani K, Maly Dustin J
Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195-1700, USA.
Mol Biosyst. 2011 Feb;7(2):447-56. doi: 10.1039/c0mb00108b. Epub 2010 Nov 9.
We recently reported a chemical genetic method for generating bivalent inhibitors of protein kinases. This method relies on the use of the DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase (AGT) to display an ATP-competitive inhibitor and a ligand that targets a secondary binding domain. With this method potent and selective inhibitors of the tyrosine kinases SRC and ABL were identified. Here, we dissect the molecular determinants of the potency and selectivity of these bivalent ligands. Systematic analysis of ATP-competitive inhibitors with varying linker lengths revealed that SRC and ABL have differential sensitivities to ligand presentation. Generation of bivalent constructs that contain ligands with differential affinities for the ATP-binding sites and SH3 domains of SRC and ABL demonstrated the modular nature of inhibitors based on the AGT scaffold. Furthermore, these studies revealed that the interaction between the SH3 domain ligand and the kinase SH3 domain is the major selectivity determinant amongst closely-related tyrosine kinases. Finally, the potency of bivalent inhibitors against distinct phospho-isoforms of SRC was determined. Overall, these results provide insight into how individual ligands can be modified to provide more potent and selective bivalent inhibitors of protein kinases.
我们最近报道了一种用于生成蛋白激酶二价抑制剂的化学遗传学方法。该方法依赖于使用DNA修复酶O(6)-烷基鸟嘌呤-DNA烷基转移酶（AGT）来展示一种ATP竞争性抑制剂和一个靶向二级结合域的配体。通过这种方法，鉴定出了酪氨酸激酶SRC和ABL的强效和选择性抑制剂。在此，我们剖析了这些二价配体的效力和选择性的分子决定因素。对具有不同连接子长度的ATP竞争性抑制剂进行系统分析表明，SRC和ABL对配体呈现具有不同的敏感性。生成包含对SRC和ABL的ATP结合位点和SH3结构域具有不同亲和力的配体的二价构建体，证明了基于AGT支架的抑制剂的模块化性质。此外，这些研究表明，SH3结构域配体与激酶SH3结构域之间的相互作用是密切相关的酪氨酸激酶之间主要的选择性决定因素。最后，确定了二价抑制剂对SRC不同磷酸异构体的效力。总体而言，这些结果为如何修饰单个配体以提供更有效和选择性的蛋白激酶二价抑制剂提供了见解。
