Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA.
Dencoda, LLC, West Lafayette, IN 47906, USA.
Molecules. 2019 Jul 30;24(15):2764. doi: 10.3390/molecules24152764.
As aberrant activity of protein kinases is observed in many disease states, these enzymes are common targets for therapeutics and detection of activity levels. The development of non-natural protein kinase substrates offers an approach to protein substrate competitive inhibitors, a class of kinase inhibitors with promise for improved specificity. Also, kinase activity detection approaches would benefit from substrates with improved activity and specificity. Here, we apply a substrate-mediated selection to a peptidomimetic DNA-encoded chemical library for enrichment of molecules that can be phosphorylated by the protein tyrosine kinase, c-Src. Several substrates were identified and characterized for activity. A lead compound () showed both the ability to serve as a substrate and to promote ATP hydrolysis by the kinase. In inhibition assays, compounds displayed ICs ranging from of 8-100 µM. NMR analysis of bound to the c-Src:ATP complex was conducted to characterize the binding mode. An ester derivative of the lead compound demonstrated cellular activity with inhibition of Src-dependent signaling in cell culture. Together, the results show the potential for substrate-mediated selections of DNA-encoded libraries to discover molecules with functions other than simple protein binding and offer a new discovery method for development of synthetic tyrosine kinase substrates.
由于蛋白激酶的异常活性在许多疾病状态中都有观察到，这些酶是治疗的常见靶点，也是检测活性水平的常见靶点。非天然蛋白激酶底物的开发提供了一种针对蛋白底物竞争性抑制剂的方法，这类激酶抑制剂有望提高特异性。此外，激酶活性检测方法也将受益于具有改善的活性和特异性的底物。在这里，我们将一种基于底物的选择应用于肽模拟 DNA 编码化学文库，以富集可被蛋白酪氨酸激酶 c-Src 磷酸化的分子。鉴定并表征了几种具有活性的底物。一种先导化合物 () 既表现出作为底物的能力，又能促进激酶的 ATP 水解。在抑制实验中，化合物的 IC 范围为 8-100µM。对与 c-Src:ATP 复合物结合的进行了 NMR 分析，以表征其结合模式。该先导化合物的酯衍生物在细胞培养中表现出抑制 Src 依赖性信号的细胞活性。总之，这些结果表明，基于底物的 DNA 编码文库选择有可能发现除简单蛋白结合以外的其他功能的分子，并为合成酪氨酸激酶底物的开发提供了一种新的发现方法。
