Novel assay to identify non-ATP competitive protein kinase inhibitors

描述（由申请人提供）：蛋白激酶在许多信号传导途径中发挥核心作用，是细胞功能的关键效应物。因此，蛋白激酶抑制剂可以潜在地开发为许多疾病的治疗剂，包括癌症、中枢神经系统病症和炎症。药物发现工作主要鉴定了与ATP结合直接竞争的小分子配体。然而，通过在高度保守的ATP位点结合来抑制一种激酶的分子通常会抑制其他激酶，从而导致潜在的药物安全性问题。我们建议开发一种新的方法，用于在体内测量蛋白质聚集通过监测信号的小荧光团，已被网站特异性地纳入蛋白质合成过程中使用无义抑制方法。我们建议开发一种新的高通量筛选方法，以确定非ATP竞争性蛋白激酶抑制剂具有潜在的更好的选择性和安全性。我们的测定将鉴定变构地抑制蛋白激酶的化合物（即稳定非活性或“关闭”状态）。我们将采用经过验证的模型系统Abelson（“Abl”）激酶进行“概念验证”研究。我们将开发和优化检测方法，以检测非活性和活性状态的β-内酰胺酶。

DESCRIPTION (provided by applicant): Protein kinases play a central role in many signaling pathways and are key effectors of cellular functions. As such, protein kinases inhibitors can potentially be developed as therapeutics for many diseases, including cancer, central nervous system disorders and inflammation. Drug discovery efforts have primarily identified small molecule ligands that compete directly with ATP binding. However, molecules that inhibit one kinase by binding at the highly conserved ATP site often inhibit other kinases, resulting in potential drug safety issues. We propose to develop a novel method for in vivo measurement of protein aggregation by monitoring the signals from small fluorophores that have been site-specifically incorporated into the protein during protein synthesis using nonsense suppression methodology. We propose to develop a novel high throughput screening assay to identify non-ATP competitive protein kinase inhibitors with potentially better selectivity and safety profiles. Our assay will identify compounds that allosterically inactivate protein kinases (i.e. stabilize the inactive or "off" state). We will employ a proven model system, Abelson ("Abl") kinase, for the "Proof of Concept" studies. We will develop and optimize the assay to detect inactive and active states of Abl.