Exquisitely selective turn-on probes of kinase activation and localization

激酶激活和定位的高选择性开启探针 蛋白激酶介导的磷酸化是真核细胞信号转导中重要的翻译后修饰。 由于缺乏选择性激酶， 抑制剂的几乎所有已知的激酶抑制剂都结合在高度保守的ATP口袋中。虽然力量是 容易在ATP口袋内获得，但由于这种结合的相似性质，难以获得选择性 穿过细胞质组的口袋。非ATP竞争性抑制剂通常具有比竞争性抑制剂更高的选择性。 然而，它们的ATP竞争性对应物通常缺乏效力。相比之下，两者 用双底物抑制剂可以获得高效力和高选择性。双底物激酶抑制剂 与两个结合口袋相互作用; ATP口袋内的相互作用提供效力， 衬底位置提供选择性。在这里，我们建议开发的模块化建设的方法， 双底物激酶抑制剂。我们的目标是以一种可以靶向任何激酶的方式开发这种方法， 产生有效的和高选择性的抑制剂。 这些建议的完全选择性激酶抑制剂有许多潜在的应用。这里我们 建议开发高选择性和细胞可渗透的探针，当与它们的 靶向激酶。靶向活性激酶群体的激酶探针使得能够在活细胞中进行成像应用， 这将对癌症生物学产生重要影响。我们的最终目标是提供癌症研究 社区具有（i）易于开发任何感兴趣激酶的双底物激酶探针的稳健方法 和（ii）一组适于研究致癌信号通路的高选择性激酶探针。 我们将首先开发一种方法，为任何激酶提供真正的选择性抑制剂。我们的方法论依赖于 一种混杂但有效的ATP竞争片段，与高度选择性的肽底物偶联。我们将 为激酶组的9个家族中的每一个开发一种双底物抑制剂。 大多数激酶通过活化（通常通过磷酸化）和/或亚细胞定位来调节。 这些变化通常改变激酶的功能，并可促进肿瘤发生。在此，我们将开发 可以将我们的选择性双底物抑制剂转化为高度选择性和细胞渗透性的“转向”的方法， 在成像探头上。这些探针将用于研究跨膜的活性激酶的丰度和定位。 一组实验室和患者来源的TNBC细胞系。此外，我们还将展示我们的 TNBC活检组织样本中的探针。

Exquisitely selective turn-on probes of kinase activation and localization Protein kinase-mediated phosphorylation is a vital posttranslational modification in eukaryotic cell signaling. Efforts to understand these complex signaling pathways have been hampered by a lack of selective kinase inhibitors. Nearly all known kinase inhibitors bind in the highly conserved ATP pocket. Although potency is easy to obtain within the ATP-pocket, selectivity is difficult to obtain due to the similar nature of this binding pocket across the kinome. Non-ATP-competitive inhibitors usually possess higher degrees of selectivity than their ATP-competitive counterparts, however, they generally suffer from a lack of potency. In contrast, both high potency and high selectivity can be obtained with bisubstrate inhibitors. Bisubstrate kinase inhibitors interact with both binding pockets; interactions within the ATP pocket provide potency while interactions within the substrate site provide selectivity. Here, we propose to develop methodology for the modular construction of bisubstrate kinase inhibitors. Our goal is to develop this methodology in a way that can target any kinase and yield a potent and highly selective inhibitor. There are many potential applications of these proposed perfectly selective kinase inhibitors. Here, we propose to develop highly selective and cell permeable probes with `turn-on' fluorescence when bound to their target kinase. Kinase probes that target active kinase populations enables imaging applications in live cells and tissues and will have an important impact on cancer biology. Our ultimate goal is provide the cancer research community with (i) robust methodology to readily develop bisubstrate kinase probes for any kinase of interest and (ii) a set of highly selective kinase probes amenable to the study oncogenic signaling pathways. We will first develop a method to provide truly selective inhibitors for any kinase. Our methodology relies on a promiscuous but potent ATP-competitive fragment coupled to a highly selective peptide substrate. We will develop one bisubstrate inhibitor for each of the 9 families of the kinome. Most kinases are regulated by activation (usually via phosphorylation) and/or subcellular localization. These changes typically alter the function of the kinase and can promote oncogenesis. Herein, we will develop methodology that can convert our selective bisubstrate inhibitors into highly selective and cell-permeable `turn- on' imaging probes. These probes will be used to study the abundance and localization of active kinase across a panel of both laboratory and patient-derived TNBC cell lines. In addition, we will showcase the utility of our probes in tissue samples from TNBC biopsies.