Exquisitely selective turn-on probes of kinase activation and localization

精巧选择的激酶激活和定位启动型探针 蛋白激酶介导的磷酸化是真核细胞信号转导中一种重要的翻译后修饰。 由于缺乏选择性激酶，人们对这些复杂的信号通路的理解受到了阻碍。 抑制剂。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。尽管效力是 在ATP口袋中很容易获得，由于这种结合的相似性质，选择性很难获得 口袋横跨在金字塔上。非三磷酸腺苷竞争性抑制剂通常具有比 然而，与ATP竞争的同行们普遍缺乏效力。相比之下，两者都 使用双底物抑制剂可以获得高效率和高选择性。双琥珀酸激酶抑制剂 与两个绑定口袋相互作用；在ATP口袋内的相互作用提供效力，而在 底物位置提供选择性。在这里，我们建议开发用于模块化构建的方法 双琥珀酸激酶抑制剂。我们的目标是开发一种方法，以一种可以靶向任何一种激酶和 产生一种有效的、高度选择性的抑制剂。 这些建议的完全选择性的激酶抑制剂有许多潜在的应用。在这里，我们 建议开发高选择性和细胞渗透性的探针，当结合到它们的 靶向激活剂。针对活性激酶群体的激酶探针使成像应用于活细胞和 并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 社区与(I)强大的方法学，以容易地为任何感兴趣的激酶开发双底物激酶探针 以及(Ii)一组可用于研究致癌信号通路的高度选择性的激酶探针。 我们将首先开发一种方法，为任何一种激酶提供真正选择性的抑制剂。我们的方法依赖于 一种杂乱但有效的ATP竞争性片段，与高选择性的多肽底物偶联。我们会 为9个亲缘关系家族中的每一个家族开发一种双底物抑制剂。 大多数激酶是通过激活(通常通过磷酸化)和/或亚细胞定位来调节的。 这些变化通常会改变激酶的功能，并可能促进肿瘤的发生。在这里，我们将发展 方法，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‘TURN- 关于成像探头。这些探针将被用来研究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.