Rational development of selective Inhibitors of the protein kinase DYRK1B

蛋白激酶DYRK 1B在某些肿瘤中具有抗增殖作用，并促进癌细胞进入静止期（静止期），对细胞生长抑制药物和辐射损伤的抗性增加。DYRK 1B的药理学抑制可诱导过表达DYRK 1B的癌细胞重新进入细胞周期，诱导细胞凋亡并增强细胞生长抑制药物的毒性作用。迄今为止，没有已知的激酶抑制剂显示出足够的（> 5倍）选择性DYRK 1B对密切相关的激酶DYRK 1A，它在基因表达的全球调控中发挥着普遍和重要的作用。该项目的目的是开发蛋白激酶DYRK 1B的特异性抑制剂。鉴于这两种激酶的非常高的序列相似性，与DYRK 1A相比，DYRK 1B的催化结构域的较低构象稳定性提供了选择性和不可逆失活的起点。我们正在通过两种新的策略来实现这一目标：首先，我们计划合理地开发一种II型抑制剂，该抑制剂特异性靶向酪氨酸激酶活性的未成熟构象的激酶。酪氨酸自身磷酸化稳定DYRK家族激酶（双特异性酪氨酸磷酸化调节激酶）的活性构象，未磷酸化的DYRK 1B以变性蛋白质的形式聚集在不溶性聚集体中。第二，通过结合用于结合激酶结构域的ATP竞争性片段和疏水部分（疏水标签）来设计嵌合化合物（PROTAC，PROteolysis-Targeting Chimera）。目的是通过增加疏水表面积来破坏激酶结构域的构象，并通过泛素-蛋白酶体系统诱导变性或降解。在新抑制剂的构建中，我们使用7-卤代吲哚-3-腈作为相对较小的片段来连接侧链。这些结构元件占据腺嘌呤结合口袋，在我们的前期工作中对DYRK 1A/B表现出高选择性。新化合物的验证是通过基于细胞的DYRK 1B活性测定进行的，该测定允许区分直接抑制与不可逆失活，并评估对DYRK 1A的特异性。最后，我们将研究DYRK 1B的药理学失活对增殖和对DYRK 1B过表达的癌细胞系的化疗耐药性的影响。我们的项目使用DYRK 1B选择性抑制剂的合理开发作为一种策略，利用密切相关的旁系同源靶蛋白的热力学稳定性差异来实现选择性。

The protein kinase DYRK1B has an antiproliferative effect in certain tumours and promotes the entry of cancer cells into a resting phase (quiescence) with increased resistance to cytostatic drugs and radiation damage. The pharmacological inhibition of DYRK1B may induce re-entry into the cell cycle in DYRK1B-overexpressing cancer cells, induce apoptosis and enhance toxic effects of cytostatic drugs. To date, no known kinase inhibitor has shown sufficient (> 5-fold) selectivity for DYRK1B against the closely related kinase DYRK1A, which plays a ubiquitous and important role in the global regulation of gene expression. The aim of the project is to develop a specific inhibitor of the protein kinase DYRK1B. Given the very high sequence similarity of the two kinases, the lower conformational stability of the catalytic domain of DYRK1B compared to DYRK1A provides a starting point for selective and irreversible inactivation. We are pursuing this goal with two novel strategies: first, we plan to rationally develop a type II inhibitor that specifically targets the tyrosine kinase activity of the immature conformation of the kinase. Tyrosine autophosphorylation stabilizes the active conformation of kinases of the DYRK family (Dual specificity tYrosine phosphorylation Regulated Kinase), and unphosphorylated DYRK1B accumulates as a denatured protein in insoluble aggregates. Second, a chimeric compound (PROTAC, PROteolysis-Targeting Chimera) will be designed by combining an ATP-competitive fragment for binding to the kinase domain and a hydrophobic part (hydrophobic tag). The aim is to destabilize the conformation of the kinase domain by increasing the hydrophobic surface area and to induce denaturation or degradation via the ubiquitin-proteasome system.In the construction of the new inhibitors we use 7-halogenoindole-3-carbonitriles as relatively small fragments to link side chains. Occupying the adenine binding pocket, these structural elements showed high selectivity for DYRK1A/B in our preliminary work. The validation of the new compounds is performed with cell-based assays of DYRK1B activity, which allow a differentiation of direct inhibition from irreversible inactivation and an assessment of the specificity against DYRK1A. Finally, we will investigate the effects of pharmacological inactivation of DYRK1B on proliferation and on chemoresistance of DYRK1B-overexpressing cancer cell lines.Our project uses the rational development of a DYRK1B-selective inhibitor as a strategy to exploit differences in the thermodynamic stability of closely related paralogous target proteins for achieving selectivity.