Rational development of selective Inhibitors of the protein kinase DYRK1B

蛋白激酶DYRK1B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)，增加对细胞抑制药物和辐射损伤的抵抗力。DYRK1B的药理抑制可能诱导DYRK1B过表达的癌细胞重新进入细胞周期，诱导细胞凋亡，增强抗细胞药物的毒性作用。到目前为止，还没有已知的激酶抑制剂对DYRK1B有足够的(&gt；5倍)选择性，而与DYRK1A密切相关的激酶DYRK1A在全球基因表达调控中起着普遍而重要的作用。该项目的目的是开发一种特定的蛋白激酶DYRK1B抑制剂。由于这两个激酶的序列相似性很高，DYRK1B催化结构域的构象稳定性比DYRK1A低，这为选择性和不可逆失活提供了起点。我们正在通过两种新的策略来追求这一目标：第一，我们计划合理地开发一种II型抑制剂，该抑制剂专门针对未成熟构象的酪氨酸激酶的活性。酪氨酸自动磷酸化稳定了Dyrk家族(双特异性酪氨酸磷酸化调节激酶)的活性构象，而未磷酸化的DYRK1B以变性蛋白的形式聚集在不溶性聚集体中。其次，我们将设计一种嵌合化合物(PROTAC，蛋白水解靶向嵌合体)，该嵌合化合物是通过将结合到激活区的ATP竞争片段和疏水部分(疏水标签)结合在一起而设计的。目的是通过增加疏水表面积来破坏激活域的构象稳定，并通过泛素-蛋白酶体系统诱导变性或降解。在构建新的抑制剂时，我们使用7-卤代吲哚-3-碳腈作为相对较小的片段连接侧链。在我们的初步工作中，占据了腺嘌呤结合口袋的这些结构元件对DYRK1A/B显示了高的选择性。新化合物的验证是通过基于细胞的DYRK1B活性分析进行的，这使得直接抑制和不可逆失活区分开来，并评估了对DYRK1A的特异性。最后，我们将研究DYRK1B的药物失活对高表达DYRK1B的癌细胞株的增殖和化疗耐药性的影响。我们的项目使用合理开发DYRK1B选择性抑制剂作为一种策略，利用密切相关的平行靶蛋白的热力学稳定性的差异来实现选择性。

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.