Rational development of selective Inhibitors of the protein kinase DYRK1B

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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.