Structural perspective on the design of selective DYRK1B inhibitors.

Dual-specificity tyrosine-phosphorylation-regulated kinase 1B (DYRK1B) has recently emerged as a critical therapeutic target in oncology and non-alcoholic fatty liver disease. As a kinase, DYRK1B plays key roles in regulating cellular survival pathways; however, the lack of structural information has impeded the development of selective inhibitors. In this study, we implemented multi-method framework: recombinant expression and stability profiling, cellular target engagement, enzyme inhibition, biophysical thermodynamics, cell-based pathway readout, X-ray crystallography, quantum-mechanical and molecular-dynamics analyses. We report the crystal structure of DYRK1B in complex with the small-molecule inhibitor AZ191. For comparative purposes, we also present the structure of the closely related kinase, DYRK1A, bound to the same ligand. While a structural overlay of the two kinase domains reveals overall negligible differences, detailed inspection highlights distinct features within the hinge-binding region of DYRK1B that are pivotal for achieving kinase selectivity. Moreover, detailed evaluation of the active site architecture reveals a notable difference in the accessibility of the catalytic lysine residue between DYRK1B and DYRK1A, suggesting potential strategies to distinguish selective binders. Overall, these findings provide important macromolecular insights into the DYRK1B structure and offer a structural framework to guide medicinal chemistry efforts towards improved inhibitor selectivity with minimized off-target activity.