A clinical casein kinase 2 inhibitor, CX-4945 (silmitasertib), shows significant affinity toward the DYRK1A and GSK3β kinases, involved in down syndrome phenotypes, Alzheimer’s disease, circadian clock regulation, and diabetes. This off-target activity offers an opportunity for studying the effect of the DYRK1A/GSK3β kinase system in disease biology and possible line extension. Motivated by the dual inhibition of these kinases, we solved and analyzed the crystal structures of DYRK1A and GSK3β with CX-4945. We built a quantum-chemistry-based model to rationalize the compound affinity for CK2α, DYRK1A, and GSK3β kinases. Our calculations identified a key element for CK2α’s subnanomolar affinity to CX-4945. The methodology is expandable to other kinase selectivity modeling. We show that the inhibitor limits DYRK1A- and GSK3β-mediated cyclin D1 phosphorylation and reduces kinase-mediated NFAT signaling in the cell. Given the CX-4945’s clinical and pharmacological profile, this inhibitory activity makes it an interesting candidate with potential for application in additional disease areas.

DYRK1B kinase recently emerged as a potential target in cancer, metabolic syndrome, and nonalcoholic fatty liver disease, but the lack of structural information hinders the design of selective DYRK1B inhibitors. Here, we provide a method for recombinant production, activity assays, crystallization conditions and a high resolution crystal structure of DYRK1B in complex with nonselective AZ191 inhibitor. A structure of a closely related DYRK1A kinase with AZ191 is also solved to facilitate comparative analysis. The analysis allows to identify a convenient anchor point at the hinge region of DYRK1B which should permit future development of selective inhibitors of potential advantage over currently available dual specificity DYRK1B/1A inhibitors.