Molecular Plasticity of Crystalline CK2α′ Leads to KN2, a Bivalent Inhibitor of Protein Kinase CK2 with Extraordinary Selectivity

Abstract

CK2α and CK2α′ are paralogous catalytic subunits of CK2, which belongs to the eukaryotic protein kinases. CK2 promotes tumorigenesis and the spread of pathogenic viruses like SARS-CoV-2 and is thus an attractive drug target. Efforts to develop selective CK2 inhibitors binding offside the ATP site had disclosed the αD pocket in CK2α; its occupation requires large conformational adaptations of the helix αD. As shown here, the αD pocket is accessible also in CK2α′, where the necessary structural plasticity can be triggered with suitable ligands even in the crystalline state. A CK2α′ structure with an ATP site and an αD pocket ligand guided the design of the bivalent CK2 inhibitor KN2. It binds to CK2 with low nanomolar affinity, is cell-permeable, and suppresses the intracellular phosphorylation of typical CK2 substrates. Kinase profiling revealed a high selectivity of KN2 for CK2 and emphasizes the selectivity-promoting potential of the αD pocket.

Competition between electrostatic interactions and halogen bonding in the protein–ligand system: structural and thermodynamic studies of 5,6-dibromobenzotriazole-hCK2α complexes

“…1A), indicating that the anionic form is highly preferred for both 5,6-DBBt and TBBt. In the published PDB structures (3OFM, 3RPS, 6HMQ, 7AT9) with bigger alkyl substitutions at N1 of TBBt, the steric bulk in this position does not significantly perturb the interaction with the ATP-binding site of human protein kinase CK2α/CK2αʹ [66][67][68] . This, together with modeling (Suppl.…”

AB668, a novel highly selective protein kinase CK2 inhibitor with a distinct anti-tumor mechanism as compared to CX-4945 and SGC-CK2-1

“…The flexibility of this αD helix was experimentally observed in various crystallographic structures of the kinase and was also reported in metadynamic studies of CK2 structure (Gouron et al, 2014). Two bivalent CK2 inhibitors targeting this pocket, in addition to the ATP site, have been previously published (Brear et al, 2016; Lindenblatt et al, 2022). While their impact on cell cytotoxicity has been reported, the effect of such inhibitors on cellular CK2 functions has not been explored.…”

“…However, the affinity of the inhibitor was rather weak (K D 30 μM) for further characterization. Bivalent CK2 inhibitors targeting simultaneously the ATP-binding site and the αD pocket have also been reported (Brear et al, 2016, De Fusco et al, 2017, Lindenblatt et al, 2022). The bivalent inhibitor CAM4066 (KD of 0.32 μM) reduced cell viability in HCT116, Jurkat and A549 cells with GI50 of 9, 6 and 20 μM, respectively (Brear et al, 2016).…”

Strategies of Targeting CK2 in Drug Discovery: Challenges, Opportunities, and Emerging Prospects

“…Whereas 32 has poor cell permeability, its synthetic methyl ester derivative, pro-CAM4066, as a prodrug enhances its membrane permeability and exhibits antitumoral activity in various cancer cell lines. , Notably, the previously mentioned bivalent inhibitor KN2 ( 5 ) has similar binding modes as 32 , which acts both on the ATP site and the αD pocket. Coupling two terminal synthons with amino groups, 5 does not contain a negative charge and is cell-permeable with higher potency and selectivity than 32 …”