CK2α and CK2α′
are the two isoforms of the catalytic
subunit of human protein kinase CK2, an important target for cancer
therapy. They have similar, albeit not identical functional and structural
properties, and were occasionally reported to be inhibited with distinct
efficacies by certain ATP-competitive ligands. Here, we present THN27,
an indeno[1,2-b]indole derivative, as a further inhibitor
with basal isoform selectivity. The selectivity disappears when measured
using CK2α/CK2α′ complexes with CK2β, the
regulatory CK2 subunit. Co-crystal structures of THN27 with CK2α
and CK2α′ reveal that subtle differences in the conformational
variability of the interdomain hinge region are correlated with the
observed effect. In the case of CK2α′, a crystallographically
problematic protein so far, this comparative structural analysis required
the development of an experimental strategy that finally enables atomic
resolution structure determinations with ab initio phasing of potentially
any ATP-competitive CK2 inhibitor and possibly many non-ATP-competitive
ligands as well bound to CK2α′.
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.
Selective inhibitors of protein kinase CK2 with significant cytotoxicity on tumor cells based on a 2-aminothiazole scaffold were described recently. Here, these studies are supplemented with representative CK2α/CK2α′ complex structures. They reveal that the 2-aminothiazole-based inhibitors occupy the ATP cavity, whereas preliminary data had indicated an allosteric binding site. The crystal structure findings are corroborated by subsequent enzyme kinetic studies; their atomic-resolution quality provides the basis for future optimization of these promising CK2 inhibitors.
Specific de novo mutations in the CSNK2A1 gene, which encodes CK2α, the catalytic subunit of protein kinase CK2, are considered as causative for the Okur-Chung neurodevelopmental syndrome (OCNDS). OCNDS is a rare congenital disease with a high phenotypic diversity ranging from neurodevelopmental disabilities to multi-systemic problems and characteristic facial features. A frequent OCNDS mutation is the exchange of Lys198 to Arg at the center of CK2α′s P+1 loop, a key element of substrate recognition. According to preliminary data recently made available, this mutation causes a significant shift of the substrate specificity of the enzyme. We expressed the CK2αLys198Arg recombinantly and characterized it biophysically and structurally. Using isothermal titration calorimetry (ITC), fluorescence quenching and differential scanning fluorimetry (Thermofluor), we found that the mutation does not affect the interaction with CK2β, the non-catalytic CK2 subunit, and that the thermal stability of the protein is even slightly increased. However, a CK2αLys198Arg crystal structure and its comparison with wild-type structures revealed a significant shift of the anion binding site harboured by the P+1 loop. This observation supports the notion that the Lys198Arg mutation causes an alteration of substrate specificity which we underpinned here with enzymological data.
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