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′.

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Protein kinase CK2, a member of the eukaryotic protein kinase superfamily, is associated with cancer and other human pathologies and thus an attractive drug target. The indeno[1,2-b]indole scaffold is a novel lead structure to develop ATP-competitive CK2 inhibitors. Some indeno[1,2-b]indole-based CK2 inhibitors additionally obstruct ABCG2, an ABC half transporter overexpressed in breast cancer and co-responsible for drug efflux and resistance. Comprehensive derivatization studies revealed substitutions of the indeno[1,2-b]indole framework that boost either the CK2 or the ABCG2 selectivity or even support the dual inhibition potential. The best indeno[1,2-b]indole-based CK2 inhibitor described yet (IC50 = 25 nM) is 5-isopropyl-4-(3-methylbut-2-enyl-oxy)-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (4p). Herein, we demonstrate the membrane permeability of 4p and describe co-crystal structures of 4p with CK2α and CK2α′, the paralogs of human CK2 catalytic subunit. As expected, 4p occupies the narrow, hydrophobic ATP site of CK2α/CK2α′, but surprisingly with a unique orientation: its hydrophobic substituents point towards the solvent while its two oxo groups are hydrogen-bonded to a hidden water molecule. An equivalent water molecule was found in many CK2α structures, but never as a critical mediator of ligand binding. This unexpected binding mode is independent of the interdomain hinge/helix αD region conformation and of the salt content in the crystallization medium.

The anti-apoptotic protein kinase CK2 increasingly becomes an attractive target in cancer research with great therapeutic potential. Here, we have performed an in vitro screening of the Diversity Set III of the DTP program from the NCI/NIH, comprising 1600 compounds. We have identified 1,3-Dichloro-6-[(E)-((4-methoxyphenyl)imino)methyl] dibenzo(b,d) furan-2,7-diol (referred to as D11) to be a potent and selective inhibitor of protein kinase CK2. The D11 compound was tested against 354 eukaryotic protein kinases. By setting the threshold for inhibition to <2% remaining kinase activity, only DYRK1B, IRAK1 and PIM3 were inhibited to an extent as the tetrameric CK2 holoenzyme and its catalytic subunits α and α'. The IC50 values for the CK2α and CK2α' were on average 1-2 nM in comparison to the DYRK1B, IRAK1 and PIM3 kinases, which ranged from 18 to 49 nM. Cell permeability and efficacy of D11 were tested with cells in culture. In MIA PaCa-2 cells (human pancreatic carcinoma cell line), the phosphorylation of the CK2 biomarker CDC37 at S13 was almost completely inhibited in the presence of D11. This was observed both under normoxia and hypoxia. In the case of the human non-small cell lung carcinoma cell line, H1299, increasing amounts of D11 led to an inhibition of S380/T382/383 phosphorylation in PTEN, another biomarker for CK2 activity.

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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.

Highlights  Structurally unrelated proteins can be encapsulated in chitosan-TPP nanoparticles  Chitosan-TPP nanoparticles delivered proteins to mammalian cells  Cellular uptake of chitosan-TPP nanoparticles was dependent on pH

Glycosylated human leukocyte elastase (HLE) was crystallized and structurally analysed in complex with a 1,3-thiazolidine-2,4-dione derivative that had been identified as an HLE inhibitor in preliminary studies. In contrast to previously described HLE structures with small-molecule inhibitors, in this structure the inhibitor does not bind to the S1 and S2 substrate-recognition sites; rather, this is the first HLE structure with a synthetic inhibitor in which the S2' site is blocked that normally binds the second side chain at the C-terminal side of the scissile peptide bond in a substrate protein. The inhibitor also induces the formation of crystalline HLE dimers that block access to the active sites and that are also predicted to be stable in solution. Neither such HLE dimers nor the corresponding crystal packing have been observed in previous HLE crystal structures. This novel crystalline environment contributes to the observation that comparatively large parts of the N-glycan chains of HLE are defined by electron density. The final HLE structure contains the largest structurally defined carbohydrate trees among currently available HLE structures.