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′.

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.

Isoform Specific Inhibition of Human Protein Kinase CK2α and CK2α' by an Indenoindole Derivative The human protein kinase CK2 is an emerging target not only in current cancer research but also in the pathophysiology of viral diseases, such as CoV-2 infection. Two isoforms of the catalytic subunit of human CK2, namely CK2α and CK2α' were identified, exhibiting high similarity but minor functional and structural differences. Further, they differ in their expression profiles, with CK2α being ubiquitously expressed in every tissue while CK2α' being mainly present in brain and testis. In the cell, CK2α and CK2α' exist either as free subunits or in a tetrameric complex bound to a dimer of non-catalytic CK2β subunits. Inhibitors of CK2 which selectively target specific subunits of the kinase are advantageous for the examination of the different functions of the paralogous isoforms. Here we report on THN27, an indeno[1,2-b]indole derivate that exhibits higher CK2α' inhibitory activity (IC 50 = 0.25 µM) in comparison to CK2α (IC 50 = 0.61 µM). Co-crystal structures of CK2α and CK2α' with THN27 reveled a different conformational viability in the interdomain hinge region explaining this behavior. Remarkably, this selective inhibitory behavior was eliminated by the addition of the regulatory subunit (CK2α 2 β 2 IC 50 = 0.12 µM; CK2α' 2 β 2 IC 50 = 0.12 µM). These results indicate that the preference of THN27 for CK2α' can be further utilized to study the distinct functions of free catalytic subunit paralogs.

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