Exploration and Exploitation of Secondary Sites of Protein Kinase CK2 – XPLOR_CK2

Protein kinase CK2, an acidophilic, pleiotropic and highly conserved member of the eukaryotic protein kinase superfamily, mainly exists as a heterotetrameric holoenzyme consisting of a central dimer of regulatory subunits (CK2ß) with two attached catalytic chains. In humans, two pa-ralogous isoforms of the catalytic CK2 subunit – designated as CK2a and CK2a’ – are present.
CK2 is upregulated in many types of cancer and thus an attractive pharmaceutical target. The typical target region for inhibition of CK2 and other protein kinases is the ATP cavity, therefore often referred to as “primary” site. Due to the conservation of the ATP site, ATP-competitive inhibitors are limited in their selectivity. A strategy to overcome this issue is the use of a “secondary” binding site that can be addressed either alone or in combination with the ATP site by a so-called bivalent inhibitor. Typical secondary sites are the substrate binding region and allosteric sites located remote of the active centre. Allosteric sites can be preformed or “cryptic”, i.e. not open in the apo-form and becoming accessible only in the presence of a suitable ligand.
An attractive secondary site of CK2a and CK2a’ is the aD pocket, a cryptic site accessible only after conformational adaptations of the helix aD region. In the XPLOR_CK2 project, the aD pocket will be exploited to construct highly potent, selective and cell permeable bivalent CK2 inhibitors with indenoindole-based ATP-competitive anchor groups. If possible, the selectivity shall be boosted even to a level that allows the distinction of the two isoforms CK2a and CK2a’. Selected bivalent inhibitors from these activities will be used to determine high-resolution crystal structures of the two CK2 holoenzymes based on either CK2a or CK2a’.
Further secondary sites in the focus of the XPLOR_CK2 project are the substrate recognition region, the heparin site and the N-terminal segment site. A recently described substrate peptide will be used to solve the first CK2a/CK2a’ structure with a visible peptide substrate attached to the substrate binding region. The heparin site was discovered in CK2a; now, it shall be detected in CK2a’ as well and exploited to construct indenoindole-based bivalent inhibitors, which address parts of the heparin site. The N-terminal segment site is new and cryptic: it was unexpectedly found in CK2a’ crystals after soaking with a putative bisubstrate inhibitor. In XPLOR_CK2, its existence shall be confirmed with CK2a and in a non-crystalline state; in case of validation, its overlap with the substrate recognition region shall be exploited to generate a selective substrate-competitive CK2 inhibitor.