Structural basis for the design of bisubstrate inhibitors of protein kinase CK2 provided by complex structures with the substrate-competitive inhibitor heparin.

The Ser/Thr kinase CK2, a member of the superfamily of eukaryotic protein kinases, has an acidophilic substrate profile with the substrate recognition sequence S/T-D/E-X-D/E, and it is inhibited by polyanionic substances like heparin. The latter, a highly sulphated glucosamino glycan composed mainly of repeating 2-O-sulpho-α-l-idopyranuronic acid/N,O6-disulpho-α-d-glucosamine disaccharide units, is the longest known substrate-competitive CK2 inhibitor. The structural basis of CK2's preference for anionic substrates and substrate-competitive inhibitors is only vaguely known which limits the value of the substrate-binding region for the structure-based development of CK2 bisubstrate inhibitors. Here, a tetragonal and a monoclinic co-crystal structure of CK2α, the catalytic subunit of CK2, with a decameric heparin fragment are described. In the tetragonal structure, the heparin molecule binds to the polybasic stretch at the beginning of CK2α's helix αC, whereas in the monoclinic structure it occupies the central substrate-recognition region around the P+1 loop. Together, the structures rationalize the inhibitory efficacy of heparin fragments as a function of chain length. The monoclinic CK2α/heparin structure, in which the heparin fragment is particularly well defined, is the first CK2 structure with an anionic inhibitor of considerable size at the central part of the substrate-recognition site. The bound heparin fragment is so close to the binding site of ATP-competitive inhibitors that it can guide the design of linkers and pave the way to efficient CK2 bisubstrate inhibitors in the future.

Unexpected CK2ß-antagonistic functionality of bisubstrate inhibitors targeting protein kinase CK2.

Protein kinase CK2, a heterotetrameric holoenzyme composed of two catalytic chains (CK2α) attached to a homodimer of regulatory subunits (CK2ß), is a target for drug development for cancer therapy. Here, we describe the tetraiodobenzimidazole derivative ARC-3140, a bisubstrate inhibitor addressing the ATP site and the substrate-binding site of CK2 with extraordinary affinity (Ki = 84 pM). In a crystal structure of ARC-3140 in complex with CK2α, three copies of the inhibitor are visible, one of them at the CK2ß interface of CK2α. Subsequent interaction studies based on microscale thermophoresis and fluorescence anisotropy changes revealed a significant impact of ARC-3140 and of its tetrabromo equivalent ARC-1502 on the CK2α/CK2ß interaction. A structural inspection revealed that ARC-3140, unlike CK2ß antagonists described so far, interferes with both sub-interfaces of the bipartite CK2α/CK2ß interaction. Thus, ARC-3140 is a lead for the further development of highly effective compounds perturbating the quaternary structure of the CK2α2ß2 holoenzyme.

A π-Halogen Bond of Dibenzofuranones with the Gatekeeper Phe113 in Human Protein Kinase CK2 Leads to Potent Tight Binding Inhibitors.

Human protein kinase CK2 is an emerging target for neoplastic diseases. Potent lead structures for human CK2 inhibitors are derived from dibenzofuranones. Two new derivatives, 7,9-dichloro-1,2-dihydro-8-hydroxy-4-[(4-methoxyphenylamino)-methylene]dibenzo[b,d]furan-3(2H)-one (4a) and (E)-1,3-dichloro-6-[(4-methoxyphenylimino)-methyl]dibenzo[b,d]furan-2,7-diol (5) were tested for inhibition of CK2 and induction of apoptosis in LNCaP cells. Both turned out to be tight binding inhibitors, with IC50 values of 7 nM (4a) and 5 nM (5) and an apparent Ki value of 0.4 nM for both. Compounds 4a and 5 reduced cellular CK2 activity, indicating cell permeability. Cell viability was substantially impaired in LNCaP cells, as well as apoptosis was induced, which was not appearing in non-neoplastic ARPE-19 cells. Co-crystallization of 4a and 5 revealed an unexpected π-halogen bond of the chloro substituent at C9 with the gatekeeper amino acid Phe113, leading to an inverted binding mode in comparison to parent compound 4b, with the Cl at C6 instead, which was co-crystallized as a control. This indicates that the position of the chloro substituent on ring A of the dibenzofuran scaffold is responsible for an inversion of the binding mode that enhances potency.

Protein kinase CK2 inhibition is associated with the destabilization of HIF-1α in human cancer cells.

Screening for protein kinase CK2 inhibitors of the structural diversity compound library (DTP NCI/NIH) led to the discovery of 4-[(E)-(fluoren-9-ylidenehydrazinylidene)-methyl]benzoic acid (E9). E9 induces apoptotic cell death in various cancer cell lines and upon hypoxia, the compound suppresses CK2-catalyzed HSP90/Cdc37 phosphorylation and induces HIF-1α degradation. Furthermore, E9 exerts a strong anti-tumour activity by inducing necrosis in murine xenograft models underlining its potential to be used for cancer treatment in future clinical studies. Crystal structure analysis of human and maize CK2α in complex with E9 reveals unique binding properties of the inhibitor to the enzyme, accounting for its affinity and selectivity.

The protein kinase CK2(Andante) holoenzyme structure supports proposed models of autoregulation and trans-autophosphorylation.

Eukaryotic protein kinases are typically strictly controlled by second messenger binding, protein/protein interactions, dephosphorylations or similar processes. None of these regulatory mechanisms is known to work for protein kinase CK2 (former name "casein kinase 2"), an acidophilic and constitutively active eukaryotic protein kinase. CK2 predominantly exists as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) complexed to a dimer of non-catalytic subunits (CK2ß). One model of CK2 regulation was proposed several times independently by theoretical docking of the first CK2 holoenzyme structure. According to this model, the CK2 holoenzyme forms autoinhibitory aggregates correlated with trans-autophosphorylation and driven by the down-regulatory affinity between an acidic loop of CK2ß and the positively charged substrate binding region of CK2α from a neighboring CK2 heterotetramer. Circular trimeric aggregates in which one-half of the CK2α chains show the predicted inhibitory proximity between those regions were detected within the crystal packing of the human CK2 holoenzyme. Here, we present further in vitro support of the "regulation-by-aggregation" model by an alternative crystal form in which CK2 tetramers are arranged as approximately linear aggregates coinciding essentially with the early predictions. In this assembly, the substrate binding region of every CK2α chain is blocked by a CK2ß acidic loop from a neighboring tetramer. We found these crystals with CK2(Andante) that contains a CK2ß variant mutated in a CK2α-contact helix and described to be responsible for a prolonged circadian rhythm in Drosophila. The increased propensity of CK2(Andante) to form aggregates with completely blocked active sites may contribute to this phenotype.