Discovery and characterization of a novel class of cyclic peptidic compounds inhibiting the subunit interaction of the protein kinase CK2α2β2 holoenzyme

Abstract

CK2 belongs to the eukaryotic protein kinase superfamily and has a distinctive heterotetrameric quaternary structure. In the so-called CK2α2β2 holoenzyme, which is the main form of CK2 in human cells, two catalytic subunits (CK2α) are attached to a stable homodimer of non-catalytic subunits (CK2β). Most CK2 inhibitors described so far are ATP-competitive. Some of them are the subject of clinical studies because the enzyme is upregulated in many tumours, and CK2 activity contributes to tumour survival. An unconventional method to interfere with CK2 is to disturb the CK2α/CK2β interaction with small molecules. In a high-throughput screen against a 67,000-compound library using a fluorescence anisotropy-based displacement assay, we identified a group of derivatised cyclic pentapeptides that target the CK2β binding site of CK2α. The CK2β-competitive effect of these screening hits was verified using either human CK2α or its paralogous isoenzyme CK2α' as interaction partners. To this end, novel Förster resonance energy transfer-based CK2α/CK2β and CK2α'/CK2β interaction assays were developed and extensively tested. A result of these validation measurements was that the CK2β-antagonists bind more strongly to CK2α than to CK2α' although both paralogs do not differ in the amino acid composition at their CK2β interfaces. Co-crystallisation experiments of two high-affinity binders with CK2α and CK2α' led to three complex structures that confirm the binding of the compounds to the CK2β interface. The structures suggest a critical role of the β4β5-loop for the higher affinity of the compounds to CK2α compared to CK2α'. Furthermore, they offer suggestions on how to enhance their efficacy in the future.