Structure-based design of small peptide inhibitors of protein kinase CK2 subunit interaction

Abstract

X-ray crystallography studies, as well as live-cell fluorescent imaging, have recently challenged the traditional view of protein kinase CK2. Unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumours. Thus the potential intersubunit flexibility suggested by these studies raises the likely prospect that the CK2 holoenzyme complex is subject to disassembly and reassembly. In the present paper, we show evidence for the reversible multimeric organization of the CK2 holoenzyme complex in vitro. We used a combination of site-directed mutagenesis, binding experiments and functional assays to show that, both in vitro and in vivo, only a small set of primary hydrophobic residues of CK2beta which contacts at the centre of the CK2alpha/CK2beta interface dominates affinity. The results indicate that a double mutation in CK2beta of amino acids Tyr188 and Phe190, which are complementary and fill up a hydrophobic pocket of CK2alpha, is the most disruptive to CK2alpha binding both in vitro and in living cells. Further characterization of hotspots in a cluster of hydrophobic amino acids centred around Tyr188-Phe190 led us to the structure-based design of small-peptide inhibitors. One conformationally constrained 11-mer peptide (Pc) represents a unique CK2beta-based small molecule that was particularly efficient (i) to antagonize the interaction between the CK2 subunits, (ii) to inhibit the assembly of the CK2 holoenzyme complex, and (iii) to strongly affect its substrate preference.

The interaction of CK2α and CK2β, the subunits of protein kinase CK2, requires CK2β in a preformed conformation and is enthalpically driven

“…In the case of His193, this is probably a crystal packing artifact, since this residue additionally has different (but well defined) conformations in the two hsCK2b 1-193 monomers. Phe190, however, is known as one of the ''hot spots'' of the CK2a/CK2b interaction from the CK2 holoenzyme structure (Niefind et al 2001) and from a recent kinetic and site-directed mutagenesis study (Laudet et al 2007). This crucial role of Phe190 fits nicely to the change of its side-chain rotamer after CK2a attachment, illustrated in Figure 2B.…”

“…First attempts to develop such substances have been published recently (Laudet et al 2007;Raaf et al 2008). These efforts can be supported by structural and biophysical insights about the CK2a/CK2b interaction.…”

An Unbiased Evaluation of CK2 Inhibitors by Chemoproteomics

“…The production of peptide inhibitors specific for CK2 has been a promising avenue of research as a number of groups have successfully developed and characterized the inhibition of CK2 in cells. Inhibition of CK2 using a CK2-specific peptide resulted in the reduction of CK2 activity and of tumor growth in a mouse model, reiterating CK2 as a promising drug target (43).…”

Force et spécificité du criblage pour des molécules bioactives au CMBA-Grenoble

“…Trois dérivés de REVUES [22]. Une approche peptidique ainsi qu'une stratégie de criblage in silico ont permis d'identifier les premières molécules qui, en ciblant cette interface, inhibent cette interaction protéine-protéine de haute affinité [23]. Ces molécules originales sont à l'origine d'une stratégie non conventionnelle destinée à manipuler l'activité catalytique de CK2 dans les cellules normales ou cancéreuses [9,24].…”