Binding assay for characterization of protein kinase inhibitors possessing sub-picomolar to sub-millimolar affinity.

High demand for inhibitors regulating the activity of protein kinases has stimulated the quest for high throughput and reliable compound screening assays. Here we introduce a method applying a non-metal photoluminescent probe ARC-Lum(Fluo) for determination of dissociation constants of competitive inhibitors of protein kinases. Employing a single probe instead of a combination of antibody and fluorescent tracer makes the assay simpler, cheaper, and more accurate than several other inhibitor-screening technologies. High affinity (20 pM) and low background signal of the free probe supports the determination of dissociation constants of tight-binding as well as low affinity inhibitors. The calculated lowest Kd value that can be accurately determined with the method is 60 fM. We also introduce graphical presentation of the linearized Cheng-Prusoff equation and demonstrate multiple possibilities for its application (deciding upon the assay formats, calculation of the limits of Kd determination, etc.). The open toolbox (http://www.ut.ee/medchem/toolbox-fluorescence-probes) is available for creating the map of resolvable affinities if applying the competitive probes at defined assay conditions.

Inhibition of CREB phosphorylation by conjugates of adenosine analogues and arginine-rich peptides, inhibitors of PKA catalytic subunit.

Bisubstrate inhibitors of protein kinases associate simultaneously with two substrate-binding sites of the kinase and thus potentially possess better inhibitory potency and selectivity than inhibitors binding to only the conserved ATP-site of the kinase. We have previously used conjugates of adenosine analogues and arginine-rich peptides (ARCs) to develop proteolytically stable cell plasma membrane-permeable bisubstrate inhibitors whose biochemical affinities towards several basophilic protein kinases of the AGC group are in the picomolar range. The potency of bisubstrate inhibitors to affect the phosphorylation of proteins in living cells has been described in a limited number of publications. In this study, the effect of ARCs on the protein kinase A (PKA)-catalysed cAMP response element-binding protein (CREB) phosphorylation pathway was studied in living mammalian cells. Our results demonstrate that at low micromolar extracellular concentration N-myristoylated ARCs are capable of reducing the activity of transcription factor CREB through inhibition of PKA.

Acetoxymethyl Ester of Tetrabromobenzimidazole-Peptoid Conjugate for Inhibition of Protein Kinase CK2 in Living Cells.

CK2 is a ubiquitous serine/threonine protein kinase, which has the potential to catalyze the generation of a large proportion of the human phosphoproteome. Due to its role in numerous cellular functions and general anti-apoptotic activity, CK2 is an important target of research with therapeutic potential. This emphasizes the need for cell-permeable highly potent and selective inhibitors and photoluminescence probes of CK2 for investigating the protein phosphorylation networks in living cells. Previously, we had developed bisubstrate inhibitors for CK2 (CK2-targeted ARCs) that showed remarkable affinity (KD < 1 nM) and selectivity, but lacked proteolytic stability and plasma membrane permeability. In this report, the structures of CK2-targeted ARCs were modified for the application in live cells. Based on structure-activity studies, proteolytically stable achiral oligoanionic peptoid conjugates of 4,5,6,7-tetrabromo-1H-benzimidazole (TBBz) were constructed. Affinity of the conjugates toward CK2 reached subnanomolar range. Acetoxymethyl (AM) prodrug strategy was applied for loading TBBz-peptoid conjugates into living cells. The uptake of inhibitors was visualized by live cell imaging and the reduction of the phosphorylation levels of two CK2-related phosphosites, Cdc37 pSer13 and NFκB pSer529, was demonstrated by Western blot analysis.

Competitive ligands facilitate dissociation of the complex of bifunctional inhibitor and protein kinase.

Dissociation of the complex of a ligand and a protein usually follows the kinetic profile of the first order process and the rate of dissociation is not affected by the presence of competitive ligands. We discovered that dissociation of the complex between a bifunctional ligand and a protein kinase (the catalytic subunit of cAMP-dependent protein kinase), an enzyme possessing 2 different substrate binding sites, was accelerated (facilitated) over 50-fold in the presence of competitive ligands at higher concentrations. Structurally diverse compounds revealed >10-fold different efficiency for acceleration of dissociation of the complex. These results show that the kinetic behavior of flexible biomolecular complexes possessing two spatially separated contact areas is highly dynamic. This property of biomolecular complexes should be carefully considered for effective application of bifunctional ligands for regulation of activity of target proteins in cells.