Competition Binding Assays

Protein kinases catalyze the transfer of ATP's terminal phosphate group to the hydroxyl group of a serine, threonine, or tyrosine residue of the kinase or a separate protein substrate. Since cells have high amounts of ATP, it was thought that employing ATP-competitive inhibitors would necessitate high drug concentrations, which would cause toxicity problems. For the purpose of developing novel small compounds as medications or instruments for chemical biology research, Creative Biolabs offers competitive binding assays that enable inhibitor selectivity for certain protein kinases.

Competition Binding Assays in Creative Biolabs

Through the competitive displacement of a probe, binding assays can be used to detect binding indirectly, or directly, when a ligand binds to its target. To find ATP-noncompetitive ligands for kinase targets, affinity selection mass spectrometry (ASMS) and surface plasmon resonance (SPR) have been employed in direct binding studies. Consequently, these screens bear resemblance to activity-based screens in that ATP-competitive ligands are considerably more likely to be identified by them, and more investi

gation is necessary to ascertain the binding mode of every hit. Competitive binding tests hold great potential in the search for substrate phosphorylation site inhibitors; nevertheless, to yet, these screens have not been applied in the identification process.

Fig.1. ATP-competition binding assays.¹

Kinase Structure and Selective Inhibition

The human genome contains codes for around 500 different protein kinases. The N- and C-terminal domains make up their two primary domains. The linker region's backbone, which connects the domains, is bound by the cofactor ATP. Both an active and an inactive catalytic state are present in many protein kinases. It has been observed that the catalytic domain's structures are more varied when it is inactive. The kinase takes on a form that allows it to engage ATP when it is active, with the binding site open and the required residues oriented correctly to complete the phosphate transfer.

Kinase inhibitors can target the ATP-binding site, the substrate-binding site, or the allosteric regions of the enzyme in either its active or inactive configuration. Every one of these tactics has advantages and disadvantages. Targeting the active state has the advantage of having a well-characterized ATP binding site and a large number of X-ray crystal structures available, which can help with the development of novel inhibitors.

The kinase structure can help with the development of novel inhibitors. (Smyth, et al, 2009) Fig.2. The structure of kinase with ATP.²

ATP-competitive inhibitors make up the great majority of small-molecule kinase inhibitors. More screening techniques are being developed to improve the chances of finding substrate phosphorylation site inhibitors, even though "traditional" HTS using activity-based assays typically does not identify them. These methods include NMR screening with ATP-competitive probes, competitive binding assays, and biased activity tests. Please contact us for more information about our competition binding assays for kinase drug discovery.

References

Breen, Meghan E., and Matthew B. Soellner. "Small molecule substrate phosphorylation site inhibitors of protein kinases: approaches and challenges." ACS Chemical Biology 10.1 (2015): 175-189.
Smyth, Lynette A., and Ian Collins. "Measuring and interpreting the selectivity of protein kinase inhibitors." Journal of Chemical Biology 2 (2009): 131-151.

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