Promega Target Engagement kinase profiling service...now live!
Re-energise your kinase discovery programme
- Do you want to evaluate your compound binding against a panel of kinases in a cellular environment?
- Do you want to understand the kinetic profile of your compound?
- How does your compound behave in a physiological environment (e.g. ATP concentration)?
- How selective is your compound?
Target Engagement (TE) Intracellular Kinase AssaysUsing Promega’s NanoBRET™ technology, we have implemented intracellular kinase assays designed to study the interaction of compounds on kinase targets in intact living cells. NanoBRET™ Target Engagement Assay measures compound binding at specific target kinases. This quantitative analysis can be performed in cultured cells via energy transfer between a 19-kDa luciferase (NanoLuc, Nluc)-tagged target protein and a cell-permeable fluorescent energy transfer probe introduced to the assay medium. Addition of competing compounds results in a dose-dependent decrease in NanoBRET™ signal, which allows quantification of the intracellular affinity of the target kinase for the test compound.
Some of the advantages of this technique are:
- First biophysical technique to profile compound binding for intracellular targets – truly functional pharmacology assay
- Intracellular examination of compound binding to target kinase at physiological ATP concentrations in live cells
- Expression of full-length kinase target in cells
- Examination of the K-on (association rate) and K-off (dissociation rate) with kinase:
– evaluates kinetic selectivity which may drastically differ between kinases in living cells
– equilibrium analysis may fail to predict target occupancy in an open system versus a cellular environment
- The kinase selectivity profile may differ in a biochemical assay compared to a cellular environment due to target occupancy
- Cells can be disrupted (with digitonin) to release cellular content from the cell and the assay can be performed as a pseudo biochemical binding assay
– allows comparison of biochemical versus live cell occupancy binding and affinity
- The approach can be used to look at allosteric inhibitors in addition to ATP competitive inhibitors
- The approach can be used for screening many compounds against one kinase (HTS) or many kinases against one compound (SAR)
- Assay may be used for kinome wide screening of compounds for intracellular target efficacy and off target effects
Competitive inhibition (A) of compound binding to DDR1 kinase in living cells. By studying both the association (B) and dissociation (C) rate of compounds from each of the target kinase it is possible to consider introducing kinetic binding parameter evaluation during compound SAR development.