Cell-based assays are a great choice for secondary screening after initial hit finding because of their inherent nature of testing compound action in intact, living cells.
Compounds face multiple challenges when put to work in cells such as
- they need to cross the plasma membrane when targeting intracellular kinases
- they meet an abundance of other proteins that they may bind to than the target kinase, potentially leading to off-target effects causing adverse drug reactions
- they are faced with the physiological pH, co-factors, ion concentrations in cells
- they must act on the target in its tertiary structure complete will all post-translational modifications such as glycosylations
Read more about the importance of screening compounds in cell-based assays in our blog: Testing kinase inhibitors where it matters: Drug screening in intact cells.
To facilitate science-driven kinase drug discovery, Reaction Biology has created three different cellular platforms for kinase inhibitor screening in intact cells.
How do they compare?
||NanoBRET Target Engagement Assay
||Cellular Phosphorylation Assay
||BaF3 Cell Proliferation Assay
||Compound binding to the kinase
||Activity of the kinase
||Cell transformation capability of the kinase
||Binding affinity, target residence times, target occupancy, binding kinetics
||Inhibitor potency, kinase-engaged signaling pathways, transformation capacity of the kinase
||Competitive tracer displacement
||Cell survival and proliferation
||BRET: energy transfer caused by compound-kinase proximity resulting in luminescence
||ELISA or AlphaLISA to quantify phosphorylated substrate
||Cell proliferation assay via Cell Titer Glo
|Detectable inhibitor types
||Oncogenic 'driver' kinases
||Endogenous or exogenous
|Compound incubation period
Our blog Spotlight: Cell-based kinase assay formats. describes our three assay formats in more detail. Check it out!