Your Compound Inhibits the Kinase In Vitro.
But Does It Actually Work Inside the Cell?
A single IC50 value rarely tells the complete story. Combining biochemical activity, intracellular target engagement and functional cellular signaling provides a more complete picture of how a kinase inhibitor behaves.
One IC50 Can Answer Only One Question
A compound that strongly inhibits a purified kinase does not necessarily demonstrate the same potency in an intact cell.
Membrane permeability, physiological ATP concentrations, target expression and competing cellular pathways can all influence compound activity.
This is why orthogonal assay strategies are increasingly useful for kinase drug discovery.
1. Biochemical Kinase Activity
Question: Does the compound inhibit catalytic kinase activity?
Purified-enzyme assays provide a direct way to measure biochemical potency and evaluate selectivity across kinase families.
Reaction Biology offers more than 870 kinase assays, including over 260 clinically relevant mutants, enabling both broad kinome screening and focused resistance profiling.
2. NanoBRET Intracellular Target Engagement
Question: Does the compound actually bind its target inside a living cell?
NanoBRET uses a NanoLuc-tagged target and a cell-permeable fluorescent tracer. When a test compound enters the cell and competes for the target, tracer displacement produces a measurable change in BRET signal.
This enables direct assessment of intracellular target engagement under a physiologically relevant cellular environment.
3. Cellular Phosphorylation
Question: Does target engagement translate into functional signaling inhibition?
Cellular phosphorylation assays measure kinase activity through changes in phosphorylation of the target or a physiological downstream substrate.
NanoBRET therefore measures binding, while cellular phosphorylation provides an orthogonal readout of the functional consequence of that binding.
Three Assays, Three Different Questions
| Assay | Primary Question | Typical Readout |
|---|---|---|
| Biochemical Kinase | Does it inhibit catalytic activity? | IC50 / % inhibition |
| NanoBRET | Does it bind the target inside the cell? | Intracellular affinity / occupancy |
| Cellular Phosphorylation | Does binding inhibit signaling? | Phosphorylation IC50 |
Adding Resistance Mutants to the Picture
In oncology programs, potency against wild-type kinase may not be sufficient. Clinically relevant mutations can significantly alter inhibitor binding and functional activity.
Focused NanoBRET mutant panels are available for targets including ABL1, FLT3, KIT, MET and PIK3CA, allowing direct comparison of wild-type and mutant target engagement.
Running variants in the same cellular background helps isolate potency shifts associated with the mutation itself.
Kinase Drug Discovery Checklist
✓ Have you established biochemical potency?
✓ Has kinome selectivity been evaluated?
✓ Does the compound engage the target inside living cells?
✓ Does target engagement translate into signaling inhibition?
✓ Are clinically relevant resistance mutants included?
✓ Have wild-type and mutant potency shifts been compared?
✓ Do binding and functional cellular results agree?
Planning a Kinase Screening or Drug Discovery Project?
Dana Bioscience can help identify CRO services for biochemical kinase screening,
mutant profiling, NanoBRET intracellular target engagement and
cellular phosphorylation studies.
Share your target, number of compounds, desired assay stages
and project objectives, and we can help evaluate appropriate options.