Understanding your drug-target binding kinetics is crucial for your drug-discovery project
Although the drug-target affinity is used most often for decisions in drug discovery projects, it has been postulated that a longer drug-target residence time leads to higher efficacy of the drug. In our 2020 study (ACS, 2018), we observed a correlation of longer residence times with increasing clinical stages. We therefore think it is crucial to your drug discovery project to learn more about the binding kinetics of your inhibitor-target complex. Various in vitro methods are available for determining the dissociation rate constant (koff) that can be used to calculate the residence time (r=1/koff). We want to increase the model complexity and determine your inhibitor-target half-life in living cells performing a wash-out experiment using the NanoBRET technology (r=t1/2/ln(2)).
CELLKinib – We want to determine your drug-target half-life in living cells
We start the wash-out experiment with a pre-incubated inhibitor saturated drug-target complex where 90% of the target binding sites are occupied by the inhibitor in the cells. To do that we previously need to have determined a cellular EC50 using our target engagement service. When the wash-out is performed, a saturating tracer concentration is added and the 90% occupied target quickly will show 10% signal. With increasing time the inhibitor will dissociate from the target making space for the tracer to bind – and consequently the signal will rise in an logarithmic manner. As controls, only tracer (no compound) (100% signal), no tracer (no compound) (0% signal) and a full-occupany control (no wash-out of the compound) are included. This will result in a characteristic curve that will be fitted and the half-life determined. If you want more information on the assay format, please have a look at our recent publication that describes exciting new insights into ABL1 mutant imatinib resistance via a decreased residence time of the drug – despite similar affinity (PNAS, 2021).
- Determine your drug-target half-life in living cells
- We are performing a wash-out experiment starting with 90% target occupany of your inhibitor (EC90).
- With increasing inhibitor dissociation the tracer associates and the signal rises.
- 100% Control, 0% Control and a no-wash-out (full occupany) Control are added.
- Find out more about your drug-target binding kinetics in living cells!
If you have any questions or would like us to determine your drug-target complex half-life, please contact us or get a quote.