Analysis of inhibitor residence times is increasingly being incorporated into lead development efforts because longer engagement with the target can result in improved efficacy, increased therapeutic window and reduced side effects. In this application note, BellBrook Labs scientist Meera Kumar, used a jump-dilution protocol with the Transcreener® ADP2 FP Kinase Assay to determine dissociation rates and residence times for kinase inhibitors using simple HTS-compatible methods.
The duration of residence time may be highly relevant for assessing the durable pharmacologic effects of a drug candidate. In general, longer residence time results in improved efficacy, as the extended contact between drug and enzyme results in extended inhibition of enzyme activity. This in turn allows longer pharmacological effects at lower doses, reducing off-target effects.
Residence time (τ) is the time that a drug remains bound to its target before dissociating. Residence time is the reciprocal of dissociation rate (koff).
The dissociation rate constant koff can be determined using a “jump dilution” method in which enzyme activity is monitored over time as an inhibitor dissociates. The jump dilution method is performed in four steps:
- STEP 1. Incubate the kinase with a saturating concentration of inhibitor (e.g., 10 × IC50).
- STEP 2. Dilute the kinase/inhibitor mixture into a solution containing all enzyme reaction components and Transcreener® ADP2 detection reagents.
- STEP 3. Measure fluorescent signal periodically to monitor the recovery of kinase activity over time.
- STEP 4. Determine koff by fitting enzyme progress curves to an integrated rate equation:
Residence times for Dasatinib, Imatinib, Ponatinib and Nilotinib were determined for Abl1 using the jump dilution method described above using the Transcreener® ADP2 FP Kinase Assay. For Abl, compound incubations were performed with an Abl concentration of 280 nM and inhibitor concentrations of 45 nM (Dasatinib), 45 μM (Imatinib), 25 μM (Nilotinib) or 100 nM (Ponatinib). A 100-fold jump dilution was performed by adding 19.8 μL of 10 μM Abltide and 5 μM ATP in presence of detection reagents. Tau values were calculated by taking the reciprocal of koff.
See more data. Check out “A Guide to Measuring Drug-Target Residence Times with Biochemical Assays”