A High-Throughput Method for Measuring Drug Residence Time Using the Transcreener ADP Assay

Analysis of drug-target residence times during drug development can result in improved efficacy, increased therapeutic window, and reduced side effects. Residence time can be estimated as the reciprocal of the dissociation rate ( k_off) of an inhibitor from its target. The traditional methods for measuring k_off require synthesis of labeled ligands or low-throughput label-free methods. To provide an alternative that is better suited to an automated high-throughput screening (HTS) environment, we adapted a classic "jump dilution" catalytic assay method for determination of k_off values for kinase inhibitor drugs. We used the Transcreener ADP² Kinase assay as a universal, homogenous method to monitor the recovery of kinase activity as the drugs dissociated from preformed inhibitor-kinase complexes. We measured residence times for several drugs that bind the epidermal growth factor receptor (EGFR), ABL1, and Aurora kinases and found that the rank ordering of inhibitor k_off values correlated with literature values determined using ligand binding assays. Moreover, very similar results were obtained using the Transcreener assay with fluorescence polarization (FP), fluorescence intensity (FI), and time-resolved Förster resonance energy transfer (TR-FRET) detection modes. This HTS-compatible, generic assay method should facilitate the use of residence time as a parameter for compound prioritization and optimization early in kinase drug discovery programs.