title: A High-Throughput Method for Measuring Drug Residence Time Using the Transcreener ADP Assay.
abstract: 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 ( koff) of an inhibitor from its target. The traditional methods for measuring koffrequire 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 koffvalues for kinase inhibitor drugs. We used the Transcreener ADP2Kinase 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 koffvalues 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.
authors.affiliation: 1 BellBrook Labs, Madison, WI, USA.
authors.forename: Meera
authors.initials: M
authors.lastname: Kumar
authors.affiliation: 1 BellBrook Labs, Madison, WI, USA.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Lowery
mesh_terms: D025181:ATP Synthetase Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D001681:Biological Assay; D066246:ErbB Receptors / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D008024:Ligands; D047428:Protein Kinase Inhibitors / Q000494:pharmacology
chemical_list: D008024:Ligands; D047428:Protein Kinase Inhibitors; D000244:Adenosine Diphosphate; C512478:EGFR protein, human; D066246:ErbB Receptors; D025181:ATP Synthetase Complexes
keywords: ADP; kinases; koff rates; residence times; transcreener