title: A universal homogeneous assay for high-throughput determination of binding kinetics.
abstract: There is an increasing demand for assay technologies that enable accurate, cost-effective, and high-throughput measurements of drug-target association and dissociation rates. Here we introduce a universal homogeneous kinetic probe competition assay (kPCA) that meets these requirements. The time-resolved fluorescence energy transfer (TR-FRET) procedure combines the versatility of radioligand binding assays with the advantages of homogeneous nonradioactive techniques while approaching the time resolution of surface plasmon resonance (SPR) and related biosensors. We show application of kPCA for three important target classes: enzymes, protein-protein interactions, and G protein-coupled receptors (GPCRs). This method is capable of supporting early stages of drug discovery with large amounts of kinetic information.
authors.affiliation: Assay Development-High-Throughput Screening, Department of Lead Discovery Berlin, Bayer HealthCare, 13353 Berlin, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Schiele
authors.affiliation: Protein Technologies, Global Drug Discovery, Department of Lead Discovery Berlin, Bayer HealthCare, 13353 Berlin, Germany.
authors.forename: Pelin
authors.initials: P
authors.lastname: Ayaz
authors.affiliation: Assay Development-High-Throughput Screening, Department of Lead Discovery Berlin, Bayer HealthCare, 13353 Berlin, Germany. Electronic address: amaury.fernandez@bayer.com.
authors.forename: Amaury
authors.initials: A
authors.lastname: Fernández-Montalván
mesh_terms: D015374:Biosensing Techniques / Q000379:methods; D018797:Cell Cycle Proteins; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D055808:Drug Discovery / Q000379:methods; D004798:Enzymes / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007700:Kinetics; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011869:Radioligand Assay; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014157:Transcription Factors / Q000378:metabolism; D000092468:Bromodomain Containing Proteins
chemical_list: D018797:Cell Cycle Proteins; D051357:Cyclin-Dependent Kinase 2; D004798:Enzymes; D005456:Fluorescent Dyes; D009687:Nuclear Proteins; D043562:Receptors, G-Protein-Coupled; D014157:Transcription Factors; C441289:BRD4 protein, human; D000092468:Bromodomain Containing Proteins; C495894:CDK2 protein, human
keywords: Binding kinetics; High throughput; Probe competition; TR–FRET