Single Step Determination of Unlabeled Compound Kinetics Using a Competition Association Binding Method Employing Time-Resolved FRET
The competition association binding method allows the characterization of the kinetics of unlabeled compounds and the calculation of receptor-drug affinity (Kd). The Kd value is defined as the ratio of the dissociation constant (or koff) of the receptor-bound ligand to its association rate constant (or kon) for a system at equilibrium. Traditionally, competition association binding experiments have been carried out using radiometric detection methods with limited assay throughput. Here we describe a novel method for the determination of unlabeled compound kinetics using the technique of time-resolved fluorescence resonance energy transfer (TR-FRET) performed at physiological temperature and sodium ion concentration. Based on a traditional screening format (10-point curves), up to 28 compounds can be tested on a single 384-well plate by this method.
Key wordsTime-resolved fluorescence resonance energy transfer Competition association binding kinetics Fluorescent ligand Terbium cryptate SNAP-tagged GPCR Association rate Dissociation rate
Catherine Wark (BMG Labtech Ltd.) for expert technical assistance and BMG for personal sponsorship of David Sykes. Nicolas Pierre, Louise Affleck, Thomas Roux and François Degorce from CisBio Bioassays for their continuing support. Rob Lane from the Monash Institute for Pharmaceutical Sciences for supplying the dopamine D2 agonists used in the competition association binding studies.
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