A universal homogeneous assay for high-throughput determination of binding kinetics

Anal Biochem. 2015 Jan 1;468:42-9. doi: 10.1016/j.ab.2014.09.007. Epub 2014 Sep 18.

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.

Keywords: Binding kinetics; High throughput; Probe competition; TR–FRET.

MeSH terms

  • Biosensing Techniques / methods
  • Cell Cycle Proteins
  • Cyclin-Dependent Kinase 2 / metabolism
  • Drug Discovery / methods
  • Enzymes / metabolism
  • Fluorescence Resonance Energy Transfer / methods
  • Fluorescent Dyes
  • High-Throughput Screening Assays / methods*
  • Humans
  • Kinetics
  • Nuclear Proteins / metabolism
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Radioligand Assay
  • Receptors, G-Protein-Coupled / metabolism
  • Surface Plasmon Resonance
  • Transcription Factors / metabolism

Substances

  • BRD4 protein, human
  • Cell Cycle Proteins
  • Enzymes
  • Fluorescent Dyes
  • Nuclear Proteins
  • Receptors, G-Protein-Coupled
  • Transcription Factors
  • CDK2 protein, human
  • Cyclin-Dependent Kinase 2