Quantitative, Real-Time Measurements of Intracellular Target Engagement Using Energy Transfer

Methods Mol Biol. 2019;1888:45-71. doi: 10.1007/978-1-4939-8891-4_3.


Intracellular target affinity and residence time are fundamental aspects of pharmacological mechanism (Lu and Tonge, Curr Opin Chem Biol 14:467-474, 2010). Although various robust biochemical approaches exist to measure these binding characteristics, analysis of compound binding with isolated targets may not accurately reflect engagement in the milieu of living cells. To realize the influence of cellular context, methods are needed that are capable of quantifying affinity and residence time in the presence of the intracellular factors that may impact target engagement. Bioluminescence resonance energy transfer (BRET) offers a solution for intracellular target engagement when quantitative metrics or kinetic analyses are required.

Keywords: BRET; Intracellular affinity; NanoBRET(TM); Residence time; Target engagement.

MeSH terms

  • Cell Culture Techniques
  • Cell Line
  • Drug Discovery / methods*
  • Fluorescence Resonance Energy Transfer* / methods
  • High-Throughput Screening Assays
  • Humans
  • Luminescent Measurements* / methods
  • Molecular Probes / chemistry
  • Molecular Probes / metabolism
  • Permeability
  • Reproducibility of Results


  • Molecular Probes