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Real-Time cAMP Dynamics in Live Cells Using the Fluorescent cAMP Difference Detector In Situ
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A homogeneous single-label time-resolved fluorescence cAMP assay.

Eija Martikkala1, Anita Rozwandowicz-Jansen, Pekka Hänninen

  • 1Laboratory of Biophysics and Medicity, University of Turku, Turku, Finland. emliuk@utu.fi

Journal of Biomolecular Screening
|February 24, 2011
PubMed
Summary

A new single-label assay using quenching resonance energy transfer (QRET) efficiently measures cyclic adenosine monophosphate (cAMP) levels for G-protein-coupled receptor (GPCR) drug discovery. This cost-effective method offers excellent reproducibility for high-throughput screening.

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Area of Science:

  • Biochemistry
  • Pharmacology
  • Cell Biology

Background:

  • G-protein-coupled receptors (GPCRs) are crucial pharmaceutical targets.
  • High-throughput screening (HTS) assays are vital for evaluating novel drug compounds.
  • Measuring cyclic adenosine monophosphate (cAMP) is a standard method for assessing GPCR activity via the adenylyl cyclase pathway.

Purpose of the Study:

  • To develop a novel, single-label, time-resolved fluorescence assay for measuring cAMP levels.
  • To establish a high-throughput screening (HTS)-compatible method for GPCR functional assays.
  • To assess the feasibility and performance of the quenching resonance energy transfer (QRET) technique for GPCR drug discovery.

Main Methods:

  • Development of a single-label time-resolved fluorescence assay utilizing quenching resonance energy transfer (QRET).
  • Application of the QRET assay to HEK293(i) cells expressing β(2)-adrenergic or δ-opioid receptors.
  • Homogeneous competitive assay design measuring antibody-bound, fluorescent europium(III)-labeled cAMP.

Main Results:

  • The QRET assay successfully measured cAMP changes in response to G(s) and G(i) protein-coupled receptor activation.
  • Excellent assay reproducibility with Z' values exceeding 0.7 was achieved.
  • The QRET assay demonstrated comparable performance to a commercial two-label time-resolved fluorescence resonance energy transfer (TR-FRET) assay.

Conclusions:

  • The single-label QRET assay is a robust and cost-effective method for functional GPCR screening.
  • This assay provides a valuable alternative to existing two-label TR-FRET assays, reducing optimization needs.
  • The developed QRET method facilitates efficient drug discovery targeting GPCRs.