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Related Experiment Videos

A homogeneous enzyme fragment complementation cyclic AMP screen for GPCR agonists.

Rajasree Golla1, Ramakrishna Seethala

  • 1Drug Discovery, Pharmaceutical Research Institute, Bristol-Myers Squibb, Princeton, NJ, USA.

Journal of Biomolecular Screening
|November 6, 2003
PubMed
Summary

A new nonradioactive assay accurately measures cyclic adenosine monophosphate (cAMP) levels in cells using enzyme fragment complementation (EFC) technology. This robust, sensitive, and automatable assay is ideal for high-throughput screening (HTS) drug discovery.

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Area of Science:

  • Biochemistry
  • Assay Development
  • Pharmacology

Background:

  • High-throughput screening (HTS) relies on robust, homogeneous, and automation-friendly functional assays.
  • Traditional methods for measuring cyclic adenosine monophosphate (cAMP) often involve radioactivity or are less amenable to automation.
  • G protein-coupled receptors (GPCRs) signaling pathways frequently involve modulation of intracellular cAMP levels.

Purpose of the Study:

  • To evaluate a nonradioactive homogeneous HTS assay utilizing enzyme fragment complementation (EFC) technology for measuring cAMP.
  • To assess the performance of the EFC-based cAMP assay in both adherent and suspension cells.
  • To validate the assay's utility for screening compounds targeting Galpha(s)-coupled receptors.

Main Methods:

Related Experiment Videos

  • Developed and optimized a homogeneous assay using HitHunter™ enzyme fragment complementation (EFC) technology to measure cAMP.
  • Utilized beta-galactosidase (beta-gal) donor and acceptor fragments for cAMP detection, where cAMP competes with a labeled conjugate for antibody binding, modulating enzyme activity.
  • Validated the assay using known agonists like glucagon-like peptide-1 (GLP-1), forskolin, and exendin, as well as adenylate cyclase inhibitors.

    • Main Results:

    • The EFC-cAMP assay demonstrated high sensitivity and robustness (Z' = 0.7-0.8) in both adherent and suspension cells, with results comparable to traditional methods.
    • GLP-1 stimulation of cAMP via Galpha(s) signaling was accurately measured (EC50 ~0.3 nM) in both cell formats.
    • The assay showed tolerance to DMSO (up to 10%) and minimal interference from colored compounds like tartrazine, with a positive signal output.

    Conclusions:

    • The nonradioactive EFC-based cAMP assay is a sensitive, robust, and automatable method suitable for HTS campaigns.
    • The assay's positive signal generation and tolerance to common assay components reduce false positives and simplify screening.
    • This validated assay provides a valuable tool for drug discovery targeting receptors that modulate cAMP levels.