From Assay Guidance Wiki
In functional screens, the modulation of Gi- and Gs-coupled GPCRs is typically monitored by detecting the intracellular signaling molecule 3’,5’-cyclic adenosine monophosphate (cAMP). cAMP modulates intracellular processes by binding to the regulatory units of protein kinases, resulting in activation of their catalytic subunits, and causing the initiation of protein phosphorylation that alters the function of target enzymes and transcription factors. The production of cAMP is controlled by adenylate cyclases, enzymes that are stimulated or inhibited as a result of direct interaction with G-protein alpha subunits. The role of the adenylate cyclases is to convert ATP to cAMP and inorganic pyrophosphate.
After Gs-coupled GPCR activation, Gαs positively stimulates the activity of adenylate cyclase, resulting in increased production of cAMP. In contrast, the stimulation of Gi-coupled GPCRs results in a negative regulation of the adenylate cyclase and a decrease of cAMP levels. Therefore, Gi-coupled receptors must first be stimulated non-specifically (e.g. with forskolin) in order to detect an inhibition of cAMP production following receptor stimulation. In addition, to counteract the natural degradation of cAMP to AMP that is catalyzed by phosphodiesterase (PDE) enzymes, an inhibitor of PDE (e.g. IBMX) must be present during the assay.
Overview of Available Technologies
cAMP levels are generally measured using a competition assay in which cellular cAMP competes with an introduced, labeled form of cAMP for binding to an anti-cAMP antibody. A number of techniques have been optimized and several easy-to-use kits are commercially available. Initially, assays depended on traditional radiometric assays such as scintillation proximity assays (SPA, Amersham) and Flashplate (Perkin Elmer) using I-125-labeled cAMP. More recently, these have been replaced with cheaper and more convenient non-radiometric assays. Several recent reviews provide detailed comparisons of many popular techniques used for HTS and ultraHTS (see references).
When developing a new assay, the newer approaches to be considered include:
- Enzyme Fragment Complementation (EFC; HitHunter, DiscoverX): Cellular cAMP competes with cAMP that has been labeled with a small peptide fragment of -galactosidase for binding to an anti-cAMP antibody. In the presence of free cAMP, antibody sites are occupied, leaving the labeled cAMP free to complement with the enzyme fragment, producing active B-galactosidase for substrate hydrolysis.
- AlphaScreen® (Perkin Elmer): Detects a chemiluminescent signal between donor and acceptor beads. Cellular cAMP competes with a biotinylated cAMP probe recognized by a streptavidin donor and anti-cAMP conjugated acceptor beads.
- Conventional fluorescence polarization (FP): A fluorescently-labeled cAMP is exposed to polarized light. The emission becomes depolarized due to molecular rotation in the period between excitation and emission. When the labeled cAMP is bound to an antibody, the observed depolarization is reduced relative to when it is free in solution.
- Homogeneous time-resolved fluorescence (HTRF, Cisbio): A modification of the fluorescence resonance energy transfer (FRET) method which uses novel donor-acceptor pairs for the labeled cAMP antibody and cAMP molecule designed to overcome the problem of autofluorescence resulting from unbound fluorophores. In this method, the native, cellular cAMP binds to anti-cAMP antibodies labeled with europium cryptate (donor) in the presence of competing cAMP labeled with a modified allophyocyanin dye d2 (the acceptor). The use of cryptate rather than a chelate as its donor molecule raises the activation energy of the transition state, making the complex highly resistant to varying buffer and pH, thus increasing the stability of the signal.The HTRF assay from Cisbio is sensitive and highly reproducible for measuring cAMP production in response to Gs and Gi-coupled GPCR stimulation.
Assay Optimization and Development
Variables to be evaluated include:
- Cell density
- DMSO tolerance
- Half area vs full area plates
- Incubation time and temperature in stimulation buffer
- Rinsing steps (do cells remain attached?)
- IBMX concentration
- Pre-incubation with compound
- Forskolin concentration (for Gi-coupled receptors)
- Reaction time and temperature
- Concentrations of antibody and labeled cAMP
- Plate Reader (choice and programming)
HTRF® cAMP Assay (Cisbio)
Assay system allows the measurement of agonist and antagonist effects on Gs and Gi coupled GPCRs. Cells are plated for 18-24 hours prior to the start of the assay. Following incubation of approximately 30 minutes in the presence and absence of test compound and stimulation by agonist, the cAMP-d2 conjugate and cryptate conjugate are added and the cells are lysed. Results can be determined on a plate reader after 1 hr and may be stable for several days. The energy transfer is inversely proportional to the concentration of the cellular cAMP in the sample. A standard curve is used to convert raw data to cAMP concentration in the sample. The calculation of the fluorescence ratio (665nm/620nm) normalizes the signal, correcting the results for effects due to the optical characteristics of the media caused by colored compounds, serum, etc.
See the following link for more information: http://www.htrf.com/products/gpcr/camp/
Sample Protocol for Agonist Detection
Assays may be conducted in 96-, 384- and 1536-well formats. Details of the procedure will differ for each cell/receptor of interest and must be determined empirically. Three kits are available, each optimized for a different range of cAMP concentration.
- Adherent cells are plated in black tissue culture-treated or poly-D-lysine-treated microplates 18-24 hours before assay.
- Remove media and replace with freshly-prepared stimulation buffer containing IBMX. Note: Add IBMX while buffer is being stirred to prevent the IBMX from falling out of solution. Incubate at 37C for 30 minutes.
- Remove buffer from cells and add test compounds plus forskolin (for Gi GPCRs) to cells.
- Incubate at 37C for 20 minutes.
- Add diluted HTRF reagents: d2-conjugated cAMP and europium cryptate-conjugated anti-cAMP antibody.
- Incubate at room temperature in the dark for at least 60 minutes before reading on a suitable instrument (e.g. Envision, Tecan GENios). For detailed information about plate readers, please visit http://www.htrf.com/technology/htrfmeasurement/compatible_readers/
- Assay emissions should be detected at 620nm (donor) and 665nm (acceptor) and analyzed using a patented radiometric method that corrects for signal interference. For detailed explanation and background, please visit http://www.htrf.com/technology/htrfmeasurement/radio_data_reduction/#ratio
- Signal stability: Needs to be determined empirically. In some cases, sealed plates have been shown to retain their signal for up to one week at RT.
Results and Data Analysis
Results are calculated from the ratio of absorbance at 665nm/620nm and expressed as Delta F (the standard or sample ratio minus the ratio of the negative control, divided by the ratio of the negative control, times 100). The standard curve is drawn by plotting Delta F% vs cAMP concentration using non-linear least squares fit (sigmoidal dose response variable slope, 4PL). Unknowns are read from the standard curve as pmol concentration of cAMP. Because of the inverse relationship between signal and cAMP concentration, accumulation of cAMP will result in a decreased signal.
Typical Standard Curve
- Cells are stimulated with agonist for optimum time and increase (Gs) or decrease (Gi) in cAMP produced by receptor activation is quantified.
- Max response is max or min cAMP resulting from full agonist stimulation
- Min response is cAMP produced in stimulation buffer alone.
- Results obtained include Relative EC50 and Relative Efficacy (% activity of test compound relative to a reference agonist).
- Cells can be pretreated with test antagonist for predetermined period of time, or antagonist may be added simultaneously with the agonist and forskolin. Agonist is added at EC80-EC90 concentration.
- Optimal incubation period is determined empirically (typically, 20-60 min).
- Inhibition of agonist activity is measured. Results obtained include relative IC50 and Kb.
- Gabriel D, Vernier M, Pfeifer MJ, Dasen B, Tenaillon L, and Bouhelal R. (2003) High throughput screening technologies for direct cyclic AMP measurement. Assay and Drug Development Technologies 1(2), 291-303.
- Weber M, Muthusubramaniam L, Murray J, Hudak E, Kornienko O, Johnson EN, Strulovici B, and Kunapuli P. (2007) Ultra-high-throughput screening for antagonists of a Gi-coupled receptor in a 2.2-µl 3,456-well plate format cyclicAMP assay. Assay and Drug Development Technologies 5(1), 117-125.
- Williams C. (2004) cAMP detection methods in HTS: Selecting the best from the rest. Nature Reviews Drug Discovery 3:125-135.