Homogeneous Cell-Based Fluorescence Polarization Assay for the Direct Detection of cAMP

Abstract

A fluorescence polarization-based functional assay for cyclic AMP (cAMP) production in cells has been proven effective for the detection of agonist-stimulated cAMP production in a HEK 293 recombinant cell line expressing the corticotropin-releasing factor subtype 2alpha (CRF2alpha) receptor. Assays were completed in a single well of a 384-well microplate with no transfer, separation, or wash steps incurred. The assay performance is excellent for adaptation to the high throughput screening environment in terms of speed of analysis, magnitude of displaced signal, precision, and detection limits for cAMP quantitation. Relative potencies of agonists and antagonists are maintained with respect to radiometric assays. The assay withstands up to 5% DMSO and up to 10 microM concentrations of highly colored compound. These attributes suggest that accurate assessment of drug binding can be measured using this assay.

A Flow-Through Fluorescence Polarization Detection System for Measuring GPCR-Mediated Modulation of cAMP Production

“…The limit of detection (LOD) in the plate reader FP assay was 0.8 ± 0.3 pmol cAMP (SEM; n = 6), and the average EC 50 value for cAMP was 37 ± 3 nM (SEM; n = 4). The EC 50 value was similar to the EC 50 value previously determined by Prystay et al 12 Z′ factor: The assay reproducibility and robustness of the plate reader FP cAMP assay was assessed by a Z′ factor calculation using the equation described by Zhang et al 20 In the Z′ factor equation…”

“…A pEC 50 value of 9.6 ± 4.2 µM (SEM; n = 4) was obtained for forskolin, which is consistent with pEC 50 values obtained with other assays. 12,21 Then, dose-response curves were constructed with a set of known hH 3 R agonists (immepip, imetit, imbutamine, histamine, and N-α-methylhistamine) and hH 3 R inverse agonists (clobenpropit, thioperamide, and iodophenpropit) with forskolin as a stimulator of basal cAMP formation. Direct adenylyl cyclase-mediated cAMP formation due to forskolin can be decreased by hH 3 R agonists, whereas hH 3 R inverse agonists result in the production of more cAMP due to indirect interactions with adenylyl cyclase.…”

“…The resulting pEC 50 value of forskolin was 10.1 ± 3.7 µM (SEM; n = 4), which is consistent with data previously obtained with other assay systems. 12,21 Then, dose-response curves were constructed from a set of known hH 3 R agonists (immepip, imetit, imbutamine, histamine, and N-α-methylhistamine) and inverse agonists (clobenpropit, thioperamide, and iodophenpropit). Figure 4D shows pEC 50 curves of the tested ligands and Table 1 the cumulative pEC 50 values of all tested ligands.…”

“…10 The importance of cAMP measurements is reflected by the many different assay formats developed and used for the detection of cAMP. These assay formats were reviewed in 2004 by Williams. 11 Recently, several FP-based assay formats have been reported for cAMP measurements, 4,12,13 which can be used to measure GPCR-mediated cAMP signaling. A drawback of these assays, however, is the fact that the commercially available cAMP FP assays are very expensive (ranging from $200 to $500 per 96-or 384-well plate).…”

A cell-based, high-throughput homogeneous time-resolved fluorescence assay for the screening of potential κ-opioid receptor agonists

“…The assay performance is ideal for adaptation to the HTS environment in terms of the speed of analysis, precision, and detection limits for cAMP quantification. These attributes suggest that this assay can accurately assess potential KOR agonists [24] .…”