High Throughput Quantitation of cAMP Production Mediated by Activation of Seven Transmembrane Domain Receptors

Abstract

Impairment of G protein—coupled seven-transmembrane (7 TM) receptor function has been implicated in a variety of different pathologic conditions, suggesting that the discovery of specific antagonists may lead to the development of successful therapeutic agents. The effect of different agents on receptor-ligand interaction is often measured directly in a receptor binding assay; however, this assay format can be time consuming and does not detect agents that interact at sites distal to the native ligand binding site. Cyclic adenosine monophospate (cAMP) represents a ubiquitous second messenger generated in response to ligand binding to many 7 TM receptors. The present study describes the practical adaptation of scintillation proximity methodology, using FlashPlate™ (NEN Life Sciences, Boston, MA) technology to evaluate cAMP production. The bioassay is based on competition between endogenously produced cAMP and exogenously added radiolabeled [125I]-cAMP. Cyclic AMP capture is mediated through an anti-cAMP antibody onto a microplate well surface. Removal of unbound radioligand is not necessary because only ligand within ≤20 μm of the plate surface is detected due to the proximity effect. The data indicate that the use of scintillation proximity technology allows accurate and specific evaluation of G protein—coupled receptor function as measured by cAMP production and is suitable for high throughput screening.

Identification of human dopamine D1-like receptor agonist using a cell-based functional assay1

“…Several high throughput screening methods to screen agonists and antagonists of G-protein coupled receptors (GPCR) have been developed [19][20][21][22] . Recently, we developed a universal functional assay for GPCR [23] .…”

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

“…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.…”

Evaluation of Nonradioactive Cell-Free cAMP Assays for Measuring in vitro Phosphodiesterase Activity

“…High-throughput screening campaigns that seek to identify novel PDE modulators have, until recently, depended on homogeneous radiometric assays such as scintillation proximity assays [2,3] and the Flash plate technology [4] . Recently, nonradioactive assays for cAMP detection like fluorescence polarization (FP) [5] , homogeneous time-resolved fluorescence (HTRF ) [6] , enzyme fragment complementation (EFC) [7] and testing activity coupled to cyclic nucleotide-gated ion channels [8] have been developed.…”