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GPCRs possibly represent the most important class of receptors for drug discovery (Wise et al, 2004). Here we describe a muscarinic M1 receptor assay which uses imaging instrumentation, specialised software and radioactive proximity reagents (Cook, 1996). This LEADseekerTM Imaging System consists of a charge-coupled device (CCD) camera and suspension-based europium polystyrene particles that emit light at 615nm. The instrument is capable of capturing the signal from an entire 384-well plate in approximately 5 minutes thus demonstrating the techniques’ utility for high-throughput screening pharmacology. With this assay method, human recombinant muscarinic M1 receptor membrane preparation from CHO-K1 cells was used in conjunction with L-Quinuclidinyl [phenyl-4-3H] benzilate ([3H] QNB) (49 Ci/mMol) and Wheat Germ Agglutinin (WGA)-coated Scintillation Proximity Assay (SPA) Imaging Beads. Non-specific binding (NSB) was determined in the presence of 2µM 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP). Reagents were added in the following order: buffer or buffer containing 2% (v/v) DMSO solution, unlabelled ligand (NSB wells), labelled ligand, pre-mixed bead and membrane. Wells contained 10µl (~13000dpm) of 12nM [3H] QNB (final concentration 3nM), 250µg SPA imaging bead, 2.5µg receptor preparation added together in a 20µl volume, and 10µl of assay buffer in the absence of competing ligand. For competition assays, 10µl of competing ligand prepared in buffer containing 2% (v/v) DMSO was added with [3H] QNB (10µl), pre-mixed bead and receptor (20µl), giving a total assay volume of 40µ l. NSB wells contained 10µl of 12nM [3H] QNB (final concentration 3nM), 250µg SPA bead, 2.5µg receptor preparation added together in a 20µl volume, and 10µl of 8µM unlabelled 4-DAMP (final concentration 2µM). Plates were sealed and incubated overnight at room temperature (20-25 oC). Following incubation, plates were imaged on the LEADseeker instrument for 5 minutes. Saturation binding was carried out with dilutions of [3H] QNB to give a range of concentrations from 0.0053-10nM in the assay wells giving a Kd value of 1.098nM (95% confidence intervals 0.623-1.574nM, n=6). Competition binding of 3nM (~13000dpm) [3H] QNB with unlabelled 4-DAMP and pirenzepine dihydrochloride were assessed. The IC50 value for 4-DAMP was 125.60nM (95% confidence interval range 114.7-137.6nM, n=6), Ki 4-DAMP, 96.47nM (95% confidence interval range 88.1-105.7nM, n=6), and the IC50 value for pirenzepine was 1.21µM (95% confidence interval range 1.08-1.35µM, n=6), Ki pirenzepine 0.93µM (95% confidence interval range 0.82-1.04µM, n=6), results which were similar to previously reported values (Caulfield & Birdsall, 1998). The assay was tolerant of DMSO at concentrations greater than 1% (v/v), with a stable assay signal over 18 hours. Assay robustness was confirmed by Z′ analysis (Zhang et al, 1999). (Assay Z′=0.851). The method described here is ideally suited for high-throughput GPCR screening pharmacology.
Caulfield, M.P. & Birdsall, N.J.M. (1998) Pharmacol. Rev.,50: 279-290. |
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