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The development of a calcitonin-gene related peptide (CGRP) receptor-binding assay using the LEADseeker Multimodality Imaging System

Key words: CGRP • receptor binding assay •LEADseeker • SPA Imaging Beads


Calcitonin-gene related peptide (CGRP) is believed to have an important physiological role in the central nervous and cardiovascular systems. Indeed, using CGRP receptors isolated from coronary and basilar arteries, in vitro studies have identified a number of CGRP antagonists as promising vasorelaxors (1). CGRP has also been reported to have longer-term effects on neuronal differentiation (2, 3, 4). Recently, a number of small molecule CGRP antagonists have been reported to be efficacious in the treatment of headache and migraine (5, 6).

This application note describes a 384-well CGRP receptor-binding assay which has been developed on the LEADseeker™ Multimodality Imaging System.


Materials
Reagents and instrumentation
LEADseeker Multimodality Imaging System 18-1140-71

Wheat Germ Agglutinin (WGA) PS RPNQ0260
LEADseeker SPA Imaging Beads

(2-[125I]iodohistidyl10) Calcitonin gene- IM184
related peptide (CGRP)


Other material required
Human recombinant CGRP
receptor membrane preparation (Euroscreen ES-420-M)

CGRP (Bachem, H-1470)

Amylin (Bachem, H-7905)

Adrenomedullin (Bachem, H-2932)

α-CGRP (8-37) (Bachem, H9895)

384-well white flat-bottom (Corning 3652)
polystyrene NBS™ microplate

Complete, EDTA-free protease (Roche 1873580)
inhibitor cocktail tablets

Assay buffer: 50-mM Tris:HCl, pH 7.4, containing 5-mM
MgCl2, 0.5% protease-free BSA plus protease inhibitors
(one inhibitor tablet per 50-ml of buffer)

GraphPad Prism™ software (GraphPad Software)


Protocol
Human recombinant CGRP receptor membrane preparation produced in CHO-K1 cells was used in conjunction with [125I]CGRP and WGA PS LEADseeker SPA Imaging Beads. Nonspecific binding (NSB) was determined in the presence of 500-nM CGRP. The standard assay format was as follows.

1. Reagents were added in the following order: buffer or DMSO solution, unlabeled ligand (NSB wells), labeled ligand, and premixed bead and membrane. Total assay volume was 40 µl.

2. Diluted membrane and bead were precoupled at room temperature immediately prior to assay addition.

3. Wells contained 10 µl of 320-pM [125I]CGRP (final concentration 80 pM), 50 µg of SPA Imaging Bead, 0.5 µg of receptor preparation added together in a 10-µl volume, and 20 µl of assay buffer in the absence of competing ligand. For competition assays, 10 µl of competing ligand and 10 µl of assay buffer was added to [125I]CGRP, bead, and receptor, also giving a total assay volume of 40 µl.

4. NSB wells contained 10 µl of 320-pM [125I]CGRP (final concentration 80 pM), 50 µg of SPA Imaging Bead, 0.5 µg of receptor preparation added together in a 10-µl volume, 10 µl of assay buffer, and 10 µl of 2-µM unlabeled CGRP (final concentration 500 nM).

5. For DMSO tolerance studies, DMSO was diluted in assay buffer to give final concentrations per well as shown in the results.

6. Plates were sealed and incubated overnight at room temperature (20–25 ºC).

7. Following incubation, plates were imaged on the LEADseeker Multimodality Imaging System for 5 min with quasi-coincident averaging and 3 x 3 binning.


Results
Kd determination
Saturation binding was carried out with dilutions of [125I]CGRP to give a range of concentrations from 2 to 275 pM in the assay wells. The saturation curve (Fig 1) was fitted using nonlinear regression with the data analysis package GraphPad Prism v4.0. A Kd value of 164 pM (95% confidence intervals 141 to 187 pM) was estimated directly from the curve.

DMSO tolerance
Dilutions of DMSO were prepared in assay buffer to give a range of concentrations from 0.1 to 2.0% (v/v) in the assay wells. From the results shown in Figure 2, it can be seen that the assay was tolerant of DMSO up to a final concentration of 1% (v/v).

IC50 determination
Competition binding of 125-pM [125I]CGRP with unlabeled CGRP, adrenomedullin, amylin, and CGRP (8–37) was assessed and the IC50 values calculated as shown in Figure 3. For the competition assays, the unlabeled ligands were prepared in assay buffer over the following ranges: CGRP (0.12 pM–1.172 µM), adrenomedullin (3.1 pM–30.4 µM), amylin (2.1 nM–32.8 µM), and CGRP (8–37) (3.1 pM–30 µM).

For CGRP, the IC50 value was 0.26 nM (95% confidence interval range 0.2 to 0.33 nM) and the Ki value was 0.16nM (95% confidence interval range 0.12 to 0.22 nM). For adrenomedullin, the IC50 value was 17.2 nM (95% confidence interval range 10.8 to 27.5 nM) and the Ki value was 10.5 nM (95% confidence interval range 6.6 to 16.8 nM). For amylin, the IC50 value was 563 nM (95% confidence interval range 371 to 856 nM) and the Ki value was 343 nM (95% confidence interval range 226.3 to 521.6 nM). For CGRP (837), the IC50 value was 3.86 nM (95% confidence interval range 2.7 to 5.4 nM) and the Ki value was 2.35 nM (95% confidence interval range 1.7 to 3.3 nM).

Time course
A time course experiment was carried out using reagent concentrations as described in the protocol. Measurements were made over an assay incubation time of 18 h. The assay reached equilibrium after 4 h incubation at room temperature, and the signal was stable for at least 14 h (Fig 4).

Z’ analysis
A Z’ analysis was carried out using 48 replicate values for total and NSB wells. Z’ was determined to be 0.64 (Fig 5), which confirmed the robustness of the assay (7).


Conclusions
The CGRP receptor-binding assay has been successfully miniaturized to a 384-well format on LEADseeker Multimodality Imaging System. The assay is suitable for high-throughput screening, and is tolerant up to 1% DMSO. The assay is robust exhibiting a Z’ value of 0.64 and it has a stable signal of at least 14 h.


References
1. Waugh, J.J., et al. Pharmacol. Experiment. Ther. 289, 1419–1426 (1999).
2. Beimer, L.H., et al. Biochem. J. 255, 377-390 (1988).
3. Poyner, D.R., Pharmacol. Ther. 56, 23-53 (1992).
4. Yamamoto, A.I. and Tohyama, M., Prog. Neurobiol. 33, 335–386 (1989).
5. Cutrer, F.M., Headache Currents 1, 13 (2004).
6. Edvinsson L., Cephalagia 25, 163–164 (2005).
7. Zhang, J., et al. J. Biomol. Screening 4, 67–73 (1999).



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