( Key words: ...)The development of a melanocortin MC4 receptor binding assay using the LEADseeker Multimodality Imaging System

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

Pro-melanocortins are present in high levels in the pituitary and are processed into three major peptide hormone fragments: adrenocorticotrophin (ACTH), the melanocyte stimulating hormones, and β-endorphin. The various melanocyte stimulating hormones are known to activate the MC₄ (melanocortin subtype 4) receptor. The MC₄ receptor has been linked to the regulation of appetite (1). Evidence has suggested that melanocortins are involved in mediating appetite regulation through the effects of leptin and controlling the expression of neuropeptide Y. Thus, the MC₄ receptor is a potential target for anti-obesity agents (2).

This application note describes a 384-well melanocortin MC₄ receptor binding assay, which has been developed on the LEADseeker™ Multimodality Imaging System.

Materials
Products used
LEADseeker Multimodality Imaging System 18-1140-71

Wheat Germ Agglutinin (WGA) PEI RPNQ0289
Type B PS SPA Imaging Beads

[¹²⁵I] (Nle⁴-D-phe⁷) α-Melanocyte Stimulating IM316
Hormone ([¹²⁵I] α-MSH), direct labeled

Other materials required
Human recombinant melanocortin (Euroscreen, ES-191-M)
MC₄ receptor membrane preparation*

NDP-α-MSH (Sigma, M-8764)

α-Melanocyte Stimulating Hormone (α-MSH) (Sigma, M-4135)

β-Melanocyte Stimulating Hormone (β-MSH) (Sigma, M-6513)

384-well white flat bottom polystyrene (Corning, 3705)
not treated microplates

Assay buffer: 25 mM HEPES, pH 7.4
5 mM MgCl₂
1 mM CaCl₂
1 mM 1,10 1,10-phenanthroline monohydrate 0.2% (w/v) protease-free BSA

GraphPad Prism™ software (GraphPad Software)

* Use of the receptor in this application note is not permitted in North America. For further information on the use of this receptor in restricted territories, please contact Euroscreen’s Business Development and Licensing Manager at vlannoy@euroscreen.be

Protocol
Human recombinant melanocortin MC₄ receptor membrane preparation produced in CHO-K1 cells was used in conjunction with [¹²⁵I] NDP-α-MSH (2000 Ci/mmol) and PEI-treated, WGA-coupled SPA Imaging Beads, Type B. Non-specific binding (NSB) was determined in the presence of 2-µM NDP-α-MSH. The standard assay format was as follows:

1. Reagents were added in the following order: assay buffer or buffer containing 2% (v/v) DMSO solution, unlabeled ligand (NSB wells), labeled ligand, premixed bead and membrane*. Total assay volume was 40 µl.

2. Wells contained 10 µl (~ 88 000 dpm) of 2-nM [¹²⁵I] NDP-α-MSH (final concentration 0.5 nM), 31.25 µg of SPA Imaging Bead, 250 ng of receptor preparation added together in a 20-µl volume, and 10 µl of assay buffer in the absence of competing ligand. For competition assays, 10 µl of competing ligand prepared in assay buffer containing 2% (v/v) DMSO was added with [¹²⁵I] NDP-α-MSH (10 µl, as above), precoupled bead and receptor (20 µl), also giving a total assay volume of 40 µl.

3. NSB wells contained 10 µl of 2-nM [¹²⁵I] NDP-α-MSH (final concentration 0.5 nM), 31.25 µg of SPA Imaging Bead, 250 ng of receptor preparation added together in a 20 µl volume, and 10 µl of 8-µM unlabeled NDP-α-MSH (final concentration 2 µM).

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

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

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

7. For background estimation, bead-only wells (31.25 µg of bead/well, 20 µl), plus assay buffer (20 µl) were included in all experiments. Typically backgrounds exhibited from bead-only wells were less than 10 integrated optical density (IOD) units, and were routinely subtracted from total and NSB values during data analyses.

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

Results
K_d determination
Saturation binding was carried out with dilutions of [¹²⁵I] NDP-α-MSH to give a range of concentrations from 4.05 pM to 10.43 nM in the assay wells. Figure 1 shows the saturation curve which was fitted using non-linear regression with the data analysis package GraphPad Prism v4.0. A K_d value of 0.931 nM (95% confidence intervals 0.744 to 1.117 nM) was estimated directly from the curve.

DMSO tolerance
Dilutions of DMSO were prepared in assay buffer to give a range of concentrations from 0.5 to 32% (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 2% (v/v).

IC₅₀ determination
Competition binding of 0.5-nM (~ 88 000 dpm) [¹²⁵I] NDP-αMSH with unlabeled α- and β-MSH, were assessed and the IC₅₀ values calculated as shown in Figure 3. For the competition assays, the unlabeled ligands were prepared in assay buffer containing 2% (v/v) DMSO over the range of 12.2 nM to 100 µM. The IC₅₀ value for α-MSH was 1.13 µM (95% confidence interval range 0.804 to 1.595 µM), K_i, α-MSH, 0.737 µM (95% confidence interval range 0.524 to 1.04 µM), the IC₅₀ value for α-MSH was 0.567 µM (95% confidence interval range 0.497 to 0.647 µM), K_i, α-MSH 0.369 µM (95% confidence interval range 0.324 to 0.422 µM).

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 20.5 h. The assay reached equilibrium after 8-h incubation at room temperature and the signal was stable for at least another 12.5 h (Fig 4).

Z’ analysis
A Z’ analysis was carried out using 48 replicate values for total and NSB wells. Z’ was 0.796 (Fig 5), which confirmed the robustness of the assay (3).

Conclusion
The melanocortin MC₄ receptor binding assay has been successfully miniaturized to a 384-well format on LEADseeker Multimodality Imaging System and is suitable for high-throughput screening formats. The assay is tolerant up to 2% DMSO (v/v); is robust, exhibiting a Z’ value of 0.789, and has a stable signal of at least 12.5 h.

References
1. Chiesi, M. et al. Trends in Pharmacol. Sci. 22, 247–254 (2001).
2. Schwartz, M.W. et al. Nature 404, 661–671 (2000).
3. Zhang, J. et al. J. Biomol. Screening 4, 67–73 (1999).

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