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The development of a melanocortin MC5 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 MC5 (melanocortin subtype 5) receptor. The melanocortin 5 receptor (MC5R) in particular is expressed in numerous human peripheral tissues including adrenal gland, leukocytes, and many others. The only firmly established function of MC5R is its participation in exocrine function particularly sebaceous gland secretion. The role of MC5R in exocrine secretion has the potential to be exploited for the treatment of skin disorders such as acne and dermatitis (1).

This application note describes a 384-well melanocortin MC5 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 IM316
Stimulating Hormone ([125I]α-MSH), direct labeled


Other materials required
Human recombinant melanocortin (Euroscreen, ES-194-M)
MC5 receptor membrane preparation*

NDP-α-MSH (Sigma, M-8764)

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

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

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 phenathroline 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 and Australia. 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 MC5 receptor membrane preparation produced in CHO-K1 cells was used in conjunction with [125I] NDP-α-MSH (2000 Ci/mmol) and WGA PEI Type B PS SPA Imaging Beads. Non-specific binding (NSB) was determined in the presence of 1-µM NDP-α-MSH. The standard assay format was as follows:

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

2. Wells contained 10 µl (~ 44 000 dpm) of [125I] NDP-αMSH (0.25 nM/well), 0.125 mg of SPA Imaging Bead and 0.3125 µg of receptor preparation added tog ether 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 was added with [125I] NDP-α-MSH (10 µl, as above), premixed bead and receptor (20 µl), also giving a total assay volume of 40 µl.

3. NSB wells contained 10 µl of [125I] NDP-α-MSH (0.25-nM/well), 0.125 mg of SPA Imaging Bead and 0.3125 µg of receptor preparation added together in a 20-µl volume, and 10 µl of 4-µM unlabeled NDP-α-MSH (final concentration 1 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 (0.125 mg of bead/well, 10 µl), plus assay buffer (30 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.

* Bead and diluted membrane were premixed at room temperature prior to assay addition.


Results
Kd determination
Saturation binding was carried out with dilutions of [125I] NDP-α-MSH to give a range of concentrations from 1.53 pM to 8.62 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 Kd value of 0.518 nM (95% conf idence intervals 0.4557 to 0.5803 nM) was estimated directly from the curve.

DMSO tolerance
Dilutions of DMSO were prepared in assay buffer to give a range of concentrations from 1 to 10% DMSO (v/v) in the assay wells. The results in Figure 2 show that the assay is DMSO tolerant to 2% (v/v). Above 2% DMSO, an elevation in NSB is observed.

IC50 determination
Competition binding of 0.25-nM (~ 44 000 dpm) [125I] NDP-α-MSH with unlabeled NDP-α-MSH, and α-MSH were assessed and the IC50 values calculated as shown in Figure 3. For the competition assays, the unlabeled ligands NDP-α-MSH and α-MSH were prepared in assay buffer. The IC50 value for NDP-α-MSH was 0.1881 nM (95% confidence interval range 0.1525 to 0.2319 nM), Ki for NDP-α-MSH 0.1262 nM (95% confidence interval range 0.1024 to 0.1556 nM). The IC50 value for α-MSH was 26.66 nM (95% confidence interval range 20.76 to 34.24 nM), Ki for α-MSH was 17.89 nM (95% confidence interval range 13.93 to 22.98 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 20 h. The assay reached equilibrium after 5-h incubation at room temperature and the signal was stable for at least another 5 h (Fig 4).

Z’ analysis
A Z’ analysis was carried out using 48 replicate values for total and NSB wells. Z’ was 0.83 (Fig 5), which confirmed the robustness of the assay (2).


Conclusion
The melanocortin MC5 receptor binding a ssay has been successfully miniaturized to a 384-well format on LEADseeker Multimodality Imaging System and is suitable for high-throughput screening. The assay is tolerant up to 2% DMSO (v/v); is robust, exhibiting a Z’ value of 0.83, and has a stable signal of at least 10 h.


References
1. The Melanocortin System. Am. J. Physiol. Endocrinol. Metab. 284, E468–E474 (2003).

2. Zhang, J. et al, J. Biomol. Screening 4, 67–73 (1999).



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