Navigation Links
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).



back to top
'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. The development of a CXCR2 chemokine receptorbinding assay using the LEADseeker Multimodality Imaging System
2. The development of a melanocortin MC4 receptor binding assay using the LEADseeker Multimodality Imaging System
3. The development of a calcitonin-gene related peptide (CGRP) receptor-binding assay using the LEADseeker Multimodality Imaging System
4. The development of a somatostatin sst4 receptor-binding assay using the LEADseeker Multimodality Imaging System
5. The development of a serotonin 5-HT2C receptor-binding assay using the LEADseeker Multimodality Imaging System
6. Screening for potential beta 2-adrenergic receptor antagonists using CypHer5E and IN Cell Analyzer 1000
7. The use of CypHer5 for receptor internalization studies in both a range of GPCRs and a non-GPCR
8. The use of CypHer5E and the IN Cell Analyzer 1000 for live-cell receptor internalization studies
9. Screening for β2-adrenergic receptor agonists using the pH-sensitive dye,CypHer5, and the IN Cell Analyzer 3000
10. Development of a histamine H1 receptor binding assay using the LEADseeker Multimodality Imaging System
11. Development of a sensitive enzyme fragment complementation assay for cyclic adenosine 3, 5 monophosphate and its validation for pharmacological screening at G protein-coupled receptors
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:5/26/2016)... ... May 26, 2016 , ... ... announced several positive developments that position the Company for the future. Kinder ... transaction, Craig F. Kinghorn has been appointed Chairman of the Board, Curtis D. ...
(Date:5/25/2016)... ... May 25, 2016 , ... ... of fracture-specific plating options designed to address fractures of the distal tibia and ... , The Acumed Ankle Plating System 3 is composed of seven plate families ...
(Date:5/25/2016)... ... May 25, 2016 , ... WEDI, the nation’s leading authority on the ... Charles W. Stellar has been named by the WEDI Board of Directors as WEDI’s ... an executive leader with more than 35 years of experience in healthcare, association management ...
(Date:5/24/2016)... May 24, 2016   MedyMatch Technology Ltd ., the ... intelligence, real-time decision support tools in the emergency room, announced ... 2016 Israeli Advanced Technology Industries (IATI) BioMed Conference. ... 15th National Life Sciences and Technology Week, and ... Intercontinental Hotel in Tel Aviv, Israel . ...
Breaking Biology Technology:
(Date:4/13/2016)... , April 13, 2016  IMPOWER physicians supporting Medicaid ... setting a new clinical standard in telehealth thanks to ... leveraging the higi platform, IMPOWER patients can routinely track ... and body mass index, and, when they opt in, ... convenient visit to a local retail location at no ...
(Date:3/22/2016)... PUNE, India , March 22, 2016 ... new market research report "Electronic Sensors Market for ... Fingerprint, Proximity, & Others), Application (Communication & ... and Geography - Global Forecast to 2022", ... consumer industry is expected to reach USD ...
(Date:3/15/2016)... , March 15, 2016 --> ... published by Transparency Market Research "Digital Door Lock Systems Market ... 2015 - 2023," the global digital door lock systems market ... in 2014 and is forecast to grow at a CAGR ... micro, small and medium enterprises (MSMEs) across the world and ...
Breaking Biology News(10 mins):