Luminescence-based cell-based primary high throughput screening assay to identify biased ligands of the melanocortin 4 receptor (MC4R): agonists of MC4R
Name: Luminescence-based cell-based primary high throughput screening assay to identify biased ligands of the melanocortin 4 receptor (MC4R): agonists of MC4R. ..more
BioActive Compounds: 3470
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRISMC)
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Scott DeWire, Trevena Inc
Network: Molecular Library Probe Production Center Network (MLPCN)
Grant Proposal Number: 1 RC2 MH090877-01
Grant Proposal PI: Scott DeWire, Trevena Inc
External Assay ID: MC4R_AG_LUMI_1536_1X%ACT PRUN
Name: Luminescence-based cell-based primary high throughput screening assay to identify biased ligands of the melanocortin 4 receptor (MC4R): agonists of MC4R.
Heterotrimeric G-protein coupled receptors (GPCRs) are major targets for disease therapeutics, due in part to their broad tissue distribution, structural diversity, varied modes of action, and disease-associated mutations (1-4). However, it has recently been demonstrated that GPCRs do not only signal in this simplistic fashion, but rather activate a network of downstream effects comprised of parallel signal transduction pathways. GPCR ligands biased towards induction or blockade of specific signaling pathways may have different physiology compared with unbiased molecules, through selective engagement of a desired subset of signal cascades. For example, the melanocortin 4 receptor (MC4R) transduces its signal via coupling to Gs and adenylyl cyclase activation, and is involved in the regulation of energy homeostasis and chronic disease-associated cachexia. Recent studies indicate that classical antagonists do not mimic MC4R regulation by its endogenous ligand, Agouti-Related Protein (AgRP). Indeed, AgRP has several actions including antagonizing Gs-mediated adenylyl cyclase activation, inducing beta-arrestin recruitment and MC4R endocytosis (5), as well as stimulating Gi-mediated inhibition of adenylyl cyclase (6). As a result, the identification of small molecules that act as biased MC4R ligands, by blocking Gs protein coupling but stimulating beta-arrestin functions and/or Gi protein coupling, may lead to a better understanding of this receptor and its role in metabolic/wasting diseases.
Pan, H.L., Wu, Z.Z., Zhou, H.Y., Chen, S.R., Zhang, H.M., and Li, D.P., Modulation of pain transmission by G-protein-coupled receptors. Pharmacol Ther, 2008. 117(1): p. 141-61.
Lagerstrom, M.C. and Schioth, H.B., Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat Rev Drug Discov, 2008. 7(4): p. 339-57.
Thompson, M.D., Cole, D.E., and Jose, P.A., Pharmacogenomics of G protein-coupled receptor signaling: insights from health and disease. Methods Mol Biol, 2008. 448: p. 77-107.
Bosier, B. and Hermans, E., Versatility of GPCR recognition by drugs: from biological implications to therapeutic relevance. Trends Pharmacol Sci, 2007. 28(8): p. 438-46.
5. Breit, A., Wolff, K., Kalwa, H., Jarry, H., Buch, T., and Gudermann, T. 2006. The natural inverse agonist agouti-related protein induces arrestin-mediated endocytosis of melanocortin-3 and -4 receptors. J Biol Chem 281:37447-37456.
6. Buch, T.R., Heling, D., Damm, E., Gudermann, T., and Breit, A. 2009. Pertussis toxin-sensitive signaling of melanocortin-4 receptors in hypothalamic GT1-7 cells defines agouti-related protein as a biased agonist. J Biol Chem 284:26411-26420.
MC4R, melanocortin, melanocortin 4 receptor, receptor, GPCR, biased ligand, lumi, luminescence, agonist, agonism, activate, activator, activation, increase, cachexia, biased, ligands, primary screen, HTS, high throughput screen, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to identify compounds that activate the melanocortin 4 receptor (MC4R), resulting in membrane recruitment of beta-arrestin. The assay monitors GPCR-beta-arrestin proximity using low affinity fragment complementation of beta-galactosidase (beta-gal). This assay employs U2OS cells which express MC4R fused to a beta-gal peptide fragment (enzyme donor), and beta-arrestin fused to the complementary beta-gal fragment (enzyme acceptor). Cells are incubated with test compound, followed by measurement of well luminescence. As designed, compounds that activate MC4R will cause beta-arrestin recruitment, resulting in reconstitution of the beta-gal holoenzyme. The reconstituted holoenzyme can then catalyze the hydrolysis of a substrate which yields a chemiluminescent signal, resulting in increased well luminescence. Melanotan II will be used as the high (100% RLU) control for agonists, and wells containing cells treated with DMSO will be used as the low (0% RLU) control. Compounds were tested in singlicate at a final nominal concentration of 8.9 uM.
The MC4R cell line was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of a 1:1 mixture of Ham's F-12 Nutrient Media (F-12) and Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% v/v heat-inactivated certified fetal bovine serum, 25 mM HEPES, 250 ug/mL Geneticin, 250 ug/mL Hygromycin B, and 1X antibiotic mix (penicillin, streptomycin, and neomycin). The day before the assay 1000 cells in 3 uL of cell plating media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 C, 5% CO2, and 95 % RH for 23 hours. Next, 27 nL of test compound in DMSO, or DMSO alone were dispensed to the appropriate wells. The plates were then incubated for 3 hours at 37 C, 5% CO2, and 95 % RH. The assay was started by adding 2 uL of PathHunter reagent (prepared according to the manufacturer's protocol); followed by 1 hour incubation at room temperature. Then, Well Luminescence was read on the ViewLux plate reader. The percent activation for each compound was calculated as follows:
%_Activation = ( (Luminescence_Test_Compound - Median_ Luminescence_Low_Control ) / ( Median_ Luminescence_High_Control - Median_ Luminescence_Low_Control ) ) * 100
High_Control is defined as wells containing cells, Melanotan II and DMSO.
Test_Compound is defined as wells containing cells, test compounds and DMSO.
Low_Control is defined as wells containing cells and DMSO.
A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated for each assay plate: (1) the average percent activation of test compound wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for each plate, i.e. any compound that exhibited greater % activation than that particular plate's cutoff parameter was declared active.
PubChem Activity Outcome and Score:
The reported PubChem Activity Score has been normalized to 100% observed activation. Negative % activation values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-1, and for inactive compounds 1-0.
List of Reagents:
U2OS MC4R cell line (DiscoveRx, part 93-0211C3)
PathHunter Detection Kit (DiscoveRx, part, 93-0558C3)
Melanotan II (control agonist, Anaspec, part 61188)
Ham's F-12 media (Invitrogen, part 11765)
DMEM media (Invitrogen, part 11995)
Detachin (Genlantis, part T100100)
Heat Inactivated Fetal Bovine Serum (Invitrogen, part 10082)
Puromycin (Invitrogen, part A11138)
Hygromycin B (Invitrogen, part 10687)
Geneticin (Invitrogen, part 10131)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640)
T-175 tissue culture flasks (Nunc, part 159910)
1536-well plates (Corning, part 7298)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned 'Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that quench or emit luminescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)