Counterscreen for agonists of OPRM1-OPRD1 heterodimerization: luminescence-based cell-based full-deck high throughput screening assay to identify agonists of 5-hydroxytryptamine (serotonin) 5A receptor (HTR5A)
Name: Counterscreen for agonists of OPRM1-OPRD1 heterodimerization: luminescence-based cell-based full-deck high throughput screening assay to identify agonists of 5-hydroxytryptamine (serotonin) 5A receptor (HTR5A). ..more
BioActive Compounds: 2034
Depositor Specified Assays
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Lakshmi A. Devi, Mount Sinai School of Medicine
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: R03NS053751
Grant Proposal PI: Lakshmi A. Devi, Mount Sinai School of Medicine
External Assay ID: HTR5A_AG_LUMI_1536_1X%ACT
Name: Counterscreen for agonists of OPRM1-OPRD1 heterodimerization: luminescence-based cell-based full-deck high throughput screening assay to identify agonists of 5-hydroxytryptamine (serotonin) 5A receptor (HTR5A).
Opiates such as morphine are the choice analgesic in the treatment of chronic pain due to their potent and rapid action. However, their long-term use is limited because of the development of tolerance and dependence, as well as respiratory suppression and constipation (1). Due to their clinical importance, various strategies have been considered for making opiates more effective while curbing liabilities such as addiction. One such strategy has been to use a combination of drugs to improve the effectiveness of morphine. The OPRM1 gene encodes the mu opioid receptor, which is the primary site of action for morphine (2) and other commonly used opioid such as heroin, fentanyl, and methadone. OPRM1 activation and subsequent dissociation of the Gi/Go G-proteins results in reduction of adenylyl cyclase-mediated cAMP production (3). There are at least two other types of opioid receptors: delta (OPRD1) and kappa (OPRK1), each with a distinct pharmacologic profile. In particular, delta (OPRD1) opioid receptor ligands have been useful in enhancing morphine's potency, but the underlying molecular basis is not understood (4). It has been shown that modulation of receptor function by physical association between mu and delta opioid receptors is a potential mechanism (5). The assay provider has previously found that a combination of OPRM1 agonist with OPRD1 antagonist selectively activates the OPRM1-OPRD1 heteromer (5) and recently showed that this could be blocked by antibodies that selectively recognize the heteromer (6). Since OPRD1 antagonist have anxiogenic effects, these are not ideal as therapies. Hence, the identification of compounds that selectively activate mu-delta opioid receptor heterodimerization may have potential in the treatment of pain and alleviate unwanted effects associated with opiate use.
1. Raehal KM, Bohn LM. Mu opioid receptor regulation and opiate responsiveness. AAPS J. 2005 Oct 19;7(3):E587-91.
2. Matthes H, Maldonado R, Simonin F, Valverde O, Slowe S, Kitchen I, Befort K, Dierich A, Le Meur M, Dolle P, Tzavara E, Hanoune J, Roques B, Kieffer B (1996) Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioid-receptor gene. Nature 383:819-823.
3. Maguea SD and Blendy JAOPRM1 SNP (A118G): Involvement in disease development, treatment response, and animal models. Drug and Alcohol Dependence. 2010 May 108 (3): 172-182.
4. Traynor J, Elliot J. Delta-opioid receptor subtypes and cross talk with mu-receptors. Trends Pharmacol Sci 1993 14:84-86.
5. Gomes I, Jordan BA, Gupta A, Trapaidze N, Nagy V, Devi LA. Heterodimerization of mu and delta opioid receptors: A role in opiate synergy. J Neurosci. 2000 Nov 15;20(22):RC110.
6. Gupta, A., Mulder, J., Gomes, I., Rozenfeld, R., Bushlin, I., Ong, E., Lim, M., Maillet, E., Junek, M., Cahill, C.M., Harkany, T. Devi, L.A. Increased abundance of opioid receptor heteromers after chronic morphine administration. Science Signaling 3:ra54, 2010
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The purpose of this assay is to identify compounds that activate the human serotonin receptor HTR5A, resulting in membrane recruitment of beta-arrestin. This assay also serves as a counterscreen to determine whether compounds identified as active in a previous set of experiments entitled, "Luminescence-based cell-based primary high throughput screening assay to identify agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors" (AID 504326) are non-selective GPCR activators/agonists.
The assay monitors GPCR-beta-arrestin proximity using low affinity fragment complementation of beta-galactosidase (beta-gal). This assay employs U2OS cells which express HTR5A 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 HTR5A 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. Serotonin (5HT) 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 9.3 uM.
The U2OS-HTR5A 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, 28 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 trade mark 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 = ( ( RLU_Test_Compound - Median_RLU_Low_Control ) / ( Median_RLU_High_Control - Median_RLU_Low_Control ) ) * 100
High_Control is defined as wells containing cells, 5HT 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 screen. Two values were calculated: (1) the average percent activation of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % activation than the cutoff parameter was declared active.
PubChem Activity Outcome and Score:
The reported PubChem Activity Score has been normalized to 100% observed primary activation. Negative % activation values are reported as activity score zero.
In this assay not all plates were run in the same batch. This resulted in batch-to-batch variation among the different batches of plates, thereby necessitating the use of a plate-based activity cutoff. For this reason the inactive and active scores overlap.
The PubChem Activity Score range for active compounds is 100-2, and for inactive compounds 23-0.
List of Reagents:
PathHuntertrade mark Beta-arrestin recruitment assay, containing the U2OS HTR5A Beta-arrestin cell line; (DiscoveRx, part 930001)
PathHunter Detection Kit (DiscoveRx, part 93-0700C3)
Ham's F-12 media (Invitrogen, part 11765-054)
DMEM media (Invitrogen, part 11995-073)
Detachin (Genlantis, part T100100)
Heat Inactivated Fetal Bovine Serum (Invitrogen, part 10082-147)
Puromycin (Invitrogen, part A11138-02)
Hygromycin B (Invitrogen, part 10687-010)
Geneticin (Invitrogen, part 10131-027)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
T-175 tissue culture flasks (Nunc, part 159910)
Agonist: Serotonin hydrochloride (5HT) (Sigma-Aldrich, part H9523)
1536-well plates (Greiner, part 789173)
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. 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 modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.
** Test Concentration.
Data Table (Concise)