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BioAssay: AID 504916

Luminescence-based cell-based high throughput confirmation assay for inverse agonists of 5-hydroxytryptamine (serotonin) 5A receptor (HTR5A)

Name: Luminescence-based cell-based high throughput confirmation assay for inverse agonists of 5-hydroxytryptamine (serotonin) 5A receptor (HTR5A). ..more
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AID: 504916
Data Source: The Scripps Research Institute Molecular Screening Center (HTR5A_IAG_LUMI_1536_3X%INH CRUN)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-07-05

Data Table ( Complete ):           Active    All
BioActive Compounds: 6
Depositor Specified Assays
504634Counterscreen for inverse agonists of OPRM1-OPRD1 heterodimerization: luminescence-based cell-based full-deck high throughput screening assay to identify inverse agonists of 5-hydroxytryptamine (serotonin) 5A receptor (HTR5A)screeningPrimary screen (HTR5A inverse agonists in singlicate)
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_IAG_LUMI_1536_3X%INH CRUN

Name: Luminescence-based cell-based high throughput confirmation assay for inverse 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. The goal of this project is identification of HTR5A inverse agonists.


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


receptor, GPCR, beta-arrestin, confirmation, triplicate, artifact, HTR5A, HTR5A, HTR5A, MGC138226, serotonin receptor 5A, U2OS, PathHunter, complementation, reconstitution, enzyme, beta-gal, beta-galactosidase, lumi, luminescence, luminescent, chemiluminescence, GPCR, receptor, inverse agonist, IAG, inhibit, inhibitor, dimer, heterodimer, homodimer, pain, HTS, high throughput screen, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Assay Overview:

The purpose of this assay is to confirm HTR5A inverse agonist activity of compounds identified as active in a previous set of assays entitled, "Counterscreen for inverse agonists of OPRM1-OPRD1 heterodimerization: luminescence-based cell-based full-deck high throughput screening assay to identify inverse agonists of 5-hydroxytryptamine (serotonin) 5A receptor (HTR5A)" (AID 504634). This assay identifies compounds that inhibit the HTR5A (5HT5A) serotonin receptor, thereby preventing or reducing the 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 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 inhibit HTR5A will prevent beta-arrestin recruitment, thereby reducing the reconstitution of the beta-gal holoenzyme. The decrease in reconstituted holoenzyme will reduce the hydrolysis of a substrate which yields a chemiluminescent signal, resulting in decreased well luminescence. Wells containing Cell Plating Media (No cells) and treated with DMSO will be used as the high (100% RLU) control for inverse agonists, and wells containing cells treated with DMSO will be used as the low (0% RLU) control. Compounds were tested in triplicate at a final nominal concentration of 9.3 uM.

Protocol Summary:

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 TM 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 inhibition for each compound was calculated as follows:

%_Inhibition = ( ( RLU_Test_Compound - Median_RLU_Low_Control ) / ( Median_RLU_High_Control - Median_RLU_Low_Control ) ) * 100


High_Control is defined as wells containing no cells and DMSO,
Test_Compound is defined as wells containing cells, test compounds and DMSO,
Low_Control is defined as wells containing cells and DMSO.

PubChem Activity Outcome and Score:

The average percent inhibition and standard deviation of each compound tested were calculated. Any compound that exhibited an average percent inhibition greater than the modified hit cutoff calculated for the primary screen was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-54, and for inactive compounds 43-0.

List of Reagents:

PathHunter TM B-arrestin recruitment assay, containing the U2OS HTR5A Beta-arrestin cell line; 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)
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 luminescence. 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. The MLSMR was unable to provide all samples requested for testing.
Result Definitions
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition at 9.3 uM (9.3μM**)Normalized percent inhibition of the screen at a compound concentration of 9.3 micromolar.Float%
2Standard DeviationStandard deviation derived from the normalized percent inhibition of the triplicate data for each compound.Float

** Test Concentration.
Additional Information
Grant Number: R03NS053751

Data Table (Concise)