Counterscreen for agonists of the mouse 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A): Luminescence-based cell-based high throughput screening assay to identify agonists of the mu 1 opioid receptor (OPRM1)
Name: Counterscreen for agonists of the mouse 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A): Luminescence-based cell-based high throughput screening assay to identify agonists of the mu 1 opioid receptor (OPRM1). ..more
BioActive Compounds: 120
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Laura Bohn
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: DA025158-01A1
Grant Proposal PI: Laura Bohn
External Assay ID: OPRM1_ACT_LUMI_1536_3X%ACT CSRUN (HTR2A)
Name: Counterscreen for agonists of the mouse 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A): Luminescence-based cell-based high throughput screening assay to identify agonists of the mu 1 opioid receptor (OPRM1).
Serotonin (5-hydroxytryptamine; 5-HT) is an abundant neurotransmitter synthesized from the essential amino acid L-tryptophan and plays a significant role in appetite, platelet aggregation, pain perception, sleep, hormone secretion, sexual behavior, and thermoregulation. Serotonin mediates much of its actions by binding to and activating G protein-coupled receptors (GPCR) on the surface of neurons and other cells. One of these GPCRs, the serotonin 2A receptor (5-HT2AR), is a 7-transmembrane spanning GPCR in the frontal cortex which impacts on development, anxiety, depression, perception, and cognitive function (1, 2). In neurons, the 5-HT2AR is a constitutively internalizing receptor and this trafficking is dependent upon interactions with the regulatory protein, Beta-arrestin2 (3). Studies in cell-based assays reveal that serotonin requires Beta-arrestin2 to internalize the receptors while other agonists, such as DOI, 5-MeO-DMT, and quipazine, internalize the receptor in the absence of Beta-arrestins (3, 4). All 5-HT2AR agonists seem to recruit Beta-arrestin2 to 5-HT2AR in cultured cells. However, while serotonin requires Beta-arrestins to traffic receptors, other agonists such as DOI, 5-MeO-DMT, and quipazine can internalize the receptor in the absence of Beta-arrestins (3, 4). It remains to be determined if the specific 5-HT2AR-Beta-arrestin interactions and trafficking events have physiological significance. It is conceivable that the regulation of 5-HT2AR in vivo may impact neurological sensitivity to serotonin and the responsiveness to pharmacological agents and drugs of abuse. We predict that drugs that disrupt the serotonin 2A receptor-Beta-arrestin interaction might provide a means to alter the sensitivity of the receptor to the levels of serotonin present in the brain(4). These findings may inspire the development of drugs that could be clinically useful for treating depression, schizophrenia, and drug addiction (5-7).
1. Hsieh, C.L., A.M. Bowcock, L.A. Farrer, J.M. Hebert, K.N. Huang, L.L. Cavalli-Sforza, D. Julius, and U. Francke, The serotonin receptor subtype 2 locus HTR2 is on human chromosome 13 near genes for esterase D and retinoblastoma-1 and on mouse chromosome 14. Somat Cell Mol Genet, 1990. 16(6): p. 567-74.
2. Berger, M., J.A. Gray, and B.L. Roth, The expanded biology of serotonin. Annu Rev Med, 2009. 60: p. 355-66.
3. Schmid, C.L., K.M. Raehal, and L.M. Bohn, Agonist-directed signaling of the serotonin 2A receptor depends on beta-arrestin-2 interactions in vivo. Proc Natl Acad Sci U S A, 2008. 105(3): p. 1079-84.
4. Bohn, L.M. and C.L. Schmid, Serotonin receptor signaling and regulation via beta-arrestins. Crit Rev Biochem Mol Biol, 2010. 45(6): p. 555-66.
5. Allen, J.A., P.N. Yadav, and B.L. Roth, Insights into the regulation of 5-HT2A serotonin receptors by scaffolding proteins and kinases. Neuropharmacology, 2008. 55(6): p. 961-8.
6. Raehal, K.M. and L.M. Bohn, The role of beta-arrestin2 in the severity of antinociceptive tolerance and physical dependence induced by different opioid pain therapeutics. Neuropharmacology, 2011. 60(1): p. 58-65.
7. Bohn, L.M. and P.H. McDonald, Seeking Ligand Bias: Assessing GPCR Coupling to Beta-Arrestins for Drug Discovery. Drug Discov Today Technol, 2010. 7(1): p. e37-e42.
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The purpose of this assay is to determine whether compounds identified as active in a set of experiments entitled, "Luminescence-based cell-based primary high throughput screening assay to identify agonists of the mouse 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A)" (AID 624169) activate the mu (OPRM1) opioid receptor or are nonselective activators of beta-arrestin signaling.
The assay monitors GPCR-beta-arrestin proximity using low affinity fragment complementation of beta-galactosidase (beta-gal). This assay employs U2OS cells which express OPRM1 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 OPRM1 will cause beta-arrestin recruitment, resulting in reconstitution of the beta-gal holoenzyme. The reconstituted holoenzyme can then catalyze the hydrolysis of a substrate (GalactonStarSubstrateTM) which yields a chemiluminescent signal, resulting in increased well luminescence. Compounds are tested in triplicate at a final nominal concentration of 9.3 uM.
The U2OS-OPRM1 (Mu) 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, 0.1mM NEAA, 1mM Sodium Pyruvate, 500 ug/mL Geneticin, 250 ug/mL Hygromycin B and 1X antibiotic mix (penicillin, streptomycin, and neomycin).
The day before the assay, 3 uL of cell plating media was dispensed into the first column of 1536 well microtiter plates and 1000 cells in 3 uL of cell plating media were seeded into the remaining wells. Plates were centrifuged and then incubated at 37 C, 5% CO2, and 95 % RH for 24 hours. Next, 28 nL of test compound in DMSO, DAMGO (9.3 uM final concentration) in DMSO, or DMSO alone (0.93% final concentration) 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) to all wells. Plates were centrifuged and after 1 hour of incubation at room temperature, well luminescence was read on a ViewLux microplate reader (Perkin Elmer, Turku, Finland).
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, DAMGO 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 activation and standard deviation of each compound tested were calculated. In addition, two values were calculated: (1) the average percent activation of the wells containing DMSO only, and (2) three times their standard deviation. The sum of these two values was used as a hit cutoff parameter. Any compound that exhibited an average percent activation greater than the calculated hit cutoff was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary activation. Negative % activation values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-0, and for inactive compounds 0-0.
List of Reagents:
PathHunter U2OS OPRM1 Beta-arrestin cell line (DiscoveRx, part, 93-0213C3)
PathHunter Detection Kit (DiscoveRx, part 93-0001)
PathHunter Cell Plating 5 Reagent (DiscoveRx, part 93-0563R5B)
Agonist: DAMGO (Sigma-Aldrich, part E7384)
Ham's F-12 media (Invitrogen, part 11765-054)
DMEM media (Invitrogen, part 11995-065)
Heat Inactivated Fetal Bovine Serum (Invitrogen, part 10082-147)
Hepes (Invitrogen, part 15630-080)
NEAA (Invitrogen, part 11140-050)
Sodium Pyruvate (Invitrogen, part 11360-070)
Hygromycin B (Invitrogen, part 10687-010)
Geneticin (Invitrogen, part 10131-027)
Penicillin-Streptomycin-Neomycin mix (100X) (Invitrogen, part 15640-055)
Detachin (Genlantis, part T100100)
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. 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 not able to provide all compounds selected for testing in this assay.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)