Luminescence-based cell-based high throughput dose response assay for agonists of the mouse 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A)
Name: Luminescence-based cell-based high throughput dose response assay for agonists of the mouse 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A). ..more
BioActive Compounds: 169
Depositor Specified Assays
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: HTR2A_ACT_LUMI_1536_3XEC50 DRUN
Name: Luminescence-based cell-based high throughput dose response assay for agonists of the mouse 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A).
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.
dose response, titration, DRUN, triplicate, 5HT2AR, 5-HT2AR, Htr2a, HTR2A, 5-HT-2, 5-HT-2A, 5-HT2A receptor, 5-hydroxytryptamine 2 receptor, 5-hydroxytryptamine receptor 2A, serotonin receptor 2A, mouse, Mus musculus, agonist, serotonin, mimetic, ligand, GPCR, receptor, G-protein, Gi/Go, DiscoverRx, PathHunter, complementation, reconstitution, enzyme, beta-gal, beta-galactosidase, luminescence, lumi, cell-based, U2OS, endocytosis, fusion protein, enzyme fragment complementation, EFC, ProLink, activate, activator, activation, mood, depression, anxiety, pain, appetite, schizophrenia, HTS, high throughput screen, 1536, Scripps, Scripps Florida, MLSMR, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to determine dose response curves for compounds that confirmed HTR2A activity in a set of experiments entitled, "Luminescence-based cell-based high throughput confirmation assay to identify agonists of the mouse 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A)" (AID 624381). Compounds should activate Htr2a, 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 CHO-K1 cells which express Htr2a 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 Htr2a 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 using a 10-point, 1:3 dilution series starting at a nominal concentration of 76 uM.
The CHO-K1 mHTR2A cell line was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of Ham's F-12 Nutrient Media (F-12) supplemented with 10% v/v heat-inactivated Charcoal/Dextran-treated Fetal Bovine Serum, 800 ug/mL Geneticin, 300 ug/mL Hygromycin B, and 1X Penicillin-Streptomycin-Glutamine.
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, 23 nL of test compound in DMSO, Serotonin (30 uM final concentration) in DMSO, or DMSO alone (0.76% final concentration) were dispensed to the appropriate wells. The plates were then incubated for 2.5 hours at 37 C, 5% CO2, and 95 % RH. The assay was started by adding 2 uL of PathHunterTM 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, Serotonin and DMSO.
Test_Compound is defined as wells containing cells, test compounds and DMSO.
Low_Control is defined as wells containing cells and DMSO.
For each test compound, percent activation was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc). The reported EC50 values were generated from fitted curves by solving for the X-intercept value at the 50% activation level of the Y-intercept value. In cases where the highest concentration tested (i.e. 76 uM) did not result in greater than 50% activation, the EC50 was determined manually as greater than 76 uM.
PubChem Activity Outcome and Score:
Compounds with an EC50 greater than 10 uM were considered inactive. Compounds with an EC50 equal to or less than 10 uM were considered active.
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-81, and for inactive compounds 78-0.
List of Reagents:
PathHunter CHO-K1 mHTR2A Beta-Arrestin Cell Line (DiscoveRx, part 93-0567C2)
PathHunter Detection Kit (DiscoveRx, part 93-0001)
PathHunter Cell Plating 20 Reagent (DiscoveRx, part 93-0563R20B)
Serotonin/5-HT (Control Agonist, Sigma, part H9523)
Ham's F-12 Nutrient Mixture (Invitrogen, part 11765-054)
Charcoal/Dextran-treated Feta Bovine Serum (Hyclone, part SH3006803HI)
Penicillin-Streptomycin-Glutamine (100X) (Invitrogen, 10378-016)
Hygromycin B (Invitrogen, part 10687-010)
Geneticin(R) Selective Antibiotic (Invitrogen, part 10131-027)
Detachin Cell Detachment Reagent (Genlantis, part T100100)
T-175 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.
Assay: Dictionary: Version: 0.1
Assay: CurveFit : Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: CurveFit : Mask: Excluded Points
* Activity Concentration. ** Test Concentration.
Data Table (Concise)