Fluorescence-based cell-based late stage dose response assay to identify antagonists of the human trace amine associated receptor 1 (TAAR1)
Name: Fluorescence-based cell-based late stage dose response assay to identify antagonists of the human trace amine associated receptor 1 (TAAR1). ..more
BioActive Compounds: 7
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: RTI International
Assay Provider: Brian P. Gilmour, RTI International
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1R21NS064780-01A1
Grant Proposal PI: Brian P. Gilmour, RTI International
External Assay ID: TAAR1_ANT_FLUO8_1536_3XIC50 MDRUN
Name: Fluorescence-based cell-based late stage dose response assay to identify antagonists of the human trace amine associated receptor 1 (TAAR1).
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). TAAR1 (trace amine associated receptor 1) is a G protein-coupled receptor activated by trace amines. Trace amines (TA) such as Beta-phenethylamine (Beta-PEA), p-tyramine (TYR), octopamine, and tryptamine are endogenous amine compounds that account for less than 1% of the biogenic amines in most brain regions (5), and exert pharmacological actions in humans (6,7). In addition to binding Beta-PEA and TYR, rat TAAR1 is also activated by dopamine, octopamine, tryptamine, amphetamine, and lysergic acid (8-10). TAs are of particular interest because they have been shown to modulate the activity of neurotransmitters such as dopamine (11-13) and gamma-amino butyric acid (14-16) and alterations in their brain levels are associated with schizophrenia (17-19) and depression (20, 21). Their potential to modulate dopaminergic activity suggests that they may play a role in the efficacy of L-DOPA in treating Parkinson disease4 and addiction (13). In humans, only TAAR1 has been shown to be activated by Beta-PEA and TYR, resulting in increased cyclic adenosine monophosphate (cAMP) accumulation through coupling to Gs (9, 10). Endogenous hTAAR1 activity is predominantly coupled to G-alpha-s and the accumulation of cAMP. The variety of compounds potentially acting at this receptor makes it attractive to assume that hTAAR1 is involved in a variety of integrated CNS processes such as mood and cognition. As a result, hTAAR1 is an interesting target for the development of ligands to probe the role of this receptor in CNS function and disease. For this project, the assay provider has created a cell line expressing the hTAAR1 in a parent cell line (RD-HGA16 cells, Molecular Devices) that stably over expresses the promiscuous G-protein, G-alpha-16, thereby coupling hTAAR1 activation to mobilization of internal calcium stores. Because TAs may be involved in modulating a variety of behaviors including mood, cognition, and addiction, it is of interest to discover novel ligands for TAAR1 to probe the role TAs play in brain function (22, 23).
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2. 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.
3. 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.
4. 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. Bunzow JR, Sonders MS, Arttamangkul S, Harrison LM, Zhang G, Quigley DI, Darland T, Suchland KL, Pasumamula S, Kennedy JL, Olson SB, Magenis RE, Amara SG, Grandy DK. Amphetamine, 3,4-methylenedioxymethamphetamine, lysergic acid diethylamide, and metabolites of the catecholamine neurotransmitters are agonists of a rat trace amine receptor. Mol Pharmacol. 2001 Dec;60(6):1181-8.
6. Premont RT, Gainetdinov RR, Caron MG: Following the trace of elusive amines. Proc Natl Acad Sci U S A 2001;98:9474-9475.
7. Branchek TA, Blackburn TP: Trace amine receptors as targets for novel therapeutics: legend, myth and fact. Curr Opin Pharmacol 2003;3:90-97.
8. Borowsky B, Adham N, Jones KA, Raddatz R, Artymyshyn R, Ogozalek KL, et al: Trace amines: identification of a family of mammalian G protein-coupled receptors. Proc Natl Acad Sci U S A 2001;98:8966-8971.
9. Bunzow JR, Sonders MS, Arttamangkul S, Harrison LM, Zhang G, Quigley DI, et al: Amphetamine, 3,4-methylenedioxymethamphetamine, lysergic acid diethylamide, and metabolites of the catecholamine neurotransmitters are agonists of a rat trace amine receptor. Mol Pharmacol 2001;60:1181-1188.
10. Lindemann L, Ebeling M, Kratochwil NA, Bunzow JR, Grandy DK, Hoener MC: Trace amine-associated receptors form structurally and functionally distinct subfamilies of novel G protein-coupled receptors. Genomics 2005;85:372-385.
11. Jones RS: Specific enhancement of neuronal responses to catecholamine by p-tyramine. J Neurosci Res 1981;6:49-61.
12. Mercuri NB, Bernardi G: The "magic" of L-dopa: why is it the gold standard Parkinson's disease therapy? Trends Pharmacol Sci 2005;26:341-344.
13. Miller GM, Verrico CD, Jassen A, Konar M, Yang H, Panas H, et al: Primate trace amine receptor 1 modulation by the dopamine transporter. J Pharmacol Exp Ther 2005;313:983-994.
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15. Berretta N, Giustizieri M, Bernardi G, Mercuri NB: Trace amines reduce GABA(B) receptor-mediated presynaptic inhibition at GABAergic synapses of the rat substantia nigra pars compacta. Brain Res 2005;1062:175-178.
16. Federici M, Geracitano R, Tozzi A, Longone P, Di Angelantonio S, Bengtson CP, et al: Trace amines depress GABA B response in dopaminergic neurons by inhibiting G-betagamma-gated inwardly rectifying potassium channels. Mol Pharmacol 2005;67:1283-1290.
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19. Duan J, Martinez M, Sanders AR, Hou C, Saitou N, Kitano T, et al: Polymorphisms in the trace amine receptor 4 (TRAR4) gene on chromosome 6q23.2 are associated with susceptibility to schizophrenia. Am J Hum Genet 2004;75:624-638.
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The purpose of this assay is to determine dose response curves for available powder samples of compounds identified as active in a set of previous experiments entitled, "Fluorescence-based cell-based primary high throughput dose response assay to identify antagonists of the human trace amine associated receptor 1 (TAAR1)"(AID 686994). This assay allows TAAR1 to signal predominantly through calcium mobilization by stably expressing TAAR1 in the RD-HGA16 cell line. The RD-HGA16 cell line is a CHO cell-derived cell line that stably expresses Ga16. Ga16 has been shown to couple many GPCRs to calcium mobilization regardless of their endogenous signaling mechanisms. In other words, this cell line permits the coupling of non-Gq-coupled G-protein-coupled receptors to the mobilization of internal calcium. In this assay the CHO-Ga16 cell line is incubated with a fluorescent, cell permeable calcium indicator dye (Fluo-8). The dye serves to monitor levels of intracellular calcium. Test compounds are then added to the cells, followed by the addition of the TAAR1 agonist Beta-PEA. As designed, compounds that act as TAAR1 antagonists will decrease calcium mobilization, resulting in decreased relative fluorescence of the indicator dye, and decreased well fluorescence. The compounds are tested at 29.9uM with a 10 point, 1:3 dilution series in triplicate.
The TAAR1-HGA16 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 qualified fetal bovine serum, 20 mM HEPES, 400 ug/mL Geneticin, 200 ug/mL Hygromycin and 1X antibiotic mix (penicillin and streptomycin).
The day before the assay 750 cells in 3 uL of growth media, lacking Geneticin and Hygromycin, were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 C, 5% CO2, and 95 % RH for 17-24 hours. Next, 2 uL of the fluorogenic Fluo-8 intracellular calcium indicator mixture (prepared according to the manufacturer's protocol) was added to each well. Plates were then incubated for 75 minutes at 37 C, 5% CO2, and 95 % RH, followed by 30 minute incubation at room temperature. Then, 30 nL of 100% DMSO was added to each well and incubated at room temperature for 5 minutes. Then, 15 nL of test compound in DMSO were dispensed to appropriate wells. The assay was started by performing a basal read of plate fluorescence (470-495 nm excitation and 515-575 nm emission) for 5 seconds on the FLIPR Tetra (Molecular Devices). Then a real time fluorescence measurement was immediately performed for the remaining 140 seconds of the assay. Then, a basal read of plate fluorescence for 5 seconds on the FLIPR Tetra prior to all wells being treated with EC80 of PEA in DMSO. Then a real time fluorescence measurement was immediately performed for the remaining 140 seconds of the assay (this is the read for the antagonist mode).
Hits for this assay were determined according to the following mathematical expression:
Ratio = I_Max / I_Min
I_Max represents the maximum measured fluorescence emission intensity over the 140 second read for the antagonist mode and;
I_Min represents the minimum (basal) measured fluorescence emission intensity before EC80 of PEA was added.
The percent inhibition was calculated from the median ratio as follows:
%_Inhibition = ( 1 - ( Ratio Test_Compound - Median_Ratio_High_Control ) / ( Median_Ratio_Low_Control - Median_Ratio_High_Control ) ) ) * 100
Test_Compound is defined as wells containing test compound.
High_Control is defined as wells containing DMSO.
Low_Control is defined as wells containing PEA (EC80) and DMSO.
For each test compound, percent inhibition 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 (Accelrys Inc). The reported IC50 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. 29.9 uM) did not result in greater than 50% activation, the IC50 was determined manually as greater than 29.9 uM.
PubChem Activity Outcome and Score:
Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.
Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero.
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 100-0, and for inactive compounds 0-0.
List of Reagents:
RD-HGA Cell Line (Molecular Devices)
Agonist:PEA Control (Sigma, P6513)
Calcium sensitive dye: Fluo-8 No Wash Calcium Assay Kit; (AAT Bioquest, part 36316)
Growth media: Ham's F-12; 10% FBS, 20mM HEPES, 400 ug/mL G418, 200ug/ml Hygromycin
Assay media: Ham's F-12, 10% FBS, 20 mM HEPES
Assay plates: Corning black/clear 1536well FLIPR plate; (Corning, part 7338)
Probenecid: 250mM (pH 8.0); (Sigma P8761)
Ham's F-12 Nutrient Mixture (Invitrogen, part 11765-054)
Charcoal/Dextran-treated Feta Bovine Serum (Hyclone, part SH3006803HI)
Penicillin-Streptomycin(100X) (Invitrogen, 15070-063)
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)
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.
Due to limitations in the amount of MLSMR compounds that could be selected for retesting, compounds that demonstrated selective activity in AID 624466 vs. other Fluo8 antagonist screening assays run at the SRIMSC were prioritized for testing in this assay. Compounds that were agonist hits in AID 624467 were also not selected for testing in this assay.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
Assay: CurveFit : Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: CurveFit : Mask: Excluded Points
Assay: Dictionary: Version: 0.1
* Activity Concentration. ** Test Concentration.
Data Table (Concise)