Fluorescence-based cell-based primary high throughput confirmation assay to identify antagonists of the human trace amine associated receptor 1 (TAAR1)
Name: Fluorescence-based cell-based primary high throughput confirmation assay to identify antagonists of the human trace amine associated receptor 1 (TAAR1). ..more
BioActive Compounds: 944
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_3X%INH CRUN1
Name: Fluorescence-based cell-based primary high throughput confirmation 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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The purpose of this assay is to confirm activity of compounds identified as active in a previous set of experiments entitled, "Fluorescence-based cell-based primary high throughput screening assay to identify antagonists of the human trace amine associated receptor 1 (TAAR1)"(AID 624466). 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. Compounds are tested in triplicate at a final nominal concentration of 3 uM.
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.
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated for each assay plate: (1) the average percent inhibition of test compound wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for each plate, i.e. any compound that exhibited greater % inhibition than that particular plate's cutoff parameter 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 100-47, and for inactive compounds 47-0.
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
** Test Concentration.
Data Table (Concise)