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BioAssay: AID 434936

Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Robust Characterization of cAMP Signal Transduction

Neuropeptide S receptor (NPSR), previously known as GPR154, is a recently de-orphanized G protein coupled receptor. Its endogenous ligand is the 20 amino acids peptide Neuropeptide S (NPS). Activation of NPSR induces transient increases in intracellular calcium and cAMP, suggesting coupling of this receptor to both Gs and Gq G proteins. NPS and its receptor are found in various tissues. more ..
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 Tested Compounds
 Tested Compounds
All(96)
 
 
Active(72)
 
 
Inactive(10)
 
 
Inconclusive(14)
 
 
 Tested Substances
 Tested Substances
All(97)
 
 
Active(73)
 
 
Inactive(10)
 
 
Inconclusive(14)
 
 
AID: 434936
Data Source: NCGC (NPSR005tr)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-05-21
Modify Date: 2010-10-15

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 72
Depositor Specified Assays
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AIDNameTypeProbeComment
1461qHTS Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal Transductionconfirmatory
1464Quantitative High-Throughput Screen for Antagonists of the Neuropeptide S Receptor: Summarysummary2
1491Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal Transductionconfirmatory
1489Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Calcium Signal Transductionconfirmatory
1492Counterscreen Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Muscarinic Receptor Calcium Signal Transduction.confirmatory
1493Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Radioligand Displacementconfirmatory
2566Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Radioligand Displacement, SAR for Probeconfirmatory
2567Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Calcium Signal Transduction, SAR for Probeconfirmatory
2568Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal Transduction, SAR for Probeconfirmatory
2570Counterscreen Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Vasopressin Receptor Calcium Signal Transductionconfirmatory
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production centers Network [MLPCN]

MLPCN Grant: X01-DA026210-01
Assay Submitter (PI): Heilig, Markus Alexander

NCGC Assay Overview:

Neuropeptide S receptor (NPSR), previously known as GPR154, is a recently de-orphanized G protein coupled receptor. Its endogenous ligand is the 20 amino acids peptide Neuropeptide S (NPS). Activation of NPSR induces transient increases in intracellular calcium and cAMP, suggesting coupling of this receptor to both Gs and Gq G proteins. NPS and its receptor are found in various tissues. Specifically they are highly expressed in brain areas that have been implicated in modulation of arousal, stress and anxiety. Central administration of NPS in mice produces an unusual profile of activity by inducing wakefulness and arousal, while at the same time suppressing anxiety. Therefore, NPSR may represent a novel drug target for the treatment of sleep and anxiety disorders.

To identify NPSR antagonists, we developed a cell-based cAMP assay. NPS can stimulate the production of cAMP in Chinese hamster ovary cells stably expressing NPS receptor. This change in intracellular cAMP level can be detected by a homogeneous LANCE cAMP assay based on the TR-FRET (time resolved fluorescence resonance energy transfer) between a europium-labeled cAMP tracer complex and a cAMP-specific antibody labeled with Alexa Fluor 647. The europium-labeled cAMP tracer complex is formed by the tight interaction between Biotin-cAMP (b-cAMP) and streptavidin labeled with Europium-W8044 chelate (Eu-SA). Light pulse at 320 nm excites the europium of the cAMP tracer and the energy emitted is transferred to the Alexa molecule bound to the cAMP antibody, generating a TR-FRET signal at 665 nm. Residual energy from the europium will produce a light at 620 nm. The native unlabeled cAMP from cell lysates competes with the europium-cAMP tracer for antibody binding and reversely reduces the emission signal of Alexa by interrupting FRET between the two labeled molecules. Both emission signals from the FRET donor (620 nm) and the acceptor (665 nm) can be detected by a plate reader in the TRF mode. Expression of result in fluorescence ratio (665 nm/620 nm) helps to normalize differences due to cell density and reagent dispensing. This assay was successfully optimized to a 1536-well plate format.

Select compounds from the primary screen (AID 1461) were chosen for confirmation based on potency, efficacy, SAR, and activity across other assays in PubChem and at NCGC. Other compounds are analogs of active compounds from the primary screen. This particular assay uses a high density of sampling across concentration-response space to establish more confidence estimates of potency.
Protocol
NCGC Assay Protocol Summary:

A Chinese hamster ovary (CHO) cell line stably expressing the NPS receptor (CHO-NPSR) was obtained from Dr. Heilig lab at NIAAA and maintained in F-12 Kaighn's media (Invitrogen, Carlsbad, CA, 21127) supplemented with 10 % FBS, 100 units/ml penicillin, 100 ug/ml streptomycin and 250 ug/ml geneticin at 37C, 5% CO2 in a humidified atmosphere. Before the assay, aliquots of cells were frozen and stored at -135C. The assay was performed in 1536-well format. Data are reported for both the ratio of the two emission wavelengths, and also for the component 'donor' channel, Em2=620. Data were normalized to the controls for basal activity (DMSO only) and 100% inhibition (No NPS control). IC50 values were determined from concentration-response data modeled with the standard Hill equation.

NPS 1536-well assay protocol:
(1) Fresh or frozen CHO-NPSR cells were suspended in F-12 Kaighn's media supplemented with 10 % FBS, 100 units/ml penicillin and 100 ug/ml streptomycin. Cells were plated at 4 ul/well (1200 cells) to white, tissue culture treated 1536-well plates, and then cultured at 37C, 5 % CO2 for 16 to 30 hours.
(2) Add 23 nl/well of compound in DMSO solution. The final titration for each compound was between 0.6 nM and 46 uM.
(3) Add 1 ul of stimulation reagent (1X HBSS buffer, 5 mM HEPES, 0.1% BSA, 500 uM RO-201724 (Sigma-Aldrich, B8279), 1.5% Alexa 647-labeled anti-cAMP antibody (from Perkin Elmer), 100 nM NPS)
(4) Incubation at 37C, 5 % CO2 for 60 min.
(5) Add 1 ul/well detection reagent. Detection reagent was prepared by adding biotin labeled cAMP (4%), streptavidin labeled with Europium-W8044 chelate (1.33%) and TritonX-100 (1%) to detection buffer. Biotin labeled cAMP, streptavidin labeled with Europium-W8044 chelate and detection buffer all came from the LANCE cAMP kit (Perkin Elmer).
(6) Incubate at room temperature for 2 hours.
(7) Detect the assay plate (Ex = 320, Em1 =665 and Em2 620) in a ViewLux plate reader.


Keywords: MLSMR, MLPCN, NIH Roadmap, qHTS, NCGC, NPS, Neuropeptide S Antagonists
Comment
Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.0000049442 uM (4.9442e-06μM**)% Activity at given concentration.Float%
15Activity at 0.0000054935 uM (5.4935e-06μM**)% Activity at given concentration.Float%
16Activity at 0.0000060429 uM (6.0429e-06μM**)% Activity at given concentration.Float%
17Activity at 0.0000098883 uM (9.8883e-06μM**)% Activity at given concentration.Float%
18Activity at 0.0000109870 uM (1.0987e-05μM**)% Activity at given concentration.Float%
19Activity at 0.0000120857 uM (1.20857e-05μM**)% Activity at given concentration.Float%
20Activity at 0.0000197767 uM (1.97767e-05μM**)% Activity at given concentration.Float%
21Activity at 0.0000219741 uM (2.19741e-05μM**)% Activity at given concentration.Float%
22Activity at 0.0000241715 uM (2.41715e-05μM**)% Activity at given concentration.Float%
23Activity at 0.0000395533 uM (3.95533e-05μM**)% Activity at given concentration.Float%
24Activity at 0.0000439481 uM (4.39481e-05μM**)% Activity at given concentration.Float%
25Activity at 0.0000483429 uM (4.83429e-05μM**)% Activity at given concentration.Float%
26Activity at 0.0000791066 uM (7.91066e-05μM**)% Activity at given concentration.Float%
27Activity at 0.0000878962 uM (8.78962e-05μM**)% Activity at given concentration.Float%
28Activity at 0.0000966858 uM (9.66858e-05μM**)% Activity at given concentration.Float%
29Activity at 0.0001582133 uM (0.000158213μM**)% Activity at given concentration.Float%
30Activity at 0.0001757925 uM (0.000175793μM**)% Activity at given concentration.Float%
31Activity at 0.0001933718 uM (0.000193372μM**)% Activity at given concentration.Float%
32Activity at 0.0003164265 uM (0.000316427μM**)% Activity at given concentration.Float%
33Activity at 0.0003515850 uM (0.000351585μM**)% Activity at given concentration.Float%
34Activity at 0.0003867435 uM (0.000386744μM**)% Activity at given concentration.Float%
35Activity at 0.0006328530 uM (0.000632853μM**)% Activity at given concentration.Float%
36Activity at 0.0007031700 uM (0.00070317μM**)% Activity at given concentration.Float%
37Activity at 0.0007734870 uM (0.000773487μM**)% Activity at given concentration.Float%
38Activity at 0.00127 uM (0.00127μM**)% Activity at given concentration.Float%
39Activity at 0.00141 uM (0.00141μM**)% Activity at given concentration.Float%
40Activity at 0.00155 uM (0.00155μM**)% Activity at given concentration.Float%
41Activity at 0.00253 uM (0.00253μM**)% Activity at given concentration.Float%
42Activity at 0.00281 uM (0.00281μM**)% Activity at given concentration.Float%
43Activity at 0.00309 uM (0.00309μM**)% Activity at given concentration.Float%
44Activity at 0.00507 uM (0.00507μM**)% Activity at given concentration.Float%
45Activity at 0.00563 uM (0.00563μM**)% Activity at given concentration.Float%
46Activity at 0.00619 uM (0.00619μM**)% Activity at given concentration.Float%
47Activity at 0.00990 uM (0.0099μM**)% Activity at given concentration.Float%
48Activity at 0.011 uM (0.011μM**)% Activity at given concentration.Float%
49Activity at 0.012 uM (0.012μM**)% Activity at given concentration.Float%
50Activity at 0.021 uM (0.021μM**)% Activity at given concentration.Float%
51Activity at 0.023 uM (0.023μM**)% Activity at given concentration.Float%
52Activity at 0.025 uM (0.025μM**)% Activity at given concentration.Float%
53Activity at 0.041 uM (0.041μM**)% Activity at given concentration.Float%
54Activity at 0.045 uM (0.045μM**)% Activity at given concentration.Float%
55Activity at 0.050 uM (0.05μM**)% Activity at given concentration.Float%
56Activity at 0.081 uM (0.081μM**)% Activity at given concentration.Float%
57Activity at 0.090 uM (0.09μM**)% Activity at given concentration.Float%
58Activity at 0.099 uM (0.099μM**)% Activity at given concentration.Float%
59Activity at 0.162 uM (0.162μM**)% Activity at given concentration.Float%
60Activity at 0.180 uM (0.18μM**)% Activity at given concentration.Float%
61Activity at 0.198 uM (0.198μM**)% Activity at given concentration.Float%
62Activity at 0.324 uM (0.324μM**)% Activity at given concentration.Float%
63Activity at 0.360 uM (0.36μM**)% Activity at given concentration.Float%
64Activity at 0.396 uM (0.396μM**)% Activity at given concentration.Float%
65Activity at 0.648 uM (0.648μM**)% Activity at given concentration.Float%
66Activity at 0.720 uM (0.72μM**)% Activity at given concentration.Float%
67Activity at 0.792 uM (0.792μM**)% Activity at given concentration.Float%
68Activity at 1.296 uM (1.296μM**)% Activity at given concentration.Float%
69Activity at 1.440 uM (1.44μM**)% Activity at given concentration.Float%
70Activity at 1.584 uM (1.584μM**)% Activity at given concentration.Float%
71Activity at 2.592 uM (2.592μM**)% Activity at given concentration.Float%
72Activity at 2.880 uM (2.88μM**)% Activity at given concentration.Float%
73Activity at 3.168 uM (3.168μM**)% Activity at given concentration.Float%
74Activity at 5.184 uM (5.184μM**)% Activity at given concentration.Float%
75Activity at 5.760 uM (5.76μM**)% Activity at given concentration.Float%
76Activity at 6.336 uM (6.336μM**)% Activity at given concentration.Float%
77Activity at 10.37 uM (10.37μM**)% Activity at given concentration.Float%
78Activity at 11.52 uM (11.52μM**)% Activity at given concentration.Float%
79Activity at 12.67 uM (12.67μM**)% Activity at given concentration.Float%
80Activity at 20.74 uM (20.74μM**)% Activity at given concentration.Float%
81Activity at 23.04 uM (23.04μM**)% Activity at given concentration.Float%
82Activity at 25.34 uM (25.34μM**)% Activity at given concentration.Float%
83Activity at 41.47 uM (41.47μM**)% Activity at given concentration.Float%
84Activity at 46.08 uM (46.08μM**)% Activity at given concentration.Float%
85Activity at 50.69 uM (50.69μM**)% Activity at given concentration.Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: DA026210-01

Data Table (Concise)
Classification
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