Counterscreen Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Vasopressin Receptor Calcium 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 ..
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 assay with transfected NPS receptor. NPS can stimulate the production of cAMP as well as release intracellular calcium in Chinese hamster ovary cells stably expressing NPS receptor. This change in intracellular calcium can be detected using a calcium indicator dye on a calcium imaging plate reader system (FDSS). To validate the specific action of compounds on the NPS receptor, a counterscreen was developed that tests compounds' antagonism of AVPR1B vasopressin receptor stimulation by vasopressin. Vasopressin shares 27% identity with the NPS receptor.
NCGC Assay Protocol Summary:
A Chinese hamster ovary (CHO) cell line stably expressing human vasopressin receptor (AVPR1B) was 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 on a FDSS-7000 kinetic plate reader in 1536-well format. The maximums of kinetic fluorescence responses were converted into text files using the instrument's software data export utility. Data for antagonist response were normalized to the controls for basal activity (DMSO only) and 100% inhibition (no signal). AC50 values were determined from concentration-response data modeled with the standard Hill equation.
CHO-V1B 1536-well FDSS assay protocol:
(1) Frozen CHO-V1B cells were thaw, washed once with fresh media and resuspended in F-12 Kaighn's media supplemented with 10 % FBS, 100 units/ml penicillin and 100 ug/ml streptomycin. Cells were plated at 3 ul/well (1200 cells) to black, clear-bottom, tissue-culture treated 1536-well plates, and then cultured at 37C, 5 % CO2 for 16 to 30 hours.
(2) Add 3 ul of calcium dye (from High Performance PBX Calcium Assay Kit, BD Biosciences). The calcium dye was prepared according to the manufactory's instruction.
(3) Incubation at 37C, 5 % CO2 for 60 min.
(4) Add 23 nl/well of compound in DMSO solution. The final titration for each compound was between 0.6 nM and 46 uM.
(5) Load plates to FDSS-7000. The following steps were performed on FDSS-7000.
(6) Record fluorescent background (Ex 480 nm, Em 520-560 nm) for 10 s.
(7) Add 2 ul of stimulation reagent (1X HBSS buffer, 0.1% BSA, 60 nM vasopressin). Record antagonist response (Ex 480 nm, Em 520-560 nm) for 180 s.
Keywords: MLSMR, MLPCN, NIH Roadmap, qHTS, NCGC, NPS, Neuropeptide S Antagonists
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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)