Counterscreen assay for antagonists of neuropeptide Y receptor Y2 (NPY-Y2): Cell-based high throughput assay to measure NPY-Y1 antagonism.
Name: Counterscreen assay for antagonists of neuropeptide Y receptor Y2 (NPY-Y2): Cell-based high throughput assay to measure NPY-Y1 antagonism. ..more
BioActive Compounds: 135
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Claes Wahlestedt, Scripps Florida
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal Number 1 R21 NS056950-01
Grant Proposal PI: Claes Wahlestedt
External Assay ID: NPY-Y1_ANT_CNGC_1536_3X%INH (CS)
Name: Counterscreen assay for antagonists of neuropeptide Y receptor Y2 (NPY-Y2): Cell-based high throughput assay to measure NPY-Y1 antagonism.
Neuropeptide Y (NPY) is a neurotransmitter with diverse physiologic roles including control of feeding behavior, regulation of cortical neural activity, heart neural activity, and emotional regulation. Importantly, NPY is implicated in human diseases such as obesity, depression and alcoholism. NPY mediates its biological effects in part through activation of the NPY-Y2 receptor, a 381-amino acid Galphai protein coupled receptor (GPCR) which decreases cytosolic cAMP production. NPY Y2 is expressed in the periventricular nucleus, amygdala, hypothalamus, hippocampus, tractus solitarius, septum and paraventricular nucleus brain regions (1, 2). Due to its expression profile and biological action, NPY Y2 is an attractive target for anxiolytic research. Additionally, Y2 is predicted to be a therapeutic target in alcoholism. Because Y2 receptors increase NPY transmission, Y2 antagonists may also mediate anxiolytic-like effects in animal models (3). Consistent with this hypothesis Y2 receptor mutant mice demonstrate reduced anxiety behavior compared with wild type controls (4). Moreover, use of the Y2 receptor antagonist BIIE0246 has been shown to suppress ethanol self-administration in rats (5). It has been reported, however, that the complex structure and high molecular weight of BIIE0246 limit its usefulness as an in vivo pharmacological tool (6). It is therefore necessary to produce high affinity selective ligands for the Y2 receptor.
1.Wahlestedt C, Ekman R, Widerlov E. Neuropeptide Y (NPY) and the central nervous system: distribution effects and possible relationship to neurological and psychiatric disorders. Prog Neuropsychopharmacol Biol Psychiatry. 1989;13(1-2):31-54.
2.Redrobe JP, Dumont Y, Quirion R. Neuropeptide Y (NPY) and depression: from animal studies to the human condition. Life Sci. 2002 Nov 8;71(25):2921-37.
3.Wahlestedt C, Yanaihara N, Hakanson R. Evidence for different pre-and post-junctional receptors for neuropeptide Y and related peptides. Regul Pept. 1986 Feb;13(3-4):307-18.
4.Redrobe JP, Dumont Y, Herzog H, Quirion R. Neuropeptide Y (NPY) Y2 receptors mediate behaviour in two animal models of anxiety: evidence from Y2 receptor knockout mice. Behav Brain Res. 2003 May 15;141(2):251-5.
5.Rimondini R, Thorsell A, Heilig M. Suppression of ethanol self-administration by the neuropeptide Y (NPY) Y2 receptor antagonist BIIE0246: evidence for sensitization in rats with a history of dependence. Neurosci Lett. 2005 Feb 28;375(2):129-33. Epub 2004 Nov 30.
6. Bonaventure P, Nepomuceno D, Mazur C, Lord B, Rudolph DA, Jablonowski JA, Carruthers NI, Lovenberg TW. Characterization of N-(1-Acetyl-2,3-dihydro-1H-indol-6-yl)-3-(3-cyano-phenyl)-N-[1-(2-cyclopentyl-ethyl)-piperidin-4yl]acrylamide (JNJ-5207787), a small molecule antagonist of the neuropeptide Y Y 2 receptor. J Pharmacol Exp Ther. 2004 Mar;308(3):1130-7.
NPY, Neuropeptide Y, NPY-Y2, NPY2R, neuropeptide Y receptor Y2, G protein coupled receptor, GPCR, Galphai, CNGC, cyclic nucleotide gated channel assay, ACTOne, membrane potential, HEK 293, HTS assay, counterscreen, Y1, antagonist, inhibition, alcoholism, depression, anxiety, fluorescence, cAMP, Scripps, Scripps Florida, Molecular Library Screening Center Network, MLSCN, 1536.
The purpose of this assay is to determine whether compounds identified as active in a previous set of experiments entitled, "Primary cell-based high-throughput screening assay for antagonists of neuropeptide Y receptor Y2 (NPY-Y2)," (PubChem AID 793) were nonselective antagonists due to inhibition of the Y1 receptor.
A cell line transfected with the NPY-Y1 receptor and a cyclic-nucleotide gated channel (CNG) were used to measure receptor antagonism. As designed, an NPY-Y1 antagonist increases the concentration of agonist-abrogated cytosolic cyclic adenosine monophosphate (cAMP), and therefore causes the opening of the CNG channel. Once open, the CNG channel changes the cell membrane potential. A fluorescent probe can be used to measure this change in membrane potential; in this assay an increase or decrease in the probe's fluorescence correlates to an increase or decrease, respectively, in cAMP concentration.
To measure NPY-Y1 antagonism, adenylate cyclase activity was activated by the beta adrenergic receptor agonist isoproterenol. Isoproterenol increased the concentration of cAMP, and therefore probe fluorescence via the activated CNG channel. The addition of the agonist NPY peptide counteracted the accumulation of cAMP induced by isoproterenol. Effective NPY-Y1 receptor antagonists therefore would reverse this reduction in NPY-mediated fluorescence, yielding higher fluorescence units. As designed, test compounds that increase probe fluorescence in Y1 cells are considered non-selective antagonists. Compounds were tested in triplicate.
The Y1 HEK293-CNG cells were routinely cultured in T-175 sq cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of Dulbecco's Modified Eagle's Media (DMEM) supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 25 mM HEPES, 5 mM L-Glutamine, 250 micrograms/mL Geneticin, 1 microgram/mL Puromycin, and 1X antibiotic mix (penicillin, streptomycin, and neomycin).
Prior to the start of the assay, 3,600 Y1 cells in a 4 microliter volume of assay media (growth media as above but without Geneticin and Puromycin) were dispensed into each well of 1536-well black clear bottom tissue culture-treated microtiter plates. Next, the plates were incubated for 24 hours at 37 degrees C, 5% CO2 and 95% RH. The assay was started by dispensing 2 microliters per well of 4.5x concentrated probe loading dye into all wells, and the plates were incubated at room temperature for 3 hours. Following incubation, the first fluorescence measurement was performed (510-545 nm excitation and 565-625 nm emission) on the FLIPR Tetra (Molecular Devices), then the cells were challenged by dispensing 2 ul of NPY (25 nM final nominal concentration; NPY's EC95) in PBS. Next, 32 nL of test compound (3.6 uM final nominal concentration) in DMSO (0.4% final concentration) or DMSO alone was added to the appropriate wells. The plates were then incubated for 60 minutes at room temperature, followed by challenge with 1 ul of a solution containing isoproterenol (1 uM final nominal concentration; isoproterenol's EC100) and the phosphodiesterase inhibitor, Ro 20-1724 (25 uM final nominal concentration) in PBS. The plates were then incubated for 45 minutes at room temperature before the final fluorescence measurement with the same instrument settings.
The following mathematical expression was used to normalize data:
Ratio = T45 / T0
Where T0 represents the measured fluorescence emission intensity before the addition of compounds and challenge and T45 represents the measured fluorescence emission intensity 45 minutes post addition of compounds and challenge. The percent inhibition for each compound was calculated as follows:
% Inhibition = [1-((RatioTest_Compound - Median Ratio High_Control)/ (Median Ratio Low_Control - Median Ratio High_Control))]*100
TestCompound is defined as wells containing test compound, NPY and isoproterenol.
LowControl is defined as wells with DMSO, NPY and isoproterenol.
HighControl is defined as wells with DMSO and isoproterenol.
Any compound that exhibited an average percent inhibition greater than the hit cutoff calculated for the Primary screen was declared active. The reported Pubchem_Activity_Score has been normalized to 100% of the highest observed inhibition. Negative % inhibition values are reported as activity score zero.
List of reagents:
Y1 HEK293-CNG cells (BD Biosciences, part 344869)
10x ACTOne Membrane Potential Assay Kit (BD Biosciences, part BD354663)
Phosphate Buffered Saline (Invitrogen, part 10010-023)
DMEM (Invitrogen, part 11965-092)
Fetal Bovine Serum (Invitrogen, part 16140-071)
Trypsin-EDTA solution (Invitrogen, part 25200-056)
Geneticin (Invitrogen, part 10131-027)
Puromycin (Sigma, part P9620)
Ro 20-1724 (Sigma, part B8279)
Isoproterenol (Sigma, part I6504)
Neuropeptide Y (American Peptide, part 60-1-11B)
1536-well plates (Greiner, part 789072)
T-175 tissue culture flasks (Corning, part 431080)
Due to the increasing size of the MLSCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon the associated Primary uHTS campaign's specific compound activity cutoff value. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that non-specifically modulate cAMP and CNG activity or membrane potential, and compounds that quench or emit fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.
The inactive compounds of this assay have activity score range of 0 to 13 and active compounds range of activity score is 13 to 100.
** Test Concentration.
Data Table (Concise)