Dose response cell-based screening assay for antagonists of neuropeptide Y receptor Y2 (NPY-Y2)
Name: Dose response cell-based screening assay for antagonists of neuropeptide Y receptor Y2 (NPY-Y2) ..more
BioActive Compounds: 72
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-Y2_ANT_CNGC_1536_IC50
Name: Dose response cell-based screening assay for antagonists of neuropeptide Y receptor Y2 (NPY-Y2)
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). Therefore, 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. PMID: 14617685.
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, dose response, 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 dose response curves for 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). The compounds selected for testing in this AID met the following criteria: 1) they were active in AID 793; 2) they confirmed activity in a set of experiments entitled, "Cell-based high-throughput confirmation assay for antagonists of neuropeptide Y receptor Y2 (NPY-Y2)" (PubChem AID 1257); and 3) they were inactive in a set of experiments entitled, "Counterscreen assay for antagonists of neuropeptide Y receptor Y2 (NPY-Y2): Cell-based high throughput assay to measure NPY-Y1 antagonism." (PubChem AID 1256).
A cell line transfected with the NPY-Y2 receptor and a cyclic-nucleotide gated channel (CNG) were used to measure receptor antagonism. As designed, an NPY-Y2 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-Y2 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-Y2 receptor antagonists therefore would reverse this reduction in NPY-mediated fluorescence, yielding higher fluorescence units. Test compounds were assayed in triplicate in a 10-point, 1:3 dilution series starting at a nominal test concentration of 35 uM.
The Y2 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, 3600 Y2 cells in a 4 ul volume of assay media (growth media as above) 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 ul 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 at its EC95 (8 nM final nominal concentration) in PBS. Next, 32 nL of either test compound, High Control (BIIE0246, 0.28 uM final nominal concentration) or Low Control (DMSO, 0.4% final concentration) 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; 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-((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 and challenge
Low_Control is defined as wells containing DMSO and challenge
High_Control is defined as wells containing BIIE0246 and challenge.
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 (MDL Information Systems). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 35 micromolar) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 35 uM. 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, with the most potent compounds assigned the highest activity scores.
List of reagents:
Neuropeptide Y receptor Y2 HEK293-CNG cells (BD Biosciences, part 344870)
10x ACTOne membrane potential Assay Kit (BD Biosciences, part BD354663)
Dulbecco's Phosphate Buffered Saline (Invitrogen, part 14190-144)
DMEM high glucose with glutamine (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)
BIIE0246 (Tocris, part 1700)
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. 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 channel 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 MLSMR was not able to provide all compounds selected for testing in this AID.
The inactive compounds of this assay have activity score range of 0 to 37 and active compounds range of activity score is 37 to 100.
Categorized Comment - additional comments and annotations
Assay Format: Cell-based
Assay Cell Type: HEK293
Assay Format: Cell-based
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)