Primary cell-based high-throughput screening assay for potentiators or agonists of NPY-Y1
Neuropeptide Y (NPY) is a neurotransmitter with 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 Galphai protein coupled receptors (GPCRs) more ..
BioActive Compounds: 798
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Claes Wahlestedt, TSRI
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal Number 1 R21 NS056950-01
Grant Proposal PI: Claes Wahlestedt
External Assay ID: NPY-Y1_POT_CNGC_1536_%ACT
Name: Primary cell-based high-throughput screening assay for potentiators or agonists of NPY-Y1
Neuropeptide Y (NPY) is a neurotransmitter with 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 Galphai protein coupled receptors (GPCRs) NPY-Y1 and Y2 receptors, which decrease cytosolic cAMP production. Recent studies have implicated these receptors in diverse biological events, including feeding (1), alcoholism (2), anxiety and depression (3), pain perception (4), immunity and inflammation (5), vascular remodeling (6), hypothermia (7), pancreatic islet cell function (8), bone and energy metabolism (9), and tumorigenesis (10). Due to the varied role of these receptors in human disease and physiology, the identification of high affinity selective probes that target each receptor subtype may provide novel tools for the study of NPY-related pathologies.
1. Ishii T, Muranaka R, Tashiro O, Nishimura M. Chronic intracerebroventricular administration of anti-neuropeptide Y antibody stimulates starvation-induced feeding via compensatory responses in the hypothalamus. Brain Res. 2007 May 4;1144:91-100.
2. Olling JD, Ulrichsen J, Haugbol S, Glenthoj B, Hemmingsen R, Woldbye DP. Decreased gene expression of neuropeptide Y and its receptors in hippocampal regions during ethanol withdrawal in rats. Neurosci Lett. 2007 Sep 13;424(3):160-4.
3. Heilig M. The NPY system in stress, anxiety and depression. Neuropeptides. 2004 Aug;38(4):213-24.
4. Taylor BK, Abhyankar SS, Vo NT, Kriedt CL, Churi SB, Urban JH. Neuropeptide Y acts at Y1 receptors in the rostral ventral medulla to inhibit neuropathic pain. Pain. 2007 Sep;131(1-2):83-95.
5. Wheway J, Herzog H, Mackay F. NPY and receptors in immune and inflammatory diseases. Curr Top Med Chem. 2007;7(17):1743-52.
6. Abe K, Tilan JU, Zukowska Z. NPY and NPY receptors in vascular remodeling. Curr Top Med Chem. 2007;7(17):1704-9.
7. Dark J, Pelz KM. NPY Y1 Receptor Antagonist Prevents NPY-Induced Torpor-Like Hypothermia in Cold-Acclimated Siberian Hamsters. Am J Physiol Regul Integr Comp Physiol. 2007 Nov 7.
8. Winzell MS, Ahren B. G-protein-coupled receptors and islet function-Implications for treatment of type 2 diabetes. Pharmacol Ther. 2007 Aug 29.
9. Baldock PA, Allison SJ, Lundberg P, Lee NJ, Slack K, Lin EJ, Enriquez RF, McDonald MM, Zhang L, During MJ, Little DG, Eisman JA, Gardiner EM, Yulyaningsih E, Lin S, Sainsbury A, Herzog H. Novel role of Y1 receptors in the coordinated regulation of bone and energy homeostasis. J Biol Chem. 2007 Jun 29;282(26):19092-102.
10. Ruscica M, Dozio E, Motta M, Magni P. Relevance of the neuropeptide Y system in the biology of cancer progression. Curr Top Med Chem. 2007;7(17):1682-91.
NPY, Neuropeptide Y, NPY-Y1, neuropeptide Y receptor Y1, G protein coupled receptor, GPCR, Galphai, cyclic nucleotide gated channel assay, CNGC, ACTOne, membrane potential, HEK 293, HTS assay, primary screen, agonist, potentiator, activation, activator, alcoholism, depression, anxiety, fluorescence, cAMP, Scripps Research Institute Molecular Screening Center, SRIMSC, MLSCN, 1536.
The purpose of this assay is to determine the ability of test compounds to act as potentiators or agonists of the NPY-Y1 receptor (Y1). A cell line transfected with Y1 and a cyclic-nucleotide gated channel (CNGC) is used to measure potentiation of the EC10 NPY response or direct agonism by test compound of the Y1 receptor. The cells are treated with isoproterenol to activate adenylate cyclase, therefore increasing cytosolic cyclic adenosine monophosphate (cAMP) concentrations, and as a consequence CNGC activity. Changes in CNGC activity also change the cell membrane potential, which is measured using a fluorescent probe. As designed, a test compound that acts as an Y1 potentiator or agonist will increase Y1 activity, leading to decreased cAMP levels, reduced CNG channel opening and therefore reduced probe fluorescence.
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, 3600 Y1 HEK293-CNG 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 cells were challenged by dispensing 2 ul of NPY at its EC10 (75 pM final concentration) in 0.1% BSA. 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 at its EC100 (1 uM final concentration) and the phosphodiesterase inhibitor, Ro 20-1724, in PBS (25 uM final concentration). The plates were then incubated for 45 minutes at room temperature before a fluorescence measurement was performed (510-545 nm excitation and 565-625 nm emission) on the FLIPR Tetra (Molecular Devices).
The percent activation for each compound was calculated using well fluorescence as follows:
% Activation = (Median_Test_Compound - Median_Low_Control)/ (Median_High_Control - Median_Low_Control)*100
Test_Compound is defined as wells containing test compound, NPY and isoproterenol.
High_Control is defined as wells with DMSO, 40 nM NPY (EC100) and isoproterenol.
Low_Control is defined as wells with DMSO, 75 pM NPY (EC10) and isoproterenol.
A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated for each assay plate: (1) the average percent activation 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 % activation than that particular plate's cutoff parameter was declared active.
The reported Pubchem_Activity_Score has been normalized to 100% of the highest observed primary activation value. Negative % activation values are reported as activity score zero.
List of reagents:
Neuropeptide Y receptor 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)
Fatty Acid-Free Bovine Serum Albumin (Calbiochem, part 126609)
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)
At the isoproterenol concentration used in this assay, the EC50 for NPY in 0.1% BSA was approximately 500 pM. 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 that 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.
Activity cutoff was different for each plate therefore activity score should not be used for ranking of this assay's compounds.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)