Primary cell-based high-throughput screening assay for antagonists 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: 1984
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_ANT_CNGC_1536_%INH
Name: Primary cell-based high-throughput screening assay for antagonists 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, Haugb#l 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, counterscreen, antagonist, inhibition, alcoholism, depression, anxiety, fluorescence, cAMP, Scripps, Scripps Florida, Molecular Library Screening Center Network, MLSCN, 1536.
In this assay test compounds from the MLSCN library were screened for their ability to act as antagonists of the NPY-Y1 receptor (Y1). A cell line transfected with Y1 and a cyclic-nucleotide gated channel (CNGC) is used to measure Y1 antagonism by test compound. The cells are treated with isoproterenol to activate adenylate cyclase, therefore increasing cytosolic cyclic adenosine monophosphate (cAMP) concentrations, and as a consequence CNGC activity. Increased CNGC activity increases the cell membrane potential, which is measured using a fluorescent probe. Addition of agonist (NPY peptide) counteracts isoproterenol action and reduces probe fluorescence. A test compound that acts as an effective Y1 antagonist will counteract NPY action, leading to increased cAMP levels, CNG channel opening, membrane potential, and therefore higher 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 ug/mL Geneticin, 1 ug/mL Puromycin, and 1X antibiotic mix (penicillin, streptomycin, and neomycin). Prior to the start of the assay, 3,600 Y1 HEK293-CNG 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% relative humidity (RH). The assay was started by dispensing two 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 at its EC95 (25 nM final nominal concentration) 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 at its EC100 (1 uM final nominal concentration) and the phosphodiesterase inhibitor, Ro 20-1724, in PBS (25 uM final nominal concentration). 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-((RatioTestCompound - MedianRatioHighControl)/ (MedianRatioLowControl - MedianRatioHighControl))]*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.
A mathematical algorithm was used to determine nominally inhibitory compounds in the counterscreen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.
The reported Pubchem_Activity_Score has been normalized to 100% of the highest observed primary activation. 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)
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)
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.
The inactive compounds of this assay have activity score range of 0 to 20 and active compounds range of activity score is 20 to 100.
** Test Concentration.
Data Table (Concise)