Late stage results from the probe development effort to identify antagonists of neuropeptide Y receptor Y2 (NPY-Y2).
Name: Late stage results from the probe development effort to identify antagonists of neuropeptide Y receptor Y2 (NPY-Y2). ..more
BioActive Compounds: 26
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
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_PROBES_LATE_STAGE
Name: Late stage results from the probe development effort to identify 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 necessary to produce high affinity selective ligands for the Y2 receptor.
Summary of Probe Development Effort:
Following primary HTS in singlicate to identify NPY-Y2 antagonists (AID793), confirmation of hit activity in triplicate (AID 1257), counterscreening in triplicate against NPY-Y1 to determine selectivity (AID 1256), as well as titration assays in triplicate against NPY-Y2 to determine potency (AID 1272) and selectivity (AID 1279), compounds were identified as possible candidates for probe development. Since receptor subtype selective antagonists were desired, compounds worth follow-up from the Primary, Confirmation and Counterscreening were required to exhibit an IC50 value of less than 10 uM at Y2R and greater than 35uM at Y1R in dose response assays. Analogs were purchased in powder form or re-ordered from the MLSMR in liquid form and tested in dose response assays against both receptors. In addition, in order to determine whether the identified Y2R antagonists were brain-penetrant, selected compounds were injected intraperitoneally into adult mice at 10 mg/kg and levels in the brain tissue and plasma were measured after thirty minutes. BIIE 0246, the current NPY Y2 antagonist probe, exhibited poor penetration (only 2% of plasma levels).
The above probe development efforts resulted in the identification of four probes. All probes demonstrated a higher brain penetrance than BIIE 0246. Compounds from four distinct chemical scaffolds were identified as probes: piperidine-carbothioamide (SID 17507305), arysulfamoyl benzamide (SID 17413392), aryl-1,2,4-oxadiazole (SID 4242079), and dimethylisoxazole (SID 22413249). Importantly, these probe compounds are inactive against NPY-Y1 and do not share structural similarities with the known NPY-Y2 antagonists BIIE 0246 or JNJ-5207787.
Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDs listed below. The results of our probe development efforts can be found at http://molscreen.florida.scripps.edu/probes.shtml. A manuscript has been published (7).
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.
7. Brothers SP, Saldanha SA, Spicer TP, Cameron M, Mercer BA, Chase P, McDonald P, Wahlestedt C, Hodder PS. Selective and Brain Penetrant Neuropeptide Y Y2 Receptor Antagonists Discovered by Whole-Cell High Throughput Screening. Mol Pharmacol. 2009 Oct 16. [Epub ahead of print]
Late stage, probes, NPY, Neuropeptide Y, NPY-Y2, NPY2R, neuropeptide Y receptor Y2, G protein coupled receptor, 1536, GPCR, Galphai, CNGC, cyclic nucleotide gated channel assay, ACTOne, membrane potential, HEK 293, HTS assay, primary, confirmation, dose response, counterscreen, antagonist, inhibition, alcoholism, depression, anxiety, fluorescence, cAMP, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center (SRIMSC), Molecular Library Screening Center Network, MLSCN.
Protocol: Please see AIDs 793, 1256, 1257, 1272, 1279, 1791, and below for all protocols performed in this probe development effort.
NPY Y2 Inhibition Assays (Assays 1 and 2):
The purpose of these assays is to determine the ability of test compounds to inhibit NPY-Y2. 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. Test compounds were assayed in singlicate at a nominal concentration of 2.8 micromolar (Assay 1) or in triplicate in a 10-point, 1:3 dilution series starting at a nominal test concentration of 35 micromolar (Assay 2).
NPY-Y1 Inhibition Counterscreen Assays (Assays 3 and 4):
The purpose of these assays is to determine the ability of test compounds to inhibit NPY-Y1. 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. Test compounds were assayed in singlicate at a nominal concentration of 3.5 micromolar (Assay 3) or in triplicate in a 10-point, 1:3 dilution series starting at a nominal test concentration of 35 micromolar (Assay 4).
Cytotoxicity Assay (Assay 5):
The purpose of this assay is to determine whether compounds of interest reduce cell viability. Y2R HEK293-CNG cells were seeded at 500 cells per well in 1536-well plates in 5 uL growth media. Compounds (in DMSO) were prepared as 10-point, 1:3 serial dilutions starting at 11 uM final concentration were added to cells. Plates were incubated for 24, 48 or 72 h at 37 C. After incubation, 5 uL of CellTiter-Glo (Promega, Madison, WI) were added to each well and the plates were allowed to incubate for 15 min at room temperature. Luminescence was then measured (ViewLux plate reader, PerkinElmer, Turku, Finland). Viability was measured as a percentage relative to control cells treated with DMSO alone (0% cytotoxicity) and cells treated with 100 uM Doxorubicin (100% cytotoxicity).
Brain Penetrance Assays (Assay 6):
Plasma and brain levels of the compounds were assessed in C57Bl6 mice 30 minutes after dosing 10 mg/kg intraperitoneally. Samples were formulated at 2 mg/mL in 10/10/80 DMSO/tween/water. Blood was collected into EDTA containing tubes at 30 min and plasma was generated using standard centrifugation techniques. Brain samples were frozen upon collection and all samples were stored at -80 C until analyzed. Brain tissue was not perfused prior to freezing to prevent diffusion of the compound out of the tissue during the process. Plasma samples were analyzed by treating 25 μL of plasma with 125 μL of acetonitrile containing an internal standard (propanolol) and filtering through a Millipore Multiscreen Solvinter 0.45 μm low binding PTFE hydrophilic filter. The filtrate was analyzed by LC-MS/MS using an API Sciex 4000. MRM methods were developed in positive ion mode and concentrations were determined using a standard curve between 2 to 2000 ng/mL. Samples with concentrations outside of the curve were diluted with blank plasma and reanalyzed. Similar conditions were used to determine brain levels except the samples were weighed and acetonitrile was added (10x, weight by volume). The samples were sonicated to extract the compound from the brain matrix and then filtered as described above. A density of 1 g/mL was used to convert compound per mg tissue into molar equivalents.
Probe Molecule Outcome was assigned based upon combined performance in all assays.
Probes were identified.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)