Summary of probe development efforts to identify antagonists of neuropeptide Y receptor Y2 (NPY-Y2)
Name: Summary of probe development efforts to identify antagonists of neuropeptide Y receptor Y2 (NPY-Y2). ..more
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_LEADS_SUMMARY Probes
Name: Summary of probe development efforts 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 (7).
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. Leads were selected for testing in additional assays performed by the SRIMSC and assay provider (AID 2142). Since receptor subtype selective antagonists were desired, compounds worth follow-up from the above Primary, Confirmation and Counterscreening were required to exhibit an IC50 value of less than 10 uM at Y2R and greater than 35 micromolar 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 the PubChem AIDs cited in the Related BioAssays section of this AID. A manuscript has been published (7). The results of our probe development efforts can be found at http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports.html#NPY-Y2. This probe development effort is now closed. A probe report for SIDs 17507305, 17413392, 4242079, and 22413249 can be found in the Molecular Libraries Bookshelf (PubMed Books) (http://www.ncbi.nlm.nih.gov/books) under ML075, ML074, ML073, and ML072, respectively.
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.
Summary, summary AID, 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, assay provider, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, MLPCN, Molecular Libraries Probe Production Center Network, Molecular Library Screening Center Network, MLSCN.
Categorized Comment - additional comments and annotations
Assay Cell Type: HEK293
From MLP Probe Report:
Probe count: 4
MLP Probe ML# for probe 1: ML075
PubChem Substance ID (SID) for probe 1: 17507305
PubChem Compound ID (CID) for probe 1: 2936384
Probe type for probe 1: Antagonist
IC50/EC50 (nM) for probe 1: 220
Target for probe 1: NPY-Y2 (gi: 4505447)
Anti-target for probe 1: NPY Y1
Fold selectivity for probe 1: >100-fold
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK47354/ (ID: 2359688)
Grant number for probe 1: NS056950-01
MLP Probe ML# for probe 2: ML072
PubChem Substance ID (SID) for probe 2: 22413249
PubChem Compound ID (CID) for probe 2: 4460128
Probe type for probe 2: Antagonist
IC50/EC50 (nM) for probe 2: 3917
Target for probe 2: NPY-Y2 (gi: 4505447)
Anti-target for probe 2: NPY Y1
Fold selectivity for probe 2: 9
MLP Probe ML# for probe 3: ML073
PubChem Substance ID (SID) for probe 3: 4242079
PubChem Compound ID (CID) for probe 3: 3236979
Probe type for probe 3: Antagonist
IC50/EC50 (nM) for probe 3: 733
Target for probe 3: NPY-Y2 (gi: 4505447)
Anti-target for probe 3: NPY Y1
Fold selectivity for probe 3: >40
MLP Probe ML# for probe 4: ML074
PubChem Substance ID (SID) for probe 4: 17413392
PubChem Compound ID (CID) for probe 4: 2228302
Probe type for probe 4: Antagonist
IC50/EC50 (nM) for probe 4: 428
Target for probe 4: NPY-Y2 (gi: 4505447)
Anti-target for probe 4: NPY Y1
Fold selectivity for probe 4: >75
NCBI Book chapter title for probe 1: Probe Report for NPY-Y2 Receptor Antagonists
Data Table (Concise)