title: Establishment of robust functional assays for the characterization of neuropeptide Y (NPY) receptors: identification of 3-(5-benzoyl-thiazol-2-ylamino)-benzonitrile as selective NPY type 5 receptor antagonist.
abstract: The human Neuropeptide Y (NPY) receptors 1 (hY1), 2 (hY2), 4 (hY4), and the mouse type 5 (mY5) receptor were expressed in human embryonic kidney 293 (HEK293) cells. The receptors bound a radioiodinated NPY ligand with high affinity and various NPY analogs competed for binding in a receptor selective-manner. Similarly, cAMP-inhibition and GTPgammaS binding assays were established. The four NPY receptors were further tested in the fluorimetric imaging plate reader (FLIPR) format, a cellular high-throughput assay, in the absence and presence of chimeric G proteins, Gqo5, Gqi5 and Gqi9. The receptors stimulated transient calcium release only in the presence of chimeric G proteins. While hY1, hY2 and hY4 receptors coupled to Gqo5, Gqi5 and Gqi9, the mY5 receptor stimulated transient calcium release only when co-expressed with Gqi9. Using an in silico screening approach we identified a small molecule 3-(5-benzoyl-thiazol-2-ylamino)-benzonitrile (compound 1), which bound to the mY5 receptor with high affinity (Ki=32.1+/-1.8 nM), competitively antagonized NPY-mediated GTPgammaS binding and calcium stimulation with high potency, and had no affinity for other NPY receptors. These data show that NPY receptors can be functionally coupled to the FLIPR readout, allowing for high throughput compound testing and identification of novel molecules.
authors.affiliation: F. Hoffmann-La Roche AG, Pharmaceutical Research, 4070 Basel, Switzerland. fdautzen@prdbe.jnj.com
authors.forename: Frank M
authors.initials: FM
authors.lastname: Dautzenberg
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Higelin
authors.forename: Philippe
authors.initials: P
authors.lastname: Pflieger
authors.forename: Werner
authors.initials: W
authors.lastname: Neidhart
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Guba
mesh_terms: D000818:Animals; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006801:Humans; D051379:Mice; D009478:Neuropeptide Y / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D009570:Nitriles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D017476:Receptors, Neuropeptide Y / Q000037:antagonists & inhibitors* / Q000502:physiology*; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*
chemical_list: C504420:3-(5-benzoylthiazol-2-ylamino)benzonitrile; D009478:Neuropeptide Y; D009570:Nitriles; D017476:Receptors, Neuropeptide Y; D013844:Thiazoles; C100568:neuropeptide Y5 receptor; C014356:benzonitrile