title: Me-talnetant and osanetant interact within overlapping but not identical binding pockets in the human tachykinin neurokinin 3 receptor transmembrane domains.
abstract: Recent clinical trials have indicated that neurokinin 3 receptor antagonists (S)-(+)-N-{{3-[1-benzoyl-3-(3,4-dichlorophenyl)-piperidin-3-yl]prop-1-yl}-4-phenylpiperidin-4-yl}-N-methylacetamine (SR142801; osanetant) and (S)-(-)-N-(alpha-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4-carboxamide (SB223412; talnetant) may treat symptoms of schizophrenia. Using site-directed mutagenesis, rhodopsin-based modeling, [(3)H](S)-(-)-N-(alpha-ethylbenzyl)-3-methoxy-2-phenylquinoline-4-carboxamide (Me-talnetant) and [(3)H]osanetant binding, and functional Schild analyses, we have demonstrated the important molecular determinants of neurokinin B (NKB), Me-talnetant, and osanetant binding pockets. The residues Asn138(2.57), Asn142(2.61), Leu232(45.49), Tyr315(6.51), Phe342(7.39), and Met346(7.43) were found to be crucial for the NKB binding site. We observed that the M134(2.53)A, V169(3.36)M, F342(7.39)M, and S341(7.38)I/F342(7.39)M mutations resulted in the complete loss of [(3)H]Metalnetant and [(3)H]osanetant binding affinities and also abolished their functional potencies in an NKB-evoked accumulation of [(3)H]inositol phosphates assay, whereas the mutations V95(1.42)A, N142(2.61)A, Y315(6.51)F, and M346(7.43)A behaved differently between the interacting modes of two antagonists. V95(1.42)A and M346(7.43)A significantly decreased the affinity and potency of Me-talnetant. Y315(6.51)F, although not affecting Me-talnetant, led to a significant decrease in affinity and potency of osanetant. The mutation N142(2.61)A, which abolished the potency and affinity of osanetant, led to a significant increase in the affinity and potency of Me-talnetant. The proposed docking mode was further validated using (S)-2-(3,5-bis-trifluoromethyl-phenyl)-N-[4-(4-fluoro-2-methyl-phenyl)-6-((S)-4-methanesulfonyl-3-methyl-piperazin-1-yl)-pyridin-3-yl]-N-methyl-isobutyramide (RO49085940), from another chemical class. It is noteworthy that the mutation F342(7.39)A caused an 80-fold gain of RO4908594 binding affinity, but the same mutation resulted in the complete loss of the affinity of Me-talnetant and partial loss of the affinity of osanetant. These observations show that the binding pocket of Me-talnetant and osanetant are overlapping, but not identical. Taken together, our data are consistent with the proposed docking modes where Me-talnetant reaches deeply into the pocket formed by transmembrane (TM)1, -2, and -7, whereas osanetant fills the pocket TM3, -5, and -6 with its phenyl-piperidine moiety.
authors.affiliation: F. Hoffmann-La Roche Ltd., Bldg. 69/333, CH-4070 Basel, Switzerland. parichehr.malherbe@roche.com
authors.forename: Pari
authors.initials: P
authors.lastname: Malherbe
authors.forename: Caterina
authors.initials: C
authors.lastname: Bissantz
authors.forename: Anne
authors.initials: A
authors.lastname: Marcuz
authors.forename: Claudia
authors.initials: C
authors.lastname: Kratzeisen
authors.forename: Marie-Thérèse
authors.initials: MT
authors.lastname: Zenner
authors.forename: Joseph G
authors.initials: JG
authors.lastname: Wettstein
authors.forename: Hasane
authors.initials: H
authors.lastname: Ratni
authors.forename: Claus
authors.initials: C
authors.lastname: Riemer
authors.forename: Will
authors.initials: W
authors.lastname: Spooren
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D016466:CHO Cells; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010880:Piperidines / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D017434:Protein Structure, Tertiary / Q000502:physiology; D011804:Quinolines / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D018042:Receptors, Neurokinin-3 / Q000235:genetics / Q000378:metabolism*
chemical_list: D010880:Piperidines; D011804:Quinolines; D018042:Receptors, Neurokinin-3; C106716:SB 223412; C091554:SR 142801