title: Neurokinin-3 receptor-specific antagonists talnetant and osanetant show distinct mode of action in cellular Ca2+ mobilization but display similar binding kinetics and identical mechanism of binding in ligand cross-competition.
abstract: Talnetant and osanetant, two structurally diverse antagonists of neurokinin-3 receptor (NK3), displayed distinct modes of action in Ca2+ mobilization. Although talnetant showed a normal Schild plot with a slope close to unity and a Kb similar to its Ki value in binding, osanetant presented an aberrant Schild with a steep slope (3.3 +/- 0.5) and a Kb value (12 nM) significantly elevated compared with its Ki value (0.8 nM) in binding. Kinetic binding experiments indicated a simple one-step binding mechanism with relatively fast on- and off-rates for both antagonists, arguing against slow onset of antagonism as the reason for abnormal Schild. This conclusion was supported by prolonged preincubation of antagonist that failed to improve the observed aberrant Schild. In ligand cross-competition binding, both talnetant and osanetant displayed linear reciprocal plots of identical slope when [MePhe7]neurokinin B (NKB) was used as the other competition partner with 125I-[MePhe7]NKB as the radioligand, indicating competitive binding of either antagonist with regard to [MePhe7]NKB. Similar patterns were obtained when talnetant was tested against osanetant, indicating competitive binding between the two antagonists as well. These results were reproduced when [3H]4-quinolinecarboxamide (SB222200), a close derivative of talnetant, was used as the radioligand. Taken together, these data strongly suggest binding of both talnetant and osanetant at the orthosteric binding site with similar kinetic properties and do not support the hypothesis that the aberrant Schild observed in functional assays for osanetant is derived from differences in the mechanism of binding for these NK3 antagonists.
authors.affiliation: Department of Lead Generation, AstraZeneca Pharmaceuticals, 1800 Concord Pike, Wilmington, DE 19803, USA. gaochao.tian@astrazeneca.com
authors.forename: Gaochao
authors.initials: G
authors.lastname: Tian
authors.forename: Dee
authors.initials: D
authors.lastname: Wilkins
authors.forename: Clay W
authors.initials: CW
authors.lastname: Scott
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D007700:Kinetics; D015287:Neurokinin B / Q000031:analogs & derivatives / Q000378:metabolism; D010880:Piperidines / Q000378:metabolism / Q000494:pharmacology*; D011804:Quinolines / Q000378:metabolism / Q000494:pharmacology*; D018042:Receptors, Neurokinin-3 / Q000037:antagonists & inhibitors*
chemical_list: D010880:Piperidines; D011804:Quinolines; D018042:Receptors, Neurokinin-3; C109112:SB 222200; C080394:neurokinin B, MePhe(7)-; C106716:SB 223412; D015287:Neurokinin B; C091554:SR 142801; D002118:Calcium