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 K_b similar to its K_i value in binding, osanetant presented an aberrant Schild with a steep slope (3.3 ± 0.5) and a K_b value (12 nM) significantly elevated compared with its K_i 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.