Optimum Cross-linking Spacer Length of Dimeric Neurokinin B Analogs for Interaction with NK-1 Tachykinin Receptors

A series of dimeric analogs of neurokinin B (NKB) COOH-terminal heptapeptide were synthesized in order to find an optimum length of cross-linking spacer for bivalent interaction with tachykinin receptors. Dimerization was carried out at the NH2-terminus of heptapeptide with succinyl bis[(Gly)n–OH], in which the number of glycine varies from 0 to 4. Dimers D–(Gly)n–NKB4—10, namely succinyl bis[(Gly)n–Asp–Ser–Phe–Val–Gly–Leu–Met–NH2] (n = 0—4), were almost inactive in the assay using rat vas deferens, indicating that they have no interaction with NK-2 receptor subtype. In contrast, these dimers were very active in guinea pig ileum (GPI) containing all of NK-1, 2, and 3 receptors. Relative activity was highest when the cross-linking spacer with oligoglycine of n = 2, and decreased sharply for dimers with shorter and longer chain lengths (n = 0 and 1; 3 and 4). In order to specify receptor subtype (NK-1 or NK-3) in GPI to which dimers bind, dimers were examined under the conditions that NK-1 receptors are desensitized by substance P methyl ester. All dimers exhibited drastically diminished contractile activity, indicating that dimers exclusively interact with NK-1. This was further confirmed by blocking the response of NK-3 receptors with atropine, which had no effect on the contractile activity of dimers. The results suggested that dimers interact bivalently with NK-1 receptors by bridging adjacent two binding sites.