Effects of substitution of hydrophobic amino acids by tryptophan on receptor binding and biological activity of neuropeptide nociceptin

Nociceptin is a neuropeptide that binds to and activates the opioid receptor-like ORL1 receptor. In order to explore the structural elements necessary for receptor recognition and activation, we designed and synthesized a series of nociceptin analogues, in which nonpolar amino acid residues such as Gly⁶, Ala⁷, Ala¹¹, Leu¹⁴, and Ala¹⁵ were substituted respectively by Trp. [Trp⁶]^- and [Trp⁷]-nociceptins exhibited rather weak activities (5-15% of nociceptin), and [Trp¹¹]^- and [Trp¹⁵]-nociceptins also showed reduced activities (40-50%). These results suggested that the space available for these particular residues are relatively restricted. By contrast, the Trp/Leu-substitution at position 14 retained full receptor binding activity, and the resulting [Trp¹⁴]nociceptin exhibited an increased biological activity in the functional assay using [³⁵S]GTPγS. This suggested that the receptor residue interacting with nociceptin-Leu¹⁴ is the aromatic amino acid.