Highly potent nociceptin analog containing the Arg-Lys triple repeat

One of the structural characteristics of a neuropeptide nociceptin is the existence of Arg-Lys (RK) residues at positions 8-9 and 12-13; both RKs have been suggested to bind to the acidic amino acid cluster in the second extracellular loop of the seven transmembrane domain receptor ORL1. With a design strategy of attempting to obtain an analog that binds more strongly to the receptor's acidic cluster, we synthesized a series of nociceptin analogs in which the RK dipeptide unit was placed at positions 6-7, 10-11, or 14-15 adjacent to the parent RKs. Among these nociceptin analogs containing the RK triple repeat, [ArgLys^6-7]- and [Arg-Lys^10-11]nociceptins exhibited weak activities (6-9 and 60-90% of nociceptin, respectively) both in the receptor binding assay and in the [³⁵S]GTPγS binding functional assay. In contrast, [Arg-Lys^14-15]nociceptin was found to be very potent in both assays (3-fold in binding and 17-fold in GTPγS functional assay). [Arg-Lys^14-15]nociceptin was the first peptide analog found to be stronger than the parent nociceptin, and structure-activity studies have suggested that the incorporated Arg-Lys^14-15 interacts with either the receptor acidic amino acid cluster or the receptor aromatic amino acid residues. (C) 2000 Academic Press.