We previously reported that the novel dynorphin A (Dyn A, Tyr-Gly-Gly-Phe-Leu-Arg-Arg-lle-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln) analog arodyn (Ac[Phe1,2,3,Arg4,D-Ala8]Dyn A-(1-11)NH2, Bennett, M.A., Murray, T.F. & Aldrich, J.V. (2002). J. Med. Chem. vol. 45, pp. 5617-5619) is a κ opioid receptor-selective peptide [Ki(κ) = 10 nM, Ki ratio (κ/μ/δ) = 1/174/583] which exhibits antagonist activity at κ opioid receptors. In this study, a series of arodyn analogs was prepared and evaluated to explore the structure-activity relationships (SAR) of this peptide; this included an alanine scan of the entire arodyn sequence, sequential isomeric D-amino acid substitution in the N-terminal 'message' sequence, NMePhe substitution individually in positions 1-3, and modifications in position 1. The results for the Ala-substituted derivatives indicated that Arg6 and Arg7 are the most important residues for arodyn's nanomolar binding affinity for κ opioid receptors. Ala substitution of the other basic residues (Arg4, Arg9 and Lys11) resulted in lower decreases in affinity for κ opioid receptors (three- to fivefold compared with arodyn). Of particular interest, while [Ala10]arodyn exhibits similar κ opioid receptor binding as arodyn, it displays higher κ vs. μ opioid receptor selectivity [Ki ratio (κ/μ) = 1/350] than arodyn because of a twofold loss in affinity at μ opioid receptors. Surprisingly, the Tyr1 analog exhibits a sevenfold decrease in κ opioid receptor affinity, indicating that arodyn displays significantly different SAR than Dyn A; [Tyr1)arodyn also unexpectedly exhibits inverse agonist activity in the adenylyl cyclase assay using Chinese hamster ovary cells stably expressing κ opioid receptors. Substitution of NMePhe in position 1 gave [NMePhe1]arodyn which exhibits high affinity [Ki(κ) = 4.56 nM] and exceptional selectivity for κ opioid receptors [Ki- ratio (κ/μ/δ) = 1/1100/>2170]. This peptide exhibits antagonistic activity in the adenylyl cyclase assay, reversing the agonism of 10 nM Dyn A-(1-13)NH2. Thus [NMePhe1]arodyn is a highly κ opioid receptor-selective antagonist that could be a useful pharmacological tool to study κ opioid receptor-mediated activities. Copyright Blackwell Munksgaard, 2005.
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