Dermorphin-based potential affinity labels for μ-opioid receptors

Dermorphin and [Lys⁷]dermorphin, selective μ-opioid receptor ligands originating from amphibian skin, have been modified with various electrophiles in either the 'message' or 'address' sequences as potential peptide-based affinity labels for μ-receptors. Introduction of the electrophilic isothiocyanate and bromoacetamide groups on the para position of Phe³ and Phe⁵ was accomplished by incorporating Fmoc-Phe(p-NHAlloc) into the peptide followed by selective deprotection and modification. The corresponding amine-containing peptides were also prepared. The pure peptides were evaluated in radioligand binding experiments using Chinese hamster ovary (CHO) cells expressing μ- and δ-opioid receptors. In dermorphin, introduction of the electrophilic groups in the 'message' domain lowered the binding affinity by > 1000-fold; only [Phe(p-NH₂)³]dermorphin retained nanomolar affinity for μ-receptors. Modifications in the 'address' region of both dermorphin and [Lys⁷]dermorphin were relatively well tolerated. In particular, [Phe(p-NH₂)⁵, Lys⁷]dermorphin showed similar affinity to dermorphin, with almost 2-fold higher selectivity for μ-receptors. [Phe(p-NHCOCH₂Br)⁵]- and [Phe(p-NHCOCH₂Br)⁵, Lys⁷]dermorphin exhibited relatively high affinity (IC₅₀ = 27.7 and 15.1 nM, respectively) for μ-receptors. However, neither of these peptides inhibited [³H]DAMGO binding in a wash-resistant manner.