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β-funaltrexamine (β-FNA) is an irreversible μ opioid (MOP) receptor antagonist and a reversible agonist of κ opioid (KOP) receptor. β-FNA binds covalently to the MOP receptor at Lys233 5.39 , which is conserved among opioid receptors. Molecular docking of β-FNA showed that K303 6.58 in the MOP receptor and E297 6.58 in the KOP receptor played distinct roles in positioning β-FNA. K303 6.58 E MOP receptor and E297 6.58 K KOP receptor mutants were generated. The mutations did not affect β-FNA affinity or efficacy. K303 6.58 E mutation in the MOP receptor greatly reduced covalent binding of [ 3 H]β-FNA; however, E297 6.58 K did not enable the KOP receptor to bind irreversibly to β-FNA. Molecular modeling demonstrated that the e-amino group of K303 6.58 in the MOP receptor interacted with C O of the acetate group of β-FNA to facilitate covalent bond formation with Lys233 5.39 . Replacement of K303 6.58 with Glu in the MOP receptor resulted in repulsion between the COOH of Glu and the C O of β-FNA and increased the distance between K233 5.39 and the fumarate group, making it impossible for covalent bond formation. These findings will be helpful for design of selective non-peptide MOP receptor antagonists.

It has been shown previously that norbinaltorphimine (norBNI) and 5′-guanidinonaltrindole (5′-GNTI), long-acting kappa opioid receptor (KOPR) antagonists, cause frenzied scratching in mice [1] , [2] . In the current study, we examined if zyklophin, a short-acting cyclic peptide KOPR antagonist, also elicited scratching behavior. When injected s.c. in the nape of the neck of male Swiss–Webster mice, zyklophin at doses of 0.1, 0.3 and 1 mg/kg induced dose-related hindleg scratching of the neck between 3 and 15 min after injection. Pretreating mice with norBNI (20 mg/kg, i.p.) at 18–20 h before challenge with zyklophin (0.3 mg/kg) did not markedly affect scratching. Additionally, KOPR−/− mice given 0.3 mg/kg of zyklophin displayed similar levels of scratching as wild-type animals. The absence of KOPR in KOPR−/− mice was confirmed with ex vivo radioligand binding using [3H]U69,593. Taken together, our data suggest that the presence of kappa receptors is not required for the excessive scratching caused by zyklophin. Thus, zyklophin, similar to the structurally different KOPR antagonist 5′-GNTI, appears to act at other targets to elicit scratching and potentially the sensation of itch.

Several investigators recently identified biased κ opioid receptor (KOP receptor) agonists. However, no comprehensive study of the functional selectivity of available KOP receptor agonists at the human and mouse KOP receptors (hKOP receptor and mKOP receptor, respectively) has been published. Here we examined the ability of over 20 KOP receptor agonists to activate G proteins and to internalize the receptor. Clonal neuro-2a mouse neuroblastoma (N2a) cells stably transfected with the hKOP receptor or mKOP receptor were used. We employed agonist-induced [(35)S]GTPγS binding and KOP receptor internalization as measures of activation of G protein and β-arrestin pathways, respectively. The method of Ehlert and colleagues was used to quantify intrinsic relative activities at G protein activation (RAi-G) and receptor internalization (RAi-I) and the degree of functional selectivity between the two [Log RAi-G - logRAi-I, RAi-G/RAi-I and bias factor]. The parameter, RAi, represents a relative estimate of agonist affinity for the active receptor state that elicits a given response. The endogenous ligand dynorphin A (1-17) was designated as the balanced ligand with a bias factor of 1. Interestingly, we found that there were species differences in functional selectivity. The most striking differences were for 12-epi-salvinorin A, U69,593, and ICI-199,441. 12-Epi-salvinorin A was highly internalization-biased at the mKOP receptor, but apparently G protein-biased at hKOP receptor. U69,593 was much more internalization-biased at mKOP receptor than hKOP receptor. ICI199,441 showed internalization-biased at the mKOP receptor and G protein-biased at the hKOP receptor. Possible mechanisms for the observed species differences are discussed.