Pseudoirreversible binding characteristics of [D-Ala², Glu⁴]deltorphin and its Cys⁴ substituted derivative to δ-opioid receptors

Following the identification of [D-Ala²,Glu⁴]deltorphin as a selective δ₂-opioid receptor agonist in vivo, we synthesized the Cys⁴-substituted analogue as a potential ligand which might bind 'irreversibly' at this site through a proposed thil-disulfide exchange mechanism. Previous studies showed that intracerebroventricular (i.c.v.) pretreatment with [D-Ala²,Cys⁴]deltorphin, 24 h prior to antinociceptive testing, produced a selective antagonism of [D-Ala²,Glu⁴]deltorphin-induced antinociception in mice. Surprisingly, however, the Ser⁴-analogue (synthesized as a control) and even the parent molecule, [D-Ala²,Glu⁴]deltorphin, had the same antagonistic effect following pretreatment in vivo, while pretreatment with an equiantinociceptive dose of [D-Ser²,Leu⁵,Thr⁶]-enkephalin, a structurally unrelated δ₂-opioid receptor agonist did not exhibit long-lasting antinociceptive actions. These data raised questions regarding the mechanism of the antagonism observed in vivo with the deltorphins; the present studies have attempted to explore these issues using radioligand binding techniques. The results demonstrate a decrease in the B_max of [tyrosyl-3′,5′-³H,D-Pen²,p-Cl-Phe⁴,D-Pen⁵]-enkephalin ([³H]p-Cl-DPDPE) (δ-opioid receptor ligand) following i.c.v. pretreatment of mice (at -24 h) with [D-Ala²,Cys⁴]deltorphin or [D-Ala²,Glu⁴]deltorphin, but not with [D-Ala²,Ser⁴] deltorphin, suggesting a difference in mechanism of antagonism seen in vivo with these compounds. Incubation of mouse whole brain homogenates in vitro with [D-Ala²,Cys⁴]deltorphin or with [D-Ala²,Glu⁴]deltorphin, also resulted in a decrease in the radioligand binding of [³H]p-Cl-DPDPE, but this effect was not prevented by coincubation with dithiothreitol, a thiol-reducing agent. Direct evaluation of binding using [³H][D-Ala²,Glu⁴]deltorphin (5 nM) showed that a portion of this ligand (i.e., about 10% of all specific binding) remained specifically and 'irreversibly' bound to mouse brain membranes following incubation in vitro and extensive washing. The 'irreversibly', specifically bound [³H][D-Ala²,Glu⁴]deltorphin could be removed, however, by brief exposure of the membranes to a low pH (2.5), high-salt (0.5 M NaCl) solution. These data suggest that [D-Ala²,Cys⁴] deltorphin and [D-Ala²,Glu⁴]deltorphin bind in a 'pseudoirreversible' (no-covalent) manner to an δ-opioid receptor via a mechanism that apparently does not involve thiol-disulfide exchange.