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

Abstract Following the identification of [D-Ala 2 ,Glu 4 ]deltorphin as a selective δ 2 -opioid receptor agonist in vivo, we synthesized the Cys 4 -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-Ala 2 ,Cys 4 ]deltorphin, 24 h prior to antinociceptive testing, produced a selective antagonism of [D-Ala 2 ,Glu 4 ]deltorphin-induced antinociception in mice. Surprisingly, however, the Ser 4 -analogue (synthesized as a control) and even the parent molecule, [D-Ala 2 ,Glu 4 ]deltorphin, had the same antagonistic effect following pretreatment in vivo, while pretreatment with an equiantinociceptive dose of [D-Ser 2 ,Leu 5 ,Thr 6 ]-enkephalin, a structurally unrelated δ 2 -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′- 3 H,D-Pen 2 ,p-Cl-Phe 4 ,D-Pen 5 ]-enkephalin ([ 3 H]p-Cl-DPDPE) (δ-opioid receptor ligand) following i.c.v. pretreatment of mice (at −24 h) with [D-Ala 2 ,Cys 4 ]deltorphin or [D-Ala 2 ,Glu 4 ]deltorphin, but not with [D-Ala 2 ,Ser 4 ] deltorphin, suggesting a difference in mechanism of antagonism seen in vivo with these compounds. Incubation of mouse whole brain homogenates in vitro with [D-Ala 2 ,Cys 4 ]deltorphin or with [D-Ala 2 ,Glu 4 ]deltorphin, also resulted in a decrease in the radioligand binding of [ 3 H]p-Cl-DPDPE, but this effect was not prevented by coincubation with dithiothreitol, a thiol-reducing agent. Direct evaluation of binding using [ 3 H][D-Ala 2 ,Glu 4 ]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 [ 3 H][D-Ala 2 ,Glu 4 ]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-Ala 2 ,Cys 4 ] deltorphin and [D-Ala 2 ,Glu 4 ]deltorphin bind in a ‘pseudoirreversible’ (no-covalent) manner to an δ-opioid receptor via a mechanism that apparently does not involve thiol-disulfide exchange.