PSEUDOIRREVERSIBLE BINDING CHARACTERISTICS OF [D-ALA(2),GLU(4)]DELTORPHIN AND ITS CYS(4) SUBSTITUTED DERIVATIVE TO DELTA-OPIOID RECEPTORS

Following the identification of [D-Ala2,Glu4]deltorphin as a selective delta2-opioid receptor agonist in vivo, we synthesized the Cys4-subst ituted analogue as a potential ligand which might bind 'irreversibly' at this site through a proposed thiol-disulfide exchange mechanism. Pr evious studies showed that intracerebroventricular (i.c.v.) pretreatme nt with [D-Ala2, Cys4]deltorphin, 24 h prior to antinociceptive testin g, produced a selective antagonism of [D-Ala2,Glu4 ]deltorphin-induced antinociception in mice. Surprisingly, however, the Ser4-analogue (sy nthesized as a control) and even the parent molecule, [D-Ala2,Glu4 ]de ltorphin, had the same antagonistic effect following pretreatment in v ivo, while pretreatment with an equiantinociceptive dose of [D-Ser2,Le u5,Thr6]-enkephalin, a structurally unrelated delta2-opoid receptor ag onist did not exhibit long-lasting antinociceptive actions. These data raised questions regarding the mechanism of the antagonism observed i n vivo with the deltorphins; the present studies have attempted to exp lore these issues using radioligand binding techniques. The results de monstrate a decrease in the B(max) of osyl-3',5'-H-3,D-Pen2,p-Cl-Phe4, D-Pen5]-enkephalin ([H-3]p-Cl-DPDPE) (delta-opioid receptor ligand) fo llowing i.c.v. pretreatment of mice (at -24 h) with [D-Ala2, Cys4]delt orphin or [D-Ala2,Glu4]deltorphin, but not with [D-Ala2, Ser4] deltorp hin, suggesting a difference in mechanism of antagonism seen in vivo w ith these compounds. Incubation of mouse whole brain homogenates in vi tro with [D-Ala2,CyS4]deltorphin or with [D-Ala,2Glu4 ]deltorphin, als o resulted in a decrease in the radioligand binding of [H-3]p-Cl-DPDPE , but this effect was not prevented by coincubation with dithiothreito l, a thiol-reducing agent. Direct evaluation of binding using [H-3][D- Ala2,Glu4]deltorphin (5 nM) showed that a portion of this ligand (i.e. , about 10% of all specific binding) remained specifically and 'irreve rsibly' bound to mouse brain membranes following incubation in vitro a nd extensive washing. The 'irreversibly', specifically bound [h-3][D-A la2,Glu4]deltorphin could be removed, however, by brief exposure of th e membranes to a low pH (2.5), high-salt (0.5 M NaCl) solution. These data suggest that [D-Ala2,Cys4] deltorphin and [D-Ala,2Glu4]deltorphin bind in a 'pseudoirreversible' (non-covalent) manner to an delta-opio id receptor via a mechanism that apparently does not involve thiol-dis ulfide exchange.