Opioid peptide receptor studies. 7. The methylfentanyl congener RTI-4614-4 and its four enantiomers bind to different domains of the rat ? opioid receptor

Abstract

Mutational analysis of opioid receptors supports the hypothesis that dissimilar receptor domains contribute to the binding affinity of different ligands. To determine whether enantiomeric ligands can serve to distinguish between different binding pockets (which focuses the analysis on asymmetric structural factors while avoiding confounding changes in physiochemical characteristics), we analyzed the binding of the 3-methylfentanyl congeners RTI-4614-4 [(+/-)-cis-N-[1-(2-hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]-N- phenylpropanamide HCl)], its four stereoisomers [(2S,3R,4S)-1a, (2R,3R,4S)-1b, (2R,3S,4R)-1c, and (2S,3S,4R)-1d], and other mu agonists with cloned rat mu opioid receptors stably expressed in HEK-293 cells and mu/kappa receptor chimeras. Chimera III (kappa[aminoacids 1-141]/mu[aminoacids 151-398]), chimera IV (mu[aminoacids 1-150]/kappa[aminoacids 142-380]), and chimera XII (kappa[aminoacids 1-262]/mu[aminoacids 269-398]) bound [(125)I]IOXY (6beta-iodo-3,14-dihydoxy-17-cyclopropylmethyl-4,5alpha++ +-epoxymorphinan) with high affinities. The Ki values of 1a, 1b, 1c, and 1d at the wild-type mu receptor were 0.55 nM, 0.66 nM, 124 nM, and 59.2 nM, respectively. When the region from the N terminal to the start of the transmembrane helix 3 (TMH3) of the mu receptor was substituted by that of the kappa receptor (chimera III), the Ki value of 1b was increased (relative to the mu receptor) 590-fold compared to a 73-fold increase for 1a. When this portion of the kappa receptor was replaced by that of the mu receptor (chimera IV), the loss of affinity was not as great: 11.7-fold for 1a and 58.5-fold for 1b. Replacement of the middle of the third intracellular loop and third extracellular loop (e3) of the kappa receptor with that of the mu receptor (chimera XII) lowered (relative to their Ki values at the kappa receptor) the Ki values of [D-Ala2,D-Leu5]enkephalin and [D-Ala2-MePhe4,Gly-ol5]enkephalin to a much greater extent than the Ki values of the isomers. The kappa/chimera XII shift was greater for isomers 1c and 1d than for 1b and 1a. Viewed collectively, these data suggest that the region from the N terminal to the start of the TMH3 of the mu opioid receptor determines the binding affinity of RTI-4614-4 and its isomers and that the e3 loop also plays a major role in determining the binding affinity of mu agonist peptides. These data also show that the stereoisomers of RTI-4614-4 probably bind to different domains of the mu receptor and suggest that manipulation of stereochemistry may be a useful tool for designing domain-specific ligands.

Molecular Docking Reveals a Novel Binding Site Model for Fentanyl at the μ-Opioid Receptor

“…These subtle differences in the ligand conformations may be a contributing factor in reducing the affinity of 5 (by 590-fold) to a κ/µ chimeric receptor in which TM-I and -II are taken from κ and TM-III-VII are taken from µ. 21 The binding affinity of 6, on the other hand, was effected much less (40-fold), lending support to the hypothesis. Similarly, the proximity of the phenethyl group of 1 to TM-III, and especially to N150 (which appears to sterically hinder binding), is corroborated by the 20-fold increase observed in fentanyl binding upon a N150A point mutation.…”

Acupuncture Inhibits GABA Neuron Activity in the Ventral Tegmental Area and Reduces Ethanol Self‐Administration

“…Unlike naloxone, naltrindole abolished the inhibitory effect of ethanol on VTA GABA neuron firing rate, suggesting the involvement of DORs for ethanol effects in the VTA. However, future studies are necessary to determine whether the effect is on DORs on accumbal GABAergic terminals on VTA GABA neurons (Margolis et al., 2008) or on NAcc DORs (Lu et al., 1998; Mansour et al., 1995; Weiner et al., 1991).…”

Opioid peptide receptor studies. 14. Stereochemistry determines agonist efficacy and intrinsic efficacy in the [35S]GTP-?-S functional binding assay

“…We explored the properties of antagonist receptor interactions to determine if they distinguished these stereoisomers. Molecular biological data indicate that DAMGO and etorphine bind to different domains of the receptor (Xu et al, 1999;Lu et al, 1996). Therefore, it was of interest to determine if the functional K i of antagonists differ as a function of the agonist used to stimulate [ 35 S]-GTP-␥-S binding.…”