title: Opioid peptide receptor studies. 7. The methylfentanyl congener RTI-4614-4 and its four enantiomers bind to different domains of the rat mu 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.
authors.affiliation: Division of Intramural Research, NIDA, NIH, Baltimore, Maryland 21224, USA.
authors.forename: Y F
authors.initials: YF
authors.lastname: Lu
authors.forename: H
authors.initials: H
authors.lastname: Xu
authors.forename: L Y
authors.initials: LY
authors.lastname: Liu-Chen
authors.forename: C
authors.initials: C
authors.lastname: Chen
authors.forename: J S
authors.initials: JS
authors.lastname: Partilla
authors.forename: G A
authors.initials: GA
authors.lastname: Brine
authors.forename: F I
authors.initials: FI
authors.lastname: Carroll
authors.forename: K C
authors.initials: KC
authors.lastname: Rice
authors.forename: J
authors.initials: J
authors.lastname: Lai
authors.forename: F
authors.initials: F
authors.lastname: Porreca
authors.forename: W
authors.initials: W
authors.lastname: Sadee
authors.forename: R B
authors.initials: RB
authors.lastname: Rothman
mesh_terms: D000345:Affinity Labels / Q000494:pharmacology; D000700:Analgesics / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D000701:Analgesics, Opioid / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D019556:COS Cells; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D016308:Enkephalin, Leucine-2-Alanine / Q000031:analogs & derivatives / Q000494:pharmacology; D004745:Enkephalins / Q000494:pharmacology; D005048:Etorphine / Q000494:pharmacology; D005283:Fentanyl / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D008558:Melphalan / Q000031:analogs & derivatives / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D051381:Rats; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013237:Stereoisomerism
chemical_list: D000345:Affinity Labels; D000700:Analgesics; D000701:Analgesics, Opioid; D004745:Enkephalins; D008565:Membrane Proteins; D017450:Receptors, Opioid, mu; D011993:Recombinant Fusion Proteins; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D005048:Etorphine; D016308:Enkephalin, Leucine-2-Alanine; C037016:F 7302; C040240:enkephalin-Leu, Ala(2)-melphalan methyl ester-; D008558:Melphalan; D005283:Fentanyl