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

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 p agonists with cloned rat μ opioid receptors stably expressed in HEK-293 cells and μ/κ receptor chimeras. Chimera III (K(aminoacids 1-141)/{aminoacids 151-398)), chimera IV (μ(aminoacids 1-150)/κ(aminoacids 142-380)), and chimera XII (κ(aminoacids 1-262)/μ(aminoacids 269-398)) bound [¹²⁵I]IOXY (6β-iodo-3,14-dihydoxy- 17-cyclopropylmethyl-4,5α-epoxymorphinan) with high affinities. The Ki values of 1a, 1b, 1c, and 1d at the wild-type μ 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 p receptor was substituted by that of the K receptor (chimera III), the Ki value of 1b was increased (relative to the μ receptor) 590-fold compared to a 73-fold increase for 1a. When this portion of the K receptor was replaced by that of the p 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 K receptor with that of the p receptor (chimera XII) lowered (relative to their Ki values at the K 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 κ/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 μ 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 μ agonist peptides. These data also show that the stereoisomers of RTI-4614-4 probably bind to different domains of the p receptor and suggest that manipulation of stereochemistry may be a useful tool for designing domain-specific ligands.