Tyrosine 7.43 is important for mu-opioid receptor downstream signaling pathways activated by fentanyl

Abstract

G protein–coupled receptors can signal through both G proteins and ß-arrestin2. For the µ-opioid receptor (MOR), early experimental evidence from a single study suggested that G protein signaling mediates analgesia and sedation, whereas ß-arrestin signaling mediates respiratory depression and constipation. Then, receptor mutations were used to clarify which residues interact with ligands to selectively regulate signals in a ligand-specific manner. However, there is no systematic study on how to determine these residues and clarify the molecular mechanism of their influence on signal pathways. We have therefore used molecular docking to predict the amino acid sites that affect the binding of ligands and MOR. Then, the corresponding sites were mutated to determine the effect of the structural determinant of MOR on Gi/o protein and ß-arrestin pathways. The pharmacological and animal behavioral experiments in combination with molecular dynamics simulations were used to elucidate the molecular mechanism of key residues governing the signaling. Without affecting ligand binding to MOR, MORY7.43A attenuated the activation of both Gi/o protein and ß-arrestin signaling pathways stimulated by fentanyl, whereas it did not change these two pathways stimulated by morphine. Likewise, the activation peak time of extracellular regulated protein kinases was significantly prolonged at MORY7.43A compared with that at MORwildtype stimulated by fentanyl, but there was no difference stimulated by morphine. In addition, MORY7.43A significantly enhanced analgesia by fentanyl but not by morphine in the mice behavioral experiment. Furthermore, the molecular dynamics simulations showed that H6 moves toward the cellular membrane. H6 of the fentanyl–Y7.43A system moved outward more than that in the morphine–Y7.43A system. Y7.43 mutation disrupted hydrophobic interactions between W6.48 and Y7.43 in the fentanyl–Y7.43A system but not in the morphine–Y7.43A system. Our results have disclosed novel mechanisms of Y7.43 mutation affecting MOR signaling pathways. Y7.43 mutation reduced the activation of the Gi/o protein pathway and blocked the ß-arrestin2 recruitment, increased the H6 outward movement of MOR, and disrupted hydrophobic interactions. This may be responsible for the enhanced fentanyl analgesia. These findings are conducive to designing new drugs from the perspective of ligand and receptor binding, and Y7.43 is also expected to be a key site to structure optimization of synthesized compounds.

Modified Akuamma Alkaloids with Increased Potency at the Mu-opioid Receptor

“…However, the relatively high efficacy of 33 ( E max = 76%) suggests that extending further into this pocket or making additional ligand interactions allows for receptor activation. Notably, the binding pose does indicate a possible pi–pi stacking interactions with Tyr326, which has been previously implicated in μOR activation. , By offering a new scaffold to probe this subpocket by engaging these and other potential ligand–receptor interactions, the akuamma alkaloids may reveal new ways to modulate μOR activation and signaling properties.…”

“…Notably, the binding pose does indicate a possible pi−pi stacking interactions with Tyr326, which has been previously implicated in μOR activation. 57,58 By offering a new scaffold to probe this subpocket by engaging these and other potential ligand−receptor interactions, the akuamma alkaloids may reveal new ways to modulate μOR activation and signaling properties.…”

Modified Akuamma Alkaloids with Increased Potency at the Mu-opioid Receptor

“…Notably, the binding pose does indicate a possible pi-pi stacking interactions with Tyr326, which has been previously implicated in µOR activation. 59,60 By offering a new scaffold to probe this subpocket by engaging these and other potential ligand-receptor interactions, the akuamma alkaloids may reveal new ways to modulate µOR activation and signaling properties.…”

Carfentanil is a β‐arrestin‐biased agonist at the μ opioid receptor

“…Determining the binding orientation of fentanyl and carfentanil has been a contentious issue, with a range of binding poses and orientations having been reported previously in the literature. These binding orientations can be grouped into either "phenethyl-down" (Ellis et al, 2018;Eshleman et al, 2020;Lipinski et al, 2019;Lipi nski et al, 2019;Subramanian et al, 2000), similar to our pose 1, or "phenethyl-up" (de Waal et al, 2020;Ricarte et al, 2021;Tian et al, 2022), similar to our pose 2. In fact, some studies (in addition to the present one) have suggested both of these potential binding orientations (Dosen-Micovic et al, 2006;Jaronczyk et al, 2017;Podlewska et al, 2020;Xie et al, 2022) and were also unable to elucidate the "correct" binding pose/orientation.…”