Mu-opioid receptor (µOR) agonists such as fentanyl have long been used for pain management, but are considered a major public health concern owing to their adverse side effects, including lethal overdose1. Here, in an effort to design safer therapeutic agents, we report an approach targeting a conserved sodium ion-binding site2 found in µOR3 and many other class A G-protein-coupled receptors with bitopic fentanyl derivatives that are functionalized via a linker with a positively charged guanidino group. Cryo-electron microscopy structures of the most potent bitopic ligands in complex with µOR highlight the key interactions between the guanidine of the ligands and the key Asp2.50 residue in the Na+ site. Two bitopics (C5 and C6 guano) maintain nanomolar potency and high efficacy at Gi subtypes and show strongly reduced arrestin recruitment—one (C6 guano) also shows the lowest Gz efficacy among the panel of µOR agonists, including partial and biased morphinan and fentanyl analogues. In mice, C6 guano displayed µOR-dependent antinociception with attenuated adverse effects, supporting the µOR sodium ion-binding site as a potential target for the design of safer analgesics. In general, our study suggests that bitopic ligands that engage the sodium ion-binding pocket in class A G-protein-coupled receptors can be designed to control their efficacy and functional selectivity profiles for Gi, Go and Gz subtypes and arrestins, thus modulating their in vivo pharmacology.
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The cryo-EM maps and corresponding coordinates have been deposited in the Electron Microscopy Data Bank (EMDB) under accession codes EMD-26314 (C5 guano–μOR–Gi–scFv16) and EMD-26313 (C6 guano–μOR–Gi) and the Protein Data Bank (PDB) under accession codes 7U2L (C5 guano–μOR–Gi–scFv16) and 7U2K (C6 guano–μOR–Gi). The authors declare that all the data supporting the findings of this study are available within the article, extended data and supplementary information files. All compounds can be made available on reasonable requests from the authors. Source data are provided with this paper.
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This work was supported by an American Heart Association Postdoctoral Fellowship (H.W.), NIH grants R33045884 (S.M.), R01DA042888 and R01DA007242 (Y.X.P.), R37DA036246 (B.K.K. and G.S.), R33DA038858 and P01DA035764 (V.K.), and R21DA048650 and R00DA038725 (R.A.-H.). B.K.K. and G.S. are additionally supported by the Mathers Foundation and R.A.-H. is supported through the Brain and Behavior Research Foundation. The State of Florida, Executive Office of the Governor’s Office of Tourism, Trade, and Economic Development provides funding to J.P.M. This research was funded in part through the NIH/NCI Cancer Center Support Grant P30 CA008748 to MSKCC. Cryo-EM data collection was performed at the Stanford-SLAC Cryo-EM Facilities, supported by Stanford University, SLAC and the National Institutes of Health S10 Instrumentation Programs. The authors thank E. Montabana and C. Zhang for their support with cryo-EM data collection; and Stanford University and the Stanford Research Computing Center for providing computational resources and support that contributed to these research results. Cryo-EM data processing for this project was performed on the Sherlock cluster. The authors acknowledge the Center for Advanced Research Computing (CARC) at the University of Southern California for providing computing resources that have contributed to the research results reported in this study. Receptor binding profiles were generously provided by the National Institute of Mental Health’s Psychoactive Drug Screening Program (NIMH PDSP), contract no. HHSN-271-2018-00023-C. B.L.R. is director of NIMH PDSP at the University of North Carolina at Chapel Hill and J.D. is project officer of NIMH PDSP at NIMH, Bethesda.
S.M. and Y.X.P. are founders of Sparian Biosciences. B.K.K. is a founder of and consultant for ConfometRx. G.S. is a cofounder of Deep Apple. All other authors declare no competing interests.
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Extended data figures and tables
Molecular docking of a fentanyl based bitopic ligand shows that the functional head group can target the Na+ pocket.
Representative micrographs, 2D classes, 3D classes and data processing procedures for (A) C5-guano and (B) C6-guano bound µOR–Gi-scFv16 complex.
(A) Gold-standard FSC curves for C5-guano and C6-guano bound μOR–Gi structures. Overall resolution is 3.2 Å for C5-guano bound μOR–Gi-scFv16 and 3.3 Å for C6-guano bound μOR–Gi using the gold Standard FSC = 0.143 criterion. (B) Local resolution map of C5 guano and C6 guano bound μOR–Gi structures. (C) Data collection, refinement, and model statistic of two structures. Extended Data Table 2. Cryo-EM data collection, refinement and validation statistics.
A, C, Side chains of μOR orthosteric pocket residues are shown for the C5-guano (A) and C6-guano (C) bound μOR–Gi complex (green) in comparison with the BU72 bound μOR (PDB code 5C1M; pink). The orthosteric pocket residues of μOR in complex with bitopic ligands and BU72 show nearly identical conformations. B, D, Side chains of μOR site-2 and Na+ site residues are shown for the C5 guano (B) and C6 guano (D) bound μOR–Gi complex (green) in comparison with the BU72 bound μOR (PDB code 5C1M; pink). The site-2 and Na+ site residues of μOR in complex with bitopic ligands and BU72 show nearly identical conformations.
Extended Data Fig. 5 Analysis of dynamics of direct and water mediated interactions of bitopic ligands.
A) Overlay of three examples of C5 guano conformations bound to active state MOR (pink cartoon/sticks) during MD simulations (B) Detailed view of the interactions between guano moiety of C5 guano (orange sticks) and D1142.50 mediated by two water molecule (C) Direct salt bridge interactions between C5 guano (light green sticks) and D1162.50 supplemented by an additional water-mediated hydrogen bond. (D) direct salt bridge interactions between C5 guano (cyan sticks) and D1142.50 (E) Probability densities of distances between guano nitrogen atoms and D1142.50 carboxylate oxygens. Each chart plots probability density for frames with two bridging waters (orange), one bridging water (green), and no bridging waters (cyan). (F) Categorization and relative proportion of D2.50 and D3.32 mediated interactions in 10 independent C5 guano-μOR MD trajectories for 1 μs each. Among the cumulative frames from the 10 μs MD runs, close to 1/3rd of the frames-maintained guano-D2.50 interactions exclusively through water-mediated hydrogen bonds, while ~57% frames formed direct salt- bridges with or without supplementary water mediated interactions. Therefore, close to 90% of the frames maintained D2.50-guano interactions. The piperidine-D3.32 interactions were observed to be even more stable, with over 96% of the frames indicating direct salt bridge or water-mediated hydrogen bonds. (G) Categorization and relative proportion of D2.50 and D3.32 mediated interactions in 5 independent C6-μOR trajectories for 1 μs each. Overall, the number of direct interactions with D2.50 increased from 57% to 85% (compared to C5), perhaps resulting from the increase in linker length by a carbon atom that decreases the overall distances to D2.50 residue.
Extended Data Fig. 6 Profiling of chemically and pharmacologically distinct μOR agonists using TRUPATH, arrestin signaling.
A) Peptides: Endomorphin-1, Leu-enkephalin, Met-enkephalin, Beta- endorphin and Dynorphin A (1-17). Dynorphin A (1-17) showed reduced arrestin recruitment while other peptides retained robust arrestin recruitment among peptides tested. B) Opioid biased agonists and partials: PZM21, TRV130, Gα- subtype selectivity and arrestin recruitment on μOR. PZM21, 7-OH and TRV130 showed <50% efficacy for arrestin1/2. Highest efficacy for all three biased agonists was seen at the Gz-subtype. μOR partial agonist pentazocine was a full agonist at the Gz subtype. C) Oxycodone and Carfentanil, Gα- subtype selectivity and arrestin recruitment on μOR. Carfentanil showed near maximal efficacy at all Gα-subtypes and arrestin1/2. Oxycodone was a full agonist at Gz and showed >50% efficacy at β-arrestin2. D) Fentanyl guano bitopics show differential G-protein and arrestin efficacy with increased chain length.
This file contains the following six sections. Section 1: Material and methods; Section 2: Procedure and synthetic schemes for fentanyl bitopics; Section 3: Spectral data of fentanyl bitopics; Section 4: Additional tables and figures; Section 5: Ethics declaration; and Section 6: References.
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Faouzi, A., Wang, H., Zaidi, S.A. et al. Structure-based design of bitopic ligands for the µ-opioid receptor. Nature 613, 767–774 (2023). https://doi.org/10.1038/s41586-022-05588-y