Abstract
The µ-opioid receptor (µOR) is a well-established target for analgesia1, yet conventional opioid receptor agonists cause serious adverse effects, notably addiction and respiratory depression. These factors have contributed to the current opioid overdose epidemic driven by fentanyl2, a highly potent synthetic opioid. µOR negative allosteric modulators (NAMs) may serve as useful tools in preventing opioid overdose deaths, but promising chemical scaffolds remain elusive. Here we screened a large DNA-encoded chemical library against inactive µOR, counter-screening with active, G-protein and agonist-bound receptor to ‘steer’ hits towards conformationally selective modulators. We discovered a NAM compound with high and selective enrichment to inactive µOR that enhances the affinity of the key opioid overdose reversal molecule, naloxone. The NAM works cooperatively with naloxone to potently block opioid agonist signalling. Using cryogenic electron microscopy, we demonstrate that the NAM accomplishes this effect by binding a site on the extracellular vestibule in direct contact with naloxone while stabilizing a distinct inactive conformation of the extracellular portions of the second and seventh transmembrane helices. The NAM alters orthosteric ligand kinetics in therapeutically desirable ways and works cooperatively with low doses of naloxone to effectively inhibit various morphine-induced and fentanyl-induced behavioural effects in vivo while minimizing withdrawal behaviours. Our results provide detailed structural insights into the mechanism of negative allosteric modulation of the µOR and demonstrate how this can be exploited in vivo.
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Data availability
All data supporting the findings of this study are available within the article, extended data, Supplementary Information or the associated data tables. Original raw data will be provided upon request for all experiments, including supporting information. The cryo-EM density map has been deposited into the Electron Microscopy Data Bank under accession code EMD-44635. Model coordinates have been deposited into the PDB under accession number 9BJK. Source data are provided with this paper.
Code availability
This manuscript does not report any new code.
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Acknowledgements
We thank staff at WuXi Apptec for providing the DEL and off-DNA synthesis of hits; J. Su for providing structure–activity relationship information for chemical families; and M. Robertson for providing the µORκ and Nb6 vectors. Cryo-EM data were collected at S2C2. K.K.K. was supported by the American Diabetes Association (ADA) Postdoctoral Fellowship. E.S.O. was supported by the American Heart Association (AHA) Postdoctoral Fellowship. S.M. and J.P.M. are supported by RO1DA057790. B.K.K. was supported by the Chan Zuckerberg Biohub and by R01DA036246.
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Contributions
E.S.O., V.A.R., S.M., J.P.M. and B.K.K. wrote the manuscript with input from all authors. E.S.O., W.H., K.K. and B.K.K. designed the DEL screening strategy. E.S.O. performed DEL selections. E.W. and E.S.O. designed, optimized and performed the radioligand-binding experiments. E.S.O., W.H. and Y.S. optimized and performed the GTP turnover assays. J.M.M. performed the in-cell cAMP experiments. E.S.O. formed the complexes for the cryo-EM studies, collected and processed cryo-EM data with assistance from H.W. and C.Z., built the structural models with assistance from K.K., and performed and analysed the molecular dynamics simulations. V.A.R. synthesized (±)-368, its pure enantiomers, developed a scale-up synthesis of (±)-368 and chemical characterization under S.M.’s supervision. K.A. carried out pharmacokinetics analysis of (±)-368 under S.M.’s supervision. A.E. and Q.J. performed TRUPATH experiments with supervision from T.C. S.O.E., H.R.H. and J.P.M. performed and analysed all behavioural pharmacology experiments. B.J.K. developed and performed the chiral analysis.
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Competing interests
B.K.K. is a founder and consultant for ConfometRx. S.M. is a founder of Sparian Biosciences. E.S.O., K.K.K., V.A.R., S.M. and B.K.K. have filed a patent around the new NAM compound acting through µOR. B.J.K. is an employee of Lotus Separations. The remaining authors declare no other competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Initial biochemical characterization of µOR allosteric modulators from DEL screen.
(a) Chemical properties of 368. MW; molecular weight (Da). logP; predicted octanol/water partition coefficient. PSA; polar surface area (Å2). ROTN; rotatable bonds. HBD; hydrogen bond donors. HBA; hydrogen bond acceptors. RCount; number of rings. ARCount; number of aromatics. CNSMPO; central nervous system multiparameter optimization. LogP was predicted using QikProp in Schrödinger, and other properties were calculated using ChemDraw. (b) Excess concentrations of 368 have no impact (P = 0.084) on Gi1 intrinsic turnover in the absence of receptor. Data are displayed as the average ± s.d. with n = 4 individual experiments. (c) We show using a direct 3H-naloxone binding experiment that increasing concentrations of 368 result in an increased antagonist affinity for µOR-containing membranes. Fitted affinity values are shown along with 95% confidence intervals in parentheses. Data are displayed as average values with error bars corresponding to the standard deviations of n = 4 measurements (d) The GTP turnover assay was used to show that 20 µM 368 inhibits turnover for a wide variety of orthosteric site conditions, ranging from slight inhibition of basal signaling (P = 0.0115) to no detectable effect on naloxone turnover (P = 0.314) to substantial inhibition of moderate partial agonist turnover (mitragynine pseudoindoxyl, MP, P = 0.005) to peptide (DAMGO, P < 0.0001) or small molecule (BU72, P < 0.0001) full agonists (all orthosteric molecules also present at 20 µM, data are displayed as the average ± s.d. with n = 4 individual experiments). P values for all of the above were calculated using an unpaired t-test (two tailed) and are denoted as follows: ns (P > 0.05), * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001), and **** (P ≤ 0.0001). Titrations of (e) morphine, (f) fentanyl, and (g) met-enkephalin result in activation of an assortment of G-proteins (Gi1, pink; Gi2, orange; Gi3, pale green; GoA, green; GoB, blue; and Gz, purple) as observed in the TRUPATH assay by a change in BRET signal as the Gα and Gβγ subunits separate. Activation by all 3 agonists was also calculated in the presence of 90 µM 368 (bottom panels). The data are displayed as the average the average ± s.d. with n = 6 individual experiments. The average log(EC50) for all 6 G-protein activation curves within each panel is shown as a black line (with dashed grey lines representing the standard deviation among different G protein subtypes). The average fold change in EC50 upon addition of 368 for morphine (14.5), fentanyl (16.2) and met-enkephalin (15.8) is shown. (h) The presence of excess (90 µM) 368 results in decreased agonist potencies for a variety of orthosteric agonists (fentanyl, circles; met-enkephalin, squares; morphine, triangles) across a series of Gi/o family G-protein effectors as observed by the TRUPATH assay for G-protein activation. The calculated log(EC50) for all agonist/G-protein combinations are right-shifted by ~1-1.5 units in the presence of 368. Data are displayed as average changes in log(EC50 values) for each condition with error bars corresponding to the additive fitted error 95% confidence intervals for EC50 values with and without 368. (i) This Gi/o family inhibition results in dampened cAMP inhibition in cells with morphine (dark blue vs. pink) and met-enkephalin (green vs. purple). Titration of 368 at EC80 concentrations of orthosteric agonists results in the reversal of cAMP inhibition, though with weaker potencies than those observed biochemically. Data are displayed as the average the average ± s.d. with n = 5 individual experiments.
Extended Data Fig. 2 CryoEM structure determination of µORκ-Nb6 bound to naloxone and 368.
(a) Schematic of µORκ purification and complex formation with Nb6. (b) Cryo-EM data collection and processing pipeline, showing representative micrographs of the µORκ-Nb6 complex, reference-free 2D cryo-EM class averages, and processing flow chart. This includes motion and CTF correction in Relion, followed by particle selection, 2D and 3D classifications, density map reconstructions, “gold standard” FSC curves in Cryosparc, and the final density map colored by local resolution. Also included is the cryo-EM density map and model for the seven transmembrane helices of the µORκ.
Extended Data Fig. 3 Stereochemistry & MD analysis of 368.
(a) Comparison of the chemical structures and raw sequencing counts in the DEL selections for a series of molecules in the enriched 368 family. S-stereoisomers are substantially more enriched than their R-counterparts. (b) Chiral chromatography trace demonstrating separation of racemic 368, conditions which were then used to calculate enantiomeric excess in (R)-368 (c) and (S)-368 (d). Calculated peak parameters for (b-d) are shown below the respective traces. (e) Comparison of the individually synthesized stereoisomers of the racemic 368 “hit”, demonstrating that the R-368 is >100 fold weaker than the S-368 isomer, consistent with the DEL enrichment data. Data are displayed as the average value ± s.d. with n = 4 individual experiments. (f) GTP turnover assay again demonstrating that S-368 retains the ability to potently inhibit fentanyl (5 µM)-induced GTP turnover like the racemic 368 “hit”, while the R-368 isomer does not display full inhibition even at 20 µM. Data are displayed as the average ± s.d. with n = 6 individual experiments. (g) Accordingly, the S-isomer of 368 was modeled and placed into the cryo-EM density map (blue), along with an alternate, sub-optimal pose (green) that fits into overlapping but distinct areas in cryoEM density. (h) Both poses were then subjected to MD simulation for three independent 200 ns simulations (without Nb6) (green). An alternate pose from the “opposite” orientation of the NAM in the binding site that (sub-optimally) fits into the cryoEM density was also subjected to three independent 200 ns simulations (blue). The overall root mean square deviations (r.m.s.d.) throughout the trajectory were calculated for protein Cα and all atoms in 368 and naloxone for comparison. The time-dependent r.m.s.d. of 368 throughout all trajectories is displayed in (h) and the average of each of the runs with error bars representing the s.d. of three independent simulation averages is displayed in (i). (j) Example simulation snapshots were overlaid by Cα alignment for both poses at 4 ns increments. For our chosen pose, the conformation of 368 remains nearly constant throughout all three simulations, with r.m.s.d. values near that for the protein Cα (h-j). While Cα and naloxone remain stable in both poses (i), the alternate 368 pose is unstable (h-j), resulting in very high r.m.s.d. values and extensive conformational sampling in the orthosteric vestibule region (j).
Extended Data Fig. 4 Mutational analysis of 368 binding site.
(a) Titration of (S)-368 against either wild type µOR (red), A323L µOR (purple) or I71W µOR (pink) expressing membranes demonstrates that both mutations substantially inhibit the ability of the NAM to enhance 3H-naloxone affinity. Data are displayed as the average ± s.d. with n = 4 individual experiments. (b) Expression levels of wild type human µOR (normalized to 100%) compared to mutations used in TRUPATH studies. Data are displayed as the average ± s.e.m. with n = 9 individual experiments. (c) Morphine dose-response curves observed by Gi1 recruitment to human µOR using the TRUPATH assay, comparing wild type receptor with a series of point mutants (A323L, I144E, H319L). The residue numbers correspond to those in the mouse µOR sequence used for the structural studies. Data are displayed as the average ± s.e.m. with n = 9 individual experiments. (d) Fentanyl dose-response curves observed by Gi1 recruitment to human µOR using the TRUPATH assay, again comparing wild type with point mutants. Data are displayed as the average ± s.e.m. with n = 9 individual experiments. Dose-response curves in c and d were fit using a three-parameter model for bottom, top, and logEC50 values. Log EC50 values for each curve, along with the 95% confidence interval range, are displayed for all curves.
Extended Data Fig. 5 Probe dependence and opioid receptor selectivity of 368.
(a) Comparison of the extracellular vestibule regions of the previous β-FNA-bound µOR and the current naloxone-NAM-bound µOR. The presence of 368 sterically restricts the ability of small molecule antagonists to enter/exit the orthosteric site. (b) Alignment of the receptor regions of various ligand-bound µOR structures demonstrate that small molecule orthosteric compounds (top; naloxone [present work], lofentanil [PDB: 7T2H], MP [PDB:7T2G]) have little steric clash with 368 (calculated as the number of atoms in the orthosteric ligand within 1.5 Å of 368), while both peptide agonists (bottom; β-endorphin [PDB: 8F7Q], DAMGO [PDB: 6DDE]) have clear and substantial steric clash when overlaid with the 368 binding site. (c-e) TRUPATH assays for Gi1 activation for three different opioid receptor/ligand pairs; (c) µOR/DAMGO, (d) δOR/DPDPE, (e) κOR/U50488. Data are displayed as the average ± s.d. with n = 6 individual experiments. Fitted agonist EC50 values for each curve are shown, along with the error values which correspond to the 95% confidence interval for the fitted values. 368 has the largest impact on DAMGO activation of µOR and shows some activity against δOR activation by DPDPE, but has no effects on κOR activation by U50488. (f) Sequence alignment of human µOR, δOR and κOR at structural elements with interaction with 368 (residues within 6 Å denoted with an *). Numbering refers to positions within the human µOR. Red asterisks denote residues with important interactions with 368 but have side chains that are predicted to clash or no longer form productive interactions with 368.
Extended Data Fig. 6 In vivo behavior of 368.
(a) Pharmacokinetics measurements of 368 at 10 mg/kg administered intravenously. 368 enters the brain with a maximum concentration of 1.66 uM, well above the observed affinity of the compound from radioligand binding (133 nM), but significantly lower than the concentration needed to inhibit agonist activity in cell assays. Data are displayed as the averages ± s.e.m. with n = 4 individuals. (b) In the absence of naloxone, 368 does not have significant effects on morphine-induced antinociception in the 55 °C warm-water tail-withdrawal assay (F(14,84) = 0.80, p = 0.67; Two-way RM ANOVA; n = 8 mice per group). This contrasts to the observed potentiated antagonism when in the presence of a low-dose (0.1 mg/kg) of naloxone (Fig. 4b). (c,d) The CLAMS assay with n = 8 mice demonstrates that in the absence of orthosteric compounds, 368 alone (10 mg/kg) has no significant impact on ambulation (F(6,108) = 0.36, p = 0.90; Two-way RM ANOVA) (c) and respiration (F(6,108) = 1.19, p = 0.32; Two-way RM ANOVA) (d). (e,f) CLAMS assay with n = 12 mice/group (n = 8 for morphine alone) demonstrating a dose- and time-dependent ability of 368 to potentiate low-dose (0.1 mg/kg) naloxone in reversing morphine-induced hyperlocomotion (F(24,306) = 4.27, p < 0.0001; Two-way RM ANOVA; e) and respiratory depression (F(24,294) = 5.00, p < 0.0001; Two-way RM ANOVA; f), with a maximal effect observed at 100 mg/kg 368 for both. (g) Antinociceptive time course experiment with n = 8 mice/group demonstrating that a 0.3 mg/kg dose of naloxone is able to partially reverse fentanyl (0.1 mg/kg)-induced antinociception on its own (factor: dose x time; Two-way RM ANOVA, F(7,98) = 3.883, p = 0.001; purple vs. orange curves), addition of 368 results in a significant enhancement of this naloxone-induced reversal (Two-way RM ANOVA,F(7,98) = 7.069, p = <0.0001; orange vs. dark red curves, n = 12 mice). Data for are displayed as the average percent antinociception ± s.e.m. of n = 8 or 12 mice as detailed above. (h) Antinociceptive time course experiment demonstrating that a 1.0 mg/kg dose of naloxone are not significantly able to reverse fentanyl (0.1 mg/kg)-induced antinociception (F(7,98) = 1.947, p = 0.070; purple vs. orange curves), addition of 368 results in a significant enhancement of this naloxone-induced reversal (F(7,98) = 7.077, p = <0.0001; orange vs. dark red curves). Data for are displayed as the average percent antinociception ± s.e.m. of n = 8 mice.
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O’Brien, E.S., Rangari, V.A., El Daibani, A. et al. A µ-opioid receptor modulator that works cooperatively with naloxone. Nature (2024). https://doi.org/10.1038/s41586-024-07587-7
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DOI: https://doi.org/10.1038/s41586-024-07587-7
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