1. Biochemistry and Chemical Biology

Controlling opioid receptor functional selectivity by targeting distinct subpockets of the orthosteric site

  1. Rajendra Uprety
  2. Tao Che
  3. Saheem A Zaidi
  4. Steven G Grinnell
  5. Balázs R Varga
  6. Abdelfattah Faouzi
  7. Samuel T Slocum
  8. Abdullah Allaoa
  9. András Varadi
  10. Melissa Nelson
  11. Sarah M Bernhard
  12. Elizaveta Kulko
  13. Valerie Le Rouzic
  14. Shainnel O Eans
  15. Chloe A Simons
  16. Amanda Hunkele
  17. Joan Subrath
  18. Ying Xian Pan
  19. Jonathan A Javitch
  20. Jay P McLaughlin
  21. Bryan L Roth  Is a corresponding author
  22. Gavril W Pasternak
  23. Vsevolod Katritch  Is a corresponding author
  24. Susruta Majumdar  Is a corresponding author
  1. Department of Neurology and Molecular Pharmacology, Memorial Sloan Kettering Cancer Center, United States
  2. Department of Pharmacology, University of North Carolina, United States
  3. Center for Clinical Pharmacology, St. Louis College of Pharmacy and Washington University School of Medicine, United States
  4. Department of Anesthesiology, Washington University in St. Louis School of Medicine, United States
  5. Department of Quantitative and Computational Biology, Department of Chemistry, Bridge Institute, Michelson Center for Convergent Bioscience, University of Southern California, United States
  6. Division of Molecular Therapeutics, New York State Psychiatric Institute and Departments of Psychiatry, Pharmacology, Columbia University Vagelos College of Physicians & Surgeons, United States
  7. Department of Pharmacodynamics, University of Florida, United States
  8. Department of Anesthesiology, Rutgers New Jersey Medical School, New Jersey, United States
https://doi.org/10.7554/eLife.56519
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Cite this article
  1. Rajendra Uprety
  2. Tao Che
  3. Saheem A Zaidi
  4. Steven G Grinnell
  5. Balázs R Varga
  6. Abdelfattah Faouzi
  7. Samuel T Slocum
  8. Abdullah Allaoa
  9. András Varadi
  10. Melissa Nelson
  11. Sarah M Bernhard
  12. Elizaveta Kulko
  13. Valerie Le Rouzic
  14. Shainnel O Eans
  15. Chloe A Simons
  16. Amanda Hunkele
  17. Joan Subrath
  18. Ying Xian Pan
  19. Jonathan A Javitch
  20. Jay P McLaughlin
  21. Bryan L Roth
  22. Gavril W Pasternak
  23. Vsevolod Katritch
  24. Susruta Majumdar
(2021)
Controlling opioid receptor functional selectivity by targeting distinct subpockets of the orthosteric site
eLife 10:e56519.
https://doi.org/10.7554/eLife.56519
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22 figures, 11 tables and 2 additional files

Figures

Figure 1
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The design concept, structures of ligands synthesized and evaluated at opioids receptors.

(A) Overview of the key hypothesis, suggesting that TM5-ECL2 engagement by morphinan ligand with ring-C chair form leads to preferred G protein signaling, whereas TM2-TM3 engagement and ring-C boat …

Figure 2
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MP1104 at both MOR and KOR and MP1202 at KOR targets the TM2-TM3 region while MP1202 at MOR targets the TM5-ECL2 region and show distinct signaling properties.

(A-B) MP1104 (red) is a full agonist in hMOR in cAMP inhibition (N = 5) and Tango-arrestin recruitment assays (N = 5) compared to DAMGO (blue). (C–D) MP1104 (red) is a full agonist in hKOR in cAMP …

Figure 3
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meta-Amino (MP1207) and meta-guanidino (MP1208) analogs prefer the chair conformation and target the TM5-ECL2 region and are G protein biased agonists at KOR and at MOR show no measurable arrestin recruitment.

(A-B) MP1207 (orange) and MP1208 (green) are full agonists at hKOR in cAMP inhibition (N = 3) and partial agonists in Tango-arrestin recruitment assays (N = 3) compared to U50, 488 (purple). (C–D) …

Figure 4
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MD simulations in hKOR with MP1207 show the guanidine group interacts with E209/D223 and analogs of MP1207 not oriented toward TM5-ECL2 region suggest G protein bias of MP1207/08 is dependent on salt-bridge formation in this region.

(A) Docking of MP1202 (green sticks) and MP1207 (yellow sticks) in wild type KOR showing MP1207 chair form engaging D223 and E209 residues in TM5-ECL2 region while MP1202 boat form not engaging this …

Figure 5
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MP1207/1208 show MOR/KOR-mediated antinociception without showing place preference or aversion.

(A) Dose-response curve: Groups of C57BL/6J mice were supraspinally (icv) administered MP1207, MP1208, morphine and U50,488h and antinociception measured using the 55°C tail withdrawal assay at peak …

Figure 6
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MP1207/08 show attenuated respiratory depression and locomotor effects compared to morphine in mice.

Mice were administered either saline (n = 15), vehicle (n = 24), morphine (30 mg/kg, IP; n = 12 or 30 nmol; n = 18 or 100 nmol, icv; n = 16), MP1207 (30 nmol; n = 26 or 100 nmol icv, n = 10), MP1208 …

Appendix 1—figure 1
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Characterization of ligands (MP1104, IBNtxA, MP1202, MP1207, MP1208, MP1305, and MP1601) at mouse mu opioid receptor (mMOR) using BRET assays-chemical structure, docking in MOR, G protein activity, and arrestin recruitment.

(A) MP1104 targets the TM2-TM3 region and is arrestin-biased at mMOR: The preferred docking pose of MP1104 (boat form, yellow stick) at an active state of MOR is shown. Ring C of MP1104 in boat form …

Appendix 1—figure 2
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Characterization of ligands (MP1104, IBNtxA, MP1202, MP1207, MP1208, MP1305 and MP1601) at rat kappa opioid receptor (rKOR) using BRET assays-chemical structures, docking in MOR, G protein activity, and arrestin recruitment.

A) MP1104 targets the TM2-TM3 region and is arrestin biased at rKOR: The preferred docking pose of MP1104 (boat form, yellow stick) at an active state of KOR is shown. The iodophenylamido moiety …

Appendix 1—figure 3
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IBNtxA is arrestin biased at KOR but shows G protein biased agonism at MOR.

(A–B) At hMOR, IBNtxA (orange) is a G protein biased agonist compared with DAMGO (blue) (N = 4). (C–D) At hKOR, IBNtxA (pink) is a full agonist in cAMP inhibition (N = 3) and Tango-arrestin …

Appendix 1—figure 4
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Bias plots for ligands at hKOR (A), hMOR (B) using cAMP and Tango assay and at rKOR /mMOR (C) using BRET assays.

Bias analysis for signaling was performed as described in Materials and methods. Data analyzed against DAMGO and/or U50,488h for each ligand using unpaired t-test with Welch’s correction. At hKOR, …

Appendix 1—figure 5
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The preferred docking pose of known KOR biased ligand 6′GNTI (chair form, orange stick) at an active state of KOR with the guanidino group engaging a region between TM5 and ECl2.

Note: possible engagement of residues D223 and E209 similar to MP1207 and MP1208 in TM5-ECL2 region.

Appendix 1—figure 6
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MP1207 is selective for opioid receptors in the GPCRome screen.

MP1207 was screened against 330 non-olfactory GPCRs for agonism in the arrestin recruitment TANGO assay. Each point shows luminescence normalized to basal level at a given GPCR at 3 µM MP1207 dose (>…

Appendix 1—figure 7
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Mutation of KOR Y312W leads to a receptor mimicking MOR arrestin recruitment.

MP1202 flips to a G protein biased agonist and arrestin recruitment for MP1207 and MP1208 is reduced. (A and C) At Y312W-hKOR, MP1202 (light green) is a G-biased agonist in cAMP inhibition (N = 3) (A…

Appendix 1—figure 8
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Methoxycarbonyl fentanyl amide analog MP102 targeting TM5 in MOR shows no arrestin recruitment compared to methoxycarbonyl fentanyl at mMOR in BRET assays.

(A-B) Methoxycarbonyl fentanyl (red) is a full agonist at hMOR in cAMP (N = 3) and arrestin recruitment assays (N = 3) compared to DAMGO (blue). (C–D) Methoxycarbonyl fentanyl (red) is a full …

Appendix 1—figure 9
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At human receptors, MP1305 is G-biased at MOR and KOR whereas MP1601 is G-biased at MOR and arrestin biased at KOR.

(A-B) MP1305 (green) is a full agonist at hKOR in cAMP inhibition (N = 6) and partial agonist in Tango-arrestin recruitment assays (N = 6) compared to U50,488H (purple) at hKOR. (C–D) MP1305 (red) …

Appendix 1—figure 10
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MP1305 is G protein biased at KOR.

Docking pose for ligands inside active state KOR shown in white carbon sticks and white ribbon representations. (A–B) MP1202 in KOR favors boat conformation. (C–D) MP1305 in KOR favors chair …

Appendix 1—figure 11
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MP1207 and 1208 show significantly less hyperlocomotion compared to morphine.

Mice were administered icv either morphine (30 nmol; n = 18), MP1207 (30 nmol; n = 26) or MP1208 (35 nmol; n = 10) and the ambulation of each group of mice monitored using the CLAMS/Oxymax system. Mo…

Appendix 1—scheme 1
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Synthesis of m-arylamido dihydromorphinans MP1202 (A), MP1207, MP1208 (B).
Appendix 1—scheme 2
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Synthesis of m-arylamido dihydromorphinans MP1210 and p-arylamido dihydromorphinan analog, MP1209.
Appendix 1—scheme 3
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Synthesis of 14-O-methyl m-iodoarylamidomorphinan MP1305.
Appendix 1—scheme 4
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Synthesis of m-iodoarylamido-4,5-deoxymorphinan MP1601.
Author response image 1
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Tables

Appendix 1—table 1
Receptor affinities of arylamidomorphinans in mouse opioid receptor transfected cell lines.
Compd. Ki [nM]a
mMOR mKOR mDOR
IBNtxA 0.11 ± 0.02 0.03 ± 0.001 0.24 ± 0.05
MP1104 0.021 ± 0.00 0.0064 ± 0.0 0.08 ± 0.01
MP1202 0.071 ± 0.031 0.11 ± 0.064 1.3 ± 0.8
MP1207 0.23 ± 0.02 0.39 ± 0.05 15.62 ± 2.64
MP1208 0.34 ± 0.01 0.28 ± 0.02 19.28 ± 6.48
MP1305 0.25 ± 0.02 2.5 ±0.3 11.7 ± 1.4
MP1601 0.2 ± 0.01 2.13 ± 0.3 5.37 ± 0.9
Morphine 4.60 ± 1.81b _ _
DAMGO 3.34 ± 0.43b _ _
U50, 488h _ 0.73 ± 0.32b _
DPDPE _ _ 1.39 ± 0.67b
Appendix 1—table 2
[35S]GTPγS Functional assays in mouse opioid receptor transfected cell lines.
Compd. mMOR mKOR mDOR
EC50 (nM) Emax (%) EC50 (nM) Emax (%) EC50 (nM) Emax (%) IC50 (nM)
IBNtxA 0.49 ± 0.12 101 ± 3 0.22 ± 0.02 102 ± 4 4.08 ± 0.67 95 ± 2 -
MP1104 0.21 ± 0.03 103 ± 2 0.027 ± 0.002 104 ± 2 0.41 ± 0.11 88 ± 0 -
MP1202 0.32 ± 0.03 68 ± 1 0.13 ± 0.02 94 ± 5 4 ± 1.6 71 ± 2 -
MP1207 1.29 ±0.65 41 ± 1 1.52 ± 0.07 39.3 ± 1.3 nd 10-15% 27.34±1.95
MP1208 1.13 ± 0.05 54 ± 0.7 1.36 ± 0.23 43 ± 0.8 nd 10-15% 11.39±0.3
MP1305 0.7 ± 0.1 81.2 ± 16 7.4 ± 1.8 42.2 ± 5.3 31.7 ± 3.6 22± 0.9 -
MP1601 0.5 ± 0.2 45 ± 4.6 3 ± 0.7 72 ± 4.5 10 ± 1.6 67 ± 3.9 -
DAMGO 3.4 ± 0.2 - - - - - -
U50,488h - - 9.5 ± 1.8 - - - - -
DPDPE - - - - 16.2 ± 5.1 - -
Morphine 14.77±3.9 102±5 - - - - -
Buprenorphineb 1.8(1.3,2.3)c Full agonistd - - - - -
Appendix 1—table 3
Energies for boat and chair conformations calculated by computational QM calculations for ligands.
Ligand (Basis set) Energy(Chair – Boat)
HF		 Energy(Chair – Boat) kcal/mol Energy(Chair – Boat) kJ/mol
MP1104 (LanL2DZ) 0.03671637 23.0396323 96.3988294
MP1104 (DGDZVP) 0.03493887 21.9242457 91.7320032
IBNtxA (LanL2DZ) −0.0014450 −0.90674184 −3.7938475
IBNtxA (DGDZVP) −0.0016583 −1.040599833 −4.353869701
MP1202 (LanL2DZ) −0.01402513 −8.8009031585 −36.822978815
MP1202 (DGDZVP) −0.011652 −7.31174652 −30.59234743997
Appendix 1—table 4
Best docking scores of each ligand with a chair or a boat conformation at active state human mu and kappa opioid receptors.
Drugs MP1104 IBNtxA MP1202 MP1207 MP1208 MP1305 MP1601 6'GNTI MP1209 MP1210
Docking score of Chair/hMOR -36.22 -52.42 -55.04 -69.74 -38.63 -40.61 -51.03 -44.17
Boat /hMOR -48.28 -31.36 -49.44 -50.81 -67.72 -28.83 -38.23 -43.67 -36.18
Chair /hKOR -46.93 -47.82 -55.99 -86.24 -46.77 -46.08 -64.86 -57.63 -53.91
Boat /hKOR -55.06 -49.51 -52.42 -45.9 -65.88 -44.87 -47.54 -60.77 -55.43
Appendix 1—table 5
Docking scores for proposed analogs of MP1202, where m-iodo group is substituted with a polar moiety (R).
Serial R Chair score Boat score Scores for chair preference
 1 -mNH2 −55.35 −58.47 +3.12
 2 -mN(CH3)2 −51.81 −48.34 −3.47
 3 -mOH −54.81 −57.34 +2.53
4 (MP1207) -mCH2NH2 −55.99 −45.9 −10.09
 5 -mCH2CH2NH2 −59.42 −51.79 −7.63
 6 -mCH2CH2CH2NH2 −64.07 −49.91 −14.16
 7 -mCH2CH2CH2CH2NH2 −52.42 −49.67 −2.75
 8 -mgaunidine −76.29 −66.34 −9.95
9 (MP1208) -mCH2guanidine −86.24 −65.88 −20.36
 10 -mCOguanidine −82.09 −77.53 −4.56
 11 -pCH2NH2 −57.63 −60.77 +3.14
 12 -mCH2OH −53.91 −55.43 +1.5
Appendix 1—table 6
Functional data at hDOR and mDOR.
Functional data at hDOR and mDOR
Compd. cAMP inhibition Arrestin recruitment
EC50,nM (pEC50± SEM) Emax%± SEM EC50,nM (pEC50± SEM) Emax%± SEM
IBNtxA 0.43 (9.3 ± 0.03) 106 ± 1 14.1(7.8 ± 0.06) 224±5
DPDPE (CTRL.) 0.69 (9.1 ± 0.07) 100 ± 2 2.99(8.5 ± 0.04) 100±1.5
MP1104 0.40 (9.4 ± 0.04) 99 ± 1.1 3.73 (8.4 ± 0.06) 189 ± 5.5
DADLE (CTRL.) 0.66(9.2 ± 0.05) 100 ± 1.3 0.349 (9.45 ± 0.10) 100 ± 3.2
MP1202 8.18(8.1 ± 0.06) 99 ± 2.2 18.14(7.7 ± 0.25) 26 ± 3.1
DADLE (CTRL.) 1.45(8.8 ± 0.06) 100 ± 2 8.41(8.1 ± 0.08) 100 ± 2.7
MP1207 11.4 (7.9 ± 0.1) 38 ± 2 64.06(7.2 ± 0.23) 34 ± 3.7
MP1208 2.49 (8.6 ± 0.13) 39 ±1.8 3624.0(5.5 ± 0.27) 62±16.7
DADLE (CTRL.) 0.48 (9.3 ± 0.05) 100 ±1.5 1.41 (8.8 ± 0.07) 100±2.3
MP1305 74.18 (7.1 ± 0.08) 71 ± 2.4 227.5(6.6 ± 0.06) 89 ± 2.8
MP1601 2.76(8.6 ± 0.06) 106 ± 2.3 86.7(7.1 ± 0.06) 203±5.4
DPDPE(CTRL.) 0.69 (9.1 ± 0.07) 100 ± 2 2.99(8.5 ± 0.04) 100±1.5
B. Functional data at mDOR using BRET assays
Compd. G protein activation Arrestin recruitment
EC50,nM (pEC50± SEM) Emax%± SEM EC50,nM (pEC50± SEM) Emax%± SEM
IBNtxA 0.48(9.3 ± 0.10) 108 ± 3.7 26.6(7.5 ± 0.2) 88 ± 6.8
DPDPE(CTRL.) 2.72(8.5 ± 0.09) 100 ± 3 184.3(6.73 ± 0.1) 100 ± 6.4
MP1104 1.4(8.9 ± 0.06) 91 ± 1.7 26.0(7.6 ± 0.11) 40 ± 1.7
DPDPE(CTRL.) 1.3(8.9 ± 0.04) 100 ± 1.2 98.0(7.0 ± 0.04) 100 ± 1.8
MP1202 7.03(8.1 ± 0.15) 103 ± 4.6 524.8(6.3 ± 0.20) 26 ± 2.3
DPDPE(CTRL.) 2.19(8.6 ± 0.10) 100 ± 3.5 109.3(6.7 ± 0.06) 100 ± 3
MP1207 116.4(6.9 ± 0.12) 52 ± 2.3 nd nd
MP1208 26.6(7.6 ± 0.20) 58 ± 3.3 nd nd
MP1305 40.4(7.4 ± 0.20) 105 ± 5.9 nd nd
MP1601 6.27(8.2 ± 0.20) 105 ± 6.7 9.0 (8.0 ± 0.33) 16 ± 1.4
DPDPE(CTRL.) 0.44(9.3 ± 0.13) 100 ± 2.5 21.8 (7.6 ± 0.04) 100 ± 1.3
Appendix 1—table 7
Gprotein & arrestin pathway potency and efficacy of ligands at hMOR, hKOR and mMOR.
Receptors Compounds cAMP inhibition (Gi) assay Arrestin recruitment (Tango) assay Figure
EC50 (pEC50± SEM) Emax%± SEM EC50 (pEC50± SEM) Emax%± SEM
hMOR MP1104 0.074 (10.13 ± 0.05) nM 94±1 0.573(9.24±0.08) nM 90±2.28 Figure 2A–B
DAMGO 0.84(9.07±0.08) nM 100 13.9 (7.86±0.06) nM 100
hKOR MP1104 0.00327 (11.49±0.035) nM 10±1.64 0.03944(10.4±0.06) nM 117 ± 2 Figure 2C–D
U50488h 0.089(10.05±0.04) nM 100 3.6(8.44±0.04) nM 100
hMOR MP1202 0.077 (10.11 ± 0.06) nM 92±1.4 26.8(7.57±0.1) nM 53±2 Figure 2G–H
DAMGO 3.78(8.4±0.06) nM 100 58.8(7.23±0.06) nM 100
hKOR MP1202 0.00139 (11.86±0.05) nM, 97.9±2.5 0.0457(10.34±0.05) nM 101±1 Figure 2I–J
U50,488h 0.006 (10.2±0.056) nM 100 3.6(8.44±0.04) nM 100
hKOR MP1207 0.11 (9.98 ± 0.07) nM 90±1.7 3.97(8.4±0.18) nM 37±2 Figure 3A–B
MP1208 0.14 (9.9 ± 0.07) nM 96±1.9 16.41(7.79±0.14) nM 48±2.4
U50,488h 0.64(9.2±0.06) nM 100 7.55(8.12±0.06) nM 100
hMOR MP1207 0.034 (10.47 ± 0.15) nM 33±1.4 nd nd Figure 3E–F
MP1208 0.008 (8.73 ± 0.12) nM 42±1.3 nd nd
U50,488h 1.86(8.73±0.06) nM 100
hKOR MP1209 0.024 (10.6 ± 0.05) nM 100±1.23 0.37(9.43 ± 0.19) nM 67±3 Figure 4E–F
MP1210 0.025 (10.6 ± 0.05) nM 101.1±1.1 1.16 (8.94 ± 0.17) nM 63±3.3
U50,488h 0.05 (10.29±0.06) nM 100 7.85 (8.11±0.1) nM. 100
hMOR MP1209 0.25(9.61 ± 0.04) nM 98.5±0.96 nd nd Figure 4G–H
MP1210 0.15(9.81 ± 0.05) nM 94.6±0.98 nd nd
DAMGO 0.2 (9.7±0.06) nM 100
hMOR Methoxycarbonyl fentanyl 0.099 (10 ± 0.06) nM 103±1.8 18.94 (7.7±0.1) nM 194±8 Appendix 1—figure 8A–B
DAMGO 2.58 (8.59±0.07) nM 100 404.1(6.39±0.05) nM 100
hMOR MP102 19.7(7.7 ± 0.08) nM 87±2.66 nd nd Appendix 1—figure 8F–G
DAMGO 2.58 (8.59±0.07) nM 100
Morphine 21.8 (7.66 ± 0.08) nM 97±3.06 888 (6.05±0.25) nM 25.32±4.4
hMOR DAMGO 8.1 (8.09 ± 0.06) nM 100 140.7 (6.85±0.08) nM 100
Buprenorphine 0.7 (10.14 ± 0.11) pM 75±2.36 1.79 (8.75±0.13) nM 43±2
DAMGO 4.36 (8.36 ± 0.08) nM 100 22 (7.64±0.063) nM 100
Receptors Compounds G protein activation (BRET) assay Arrestin recruitment (BRET) assay Figure
EC50 (pEC50± SEM) Emax%± SEM EC50 (pEC50± SEM) Emax%± SEM
mMOR MP1207 3.61 (8.44 ± 0.26) nM 42±2.6 nd nd Figure 3I–J
MP1208 2.27 (8.64 ± 0.29) nM 41±3 nd nd
DAMGO 3.27(8.49±0.08) nM 100
DAMGO 9.09 (8.04 ± 0.13) nM 100
mMOR Morphine 9.09 (8.04 ± 0.13) nM 109±3.1
Buprenorphine 1.17 (8.93 ± 0.14) nM 59±2.4
Appendix 1—table 8
Gprotein & arrestin pathway potency and efficacy of ligands at mMOR and rKOR.
Receptors Compound G protein activation (BRET) assay Arrestin recruitment (BRET) assay Figure
EC50 (pEC50± SEM) Emax%± SEM EC50 (pEC50± SEM) Emax%± SEM
mMOR MP1104 0.66 (9.2±0.11) nM 62±2 0.285 (9.55±0.12) nM 53 ± 1.6 Appendix 1—figure 1A
DAMGO 7.3 (8.1±0.07) nM 100 31.5 (7.5±0.06) nM 100
IBNtxA 0.054(10.3 ± 0.02) nM 59±2.2 11.32(4.9±0.3) µM 75±13.9 Appendix 1—figure 1B
DAMGO 12.9(7.8±0.06) nM 100 0.77(6.11±0.16) µM 100
MP1202 0.63 (9.2±0.09) nM 61±1.5 3140 (5.5±0.28) nM 53 ± 1.6 Appendix 1—figure 1C
DAMGO 6.31 (8.2±0.07) nM 100 114 (6.9±0.15) nM 100
MP1207 3.61 (8.44 ± 0.26) nM 42±2.6 nd nd Appendix 1—figure 1D
MP1208 2.27 (8.64 ± 0.29) nM 41±3 nd nd
DAMGO 3.27(8.49±0.08) nM 100
MP1305 0.74(9.1 ± 0.12) nM 72±2 nd nd Appendix 1—figure 1E
DAMGO 3.3(8.4±0.08) nM 100
MP1601 2.2(8.6 ± 0.3) nM 49±3 nd nd
DAMGO 0.9(9.0±0.1) nM 100
rKOR MP1104 0.073 (10.14±0.04) nM 93±1 1.14(8.94±0.07) nM 89 ± 2.6 Appendix 1—figure 2A
U50, 488h 1.05(8.98±0.03) nM 100 110(6.95±0.05) nM 100
IBNtxA 0.064(10.2 ± 0.1) nM 101±2.3 1.23(8.9±0.02) nM 133±7.3 Appendix 1—figure 2B
U50, 488h 27.7(7.6±0.01)nM 100 345.6(6.4±0.03) nM 100
MP1202 0.134(9.87±0.09) nM 104±2 1.44(8.84±0.25) nM 77 ± 5 Appendix 1—figure 2C
U50, 488h 4.79 (8.32±0.07) nM 100 235(6.63±0.18) nM 100
MP1207 1.13 (8.95 ± 0.31) nM 32±2.3 nd nd Appendix 1—figure 2D
MP1208 1.1(8.97 ± 0.29) nM 40±2.4 nd nd
U50, 488h 82.6(7.1±0.09) nM 100
MP1305 5.04 (8.3 ± 0.32) nM 35±3 nd nd Appendix 1—figure 2E
U50, 488h 40.3(7.4±0.07) nM 100
MP1601 8.9(8.1 ± 0.12) nM 75±3 56.5(7.2±0.23) nM 37±3
U50, 488h 26.7(7.6±0.09) nM 100 169.6(6.7±0.07) nM 100
mMOR IBNtxA 0.054(10.3 ± 0.02) nM 59±2.2 11.32(4.9±0.3) µM 75±13.9 Figure 3F–G
DAMGO 12.9(7.8±0.06) nM 100 0.77(6.11±0.16) µM 100
rKOR IBNtxA 0.064(10.2 ± 0.1) nM 101±2.3 1.23(8.9±0.02) nM 133±7.3 Figure 3H–I
U50, 488h 27.7(7.6±0.01) nM 100 345.6(6.4±0.03) nM 100
mMOR Methoxycarbonyl fentanyl 0.21(9.7 ± 0.04) nM 109±0.94 1.96(8.71±0.11) nM 63±1.8 Appendix 1—figure 7C–D
DAMGO 1.73(8.76±0.05) nM 100 110.1(6.96±0.06) nM 100
MP102 404.5 (6.39 ± 0.2) nM 78±7 nd nd Appendix 1—figure 7E
DAMGO 3.27(8.5±2) nM 100
Appendix 1—table 9
cAMP & arrestin potency and efficacy at hMOR, hKOR and Y312W-hKOR of select ligands.
Receptors Compounds cAMP inhibition (Gi) assay Arrestin recruitment (Tango) assay Figure
EC50 (pEC50± SEM) Emax%± SEM EC50 (pEC50± SEM) Emax%± SEM
hMOR IBNtxA 0.07(10.2 ± 0.006) nM 95±2 5.86(8.2±0.002) nM 29±0.02 Appendix 1—figure 3A-B
DAMGO 0.99(9.0±0.007) nM 100 14.16(7.9±0.003) nM 100
hKOR IBNtxA 2.46 (11.6 ± 0.037) pM 101±1.79 0.013(10.9±0.07) nM 109±1.8 Appendix 1—figure 3C-D
U50488h 0.089(10.05±0.042) nM 100 3.63(8.4±0.03) nM 100
Y312W-hKOR MP1202 0.21 (10.69 ± 0.07)pM 101±1.5 3.4(8.5±0.14) nM 55±2.6 Appendix 1—figure 7A,C
U50,488h 2.7(8.56±0.06)nM 100 0.63(7.2±0.06) nM 100
WT-hKOR MP1202 0.0457(10.34±0.05) nM 101±1 Appendix 1—figure 7B,D
U50,488h 3.6(8.44±0.04) nM 100
WT-hMOR MP1202 26.8(7.57±0.1) nM 53±2
DAMGO 58.8(7.23±0.06) nM 100
Y312W-hKOR MP1207 0.16 (9.8 ± 0.08) nM 95±1.8 nd nd Appendix 1—figure 7E,G
MP1208 0.36 (10.44 ± 0.05) pM 97±1 nd nd
U50,488h 0.92(9.04±0.04)nM 100 14(7.85±0.1) nM 100
WT-hKOR MP1207 3.97(8.4±0.18) nM 37±2 nd nd Appendix 1—figure 7F
MP1208 16.41(7.79±0.14) nM 48±2.4 nd nd
U50,488h 7.55(8.12±0.06) nM 100
hKOR MP1305 0.72 (9.14 ± 0.05) nM 102±1.2 25.72(7.6±0.04) nM 75±1 Appendix 1—figure 9A-B
U50, 488h 0.076(10.11±0.04) nM 100 3.6(8.44±0.04) nM 100
hMOR MP1305 0.12(9.9 ± 0.12) nM 69±1.8 16.4(7.8±0.3) nM 21±3 Appendix 1—figure 9C-D
DAMGO 3.88(8.4±0.05) nM 100 168.1(6.77±0.09) nM 100
hKOR MP1601 0.17(9.76 ± 0.05) nM 109±1 3.23(8.49±0.07) nM 107±3 Appendix 1—figure 9E-F
U50, 488h 0.077(10.11±0.04) nM 100 3.6(8.44±0.04) nM 100
hMOR MP1601 0.1(9.99 ± 0.09) nM 69±1 4.02(8.4±0.2) nM 27±2 Appendix 1—figure 9G-H
DAMGO 3.88(8.4±0.05) nM 100 168.1(6.77±0.09) nM 100
Appendix 1—table 10
Bias analysis of ligands at human opioid receptors.
Drug Receptor cAMP Tango ΔlogRA ΔlogRA ΔΔlog RA Bias factor Figure
Log RA LogRA cAMP Tango (cAMP-Tango) (cAMP-Tango)
U50,488h hKOR 10.09±0.035 8.29±0.019 0±0.049 0±0.027 0±0.056 1 Appendix 1—figure 4A
MP1104 hKOR 11.44±0.034 10.45±0.019 1.35±0.048 2.16±0.027 -0.81±0.055 0.15
U50,488h hKOR 10.22±0.051 8.44±0.004 0±0.072 0±0.006 0±0.070 1 Appendix 1—figure 4A
MP1202 hKOR 11.88±0.050 10.30±0.004 1.66±0.072 1.856±0.006 -0.19±0.072 nd
U50,488h hKOR 10.06±0.036 8.36±0.011 0±0.051 0±0.015 0±0.050 1 Appendix 1—figure 4A
IBNtxA hKOR 11.59±0.036 10.89±0.010 1.53±0.050 2.53±0.015 -1.002±0.052 0.10
U50,488h hKOR 9.175±0.004 8.14±0.007 0±0.005 0±0.010 0±0.012 1 Appendix 1—figure 4A
MP1207 hKOR 9.902±0.004 7.97±0.024 0.73±0.006 -0.176±0.025 0.903±0.026 8.00
U50,488h hKOR 9.176±0.004 8.14±0.008 0±0.005 0±0.011 0±0.012 1 Appendix 1—figure 4A
MP1208 hKOR 9.83±0.004 7.447±0.020 0.65±0.005 -0.697±0.022 1.35±0.022 22
U50,488h hKOR 10.42±0.06 8.37±0.22 0±0.084 0±0.31 0±0.32 1 Appendix 1—figure 4A
MP1209 hKOR 10.55±0.06 8.735±0.220 0.13±0.084 0.368±0.31 -0.238±0.32 nd
U50,488h hKOR 10.39±0.06 8.096±0.084 0±0.081 0±0.12 0±0.144 1 Appendix 1—figure 4A
MP1210 hKOR 10.59±0.06 8.576±0.156 0.2±0.081 0.48±0.18 -0.28±0.19 nd
U50,488h hKOR 10.010±0.012 8.507±0.026 0±0.017 0±0.037 0±0.041 1 Appendix 1—figure 4A
MP1305 hKOR 9.22±0.012 7.17±0.025 -0.79±0.017 -1.341±0.036 0.55±0.04 4
U50,488h hKOR 10.03±0.014 8.414±0.009 0±0.020 0±0.012 0±0.023 1 Appendix 1—figure 4A
MP1601 hKOR 9.85±0.014 8.51±0.009 -0.19±0.020 0.097±0.012 -0.282±0.023 0.52
DAMGO hMOR 9.102±0.009 7.925±0.013 0±0.012 0±0.019 0±0.022 1 Appendix 1—figure 4B
MP1104 hMOR 10.12±0.009 9.18±0.013 1.018±0.012 1.26±0.019 -0.24±0.022 0.58
DAMGO hMOR 8.44±0.011 7.18±0.001 0±0.015 0±0.001 0±0.015 1 Appendix 1—figure 4B
MP1202 hMOR 9.99±0.012 7.24±0.002 1.55±0.016 0.058±0.002 1.49±0.016 31
DAMGO hMOR 8.86±0.075 7.82±0.060 0±0.106 0±0.084 0±0.135 1 Appendix 1—figure 4B
IBNtxA hMOR 9.99±0.081 7.572±0.25 1.14±0.11 -0.25±0.25 1.38±0.28 24
DAMGO hMOR 8.62±0.054 6.78±0.010 0±0.076 0±0.014 0±0.077 1 Appendix 1—figure 4B
MP1305 hMOR 9.79±0.053 6.89±0.048 1.17±0.076 0.109±0.049 1.059±0.090 11
DAMGO hMOR 8.40±0.001 6.78±0.009 0±0.002 0±0.012 0±0.013 1 Appendix 1—figure 4B
MP1601 hMOR 9.84±0.002 7.65±0.035 1.44±0.002 0.87±0.036 0.57±0.036 4
U50,488h WT hKOR 10.22±0.051 8.44±0.004 0±0.072 0±0.006 0±0.070 1 Appendix 1—figure 7L
MP1202 WT hKOR 11.88±0.050 10.30±0.004 1.66±0.072 1.856±0.006 -0.19±0.072 nd
U50,488h Y312W-hKOR 8.568±0.005 7.559±0.051 0±0.007 0±0.072 0±0.073 1 Appendix 1—figure 7L
MP1202 Y312W-hKOR 10.63±0.005 8.091±0.051 2.062±0.007 0.532±0.073 1.53±0.073 34
DAMGO hMOR 8.563±0.004 5.037±0.176 0±0.006 0±0.249 0±0.249 1 Appendix 1—figure 8J
Methoxycarbonyl hMOR 10.020±0.004 7.546±0.184 1.457±0.006 2.509±0.255 -1.052±0.255 0.09
fentanyl
Appendix 1—table 11
Bias analysis of ligands at rodent opioid receptors.
Drug Receptor BRET-G BRET-Arr ΔlogRA ΔlogRA ΔΔlog RA Bias factor Figure
Log RA LogRA BRET-G BRET-arr (BRETG-BRETArr) (BRETG-BRETArr)
U50,488h rKOR 9.003±0.044 7.14±0.034 0±0.062 0±0.047 0±0.078 1 Appendix 1—figure 4C
MP1104 rKOR 10.07±0.047 8.84±0.033 1.067±0.064 1.702±0.047 -0.64±0.079 0.23
U50,488h rKOR 8.312±0.077 6.798±0.168 0±0.109 0±0.238 0±0.261 1 Appendix 1—figure 4C
MP1202 rKOR 9.906±0.088 8.791±0.171 1.594±0.117 1.993±0.239 -0.399±0.27 nd
U50,488h rKOR 7.55±0.103 5.988±0.222 0±0.146 0±0.313 0±0.345 1 Appendix 1—figure 4C
IBNtxA rKOR 10.19±0.141 8.846±0.266 2.64±0.175 2.858±0.346 -0.218±0.388 nd
U50,488h rKOR 7.593±0.012 6.783±0.005 0±0.016 0±0.007 0±0.018 1 Appendix 1—figure 4C
MP1601 rKOR 7.917±0.018 7.113±0.052 0.324±0.052 0.33±0.052 -0.006±0.057 nd
DAMGO mMOR 8.56±0.12 7.873±0.06 0±0.176 0±0.09 0±0.197 1 Appendix 1—figure 4C
MP1104 mMOR 8.917±0.12 9.196±0.06 0.357±0.173 1.323±0.09 -0.966±0.195 0.11
DAMGO mMOR 8.713±0.013 7.291±0.044 0±0.019 0±0.063 0±0.066 1 Appendix 1—figure 8J
Methoxycarbonyl mMOR 9.697±0.013 8.464±0.045 0.984±0.019 1.173±0.063 -0.189±0.066 nd
fentanyl

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Analgesia and CLAMS data.

https://cdn.elifesciences.org/articles/56519/elife-56519-data1-v2.zip
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