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 LeRouzic
  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. Memorial Sloan Kettering Cancer Center, United States
  2. University of North Carolina, United States
  3. The Bridge Institute, University of Southern California, United States
  4. Columbia University, United States
  5. St. Louis College of Pharmacy and Washington University School of Medicine, United States
  6. Washington University in St. Louis, United States
  7. Washington University, United States
  8. University of Florida, United States
  9. University of Southern California, United States

Abstract

Controlling receptor functional selectivity profiles for opioid receptors is a promising approach for discovering safer analgesics; however, the structural determinants conferring functional selectivity are not well understood. Here we used crystal structures of opioid receptors, including the recently solved active state kappa opioid complex with MP1104, to rationally design novel mixed mu (MOR) and kappa (KOR) opioid receptor agonists with reduced arrestin signaling. Analysis of structure-activity relationships for new MP1104 analogs points to a region between transmembrane 5 (TM5) and extracellular loop (ECL2) as key for modulation of arrestin recruitment to both MOR and KOR. The lead compounds, MP1207 and MP1208, displayed MOR/KOR Gi-partial agonism with diminished arrestin signaling, showed efficient analgesia with attenuated liabilities, including respiratory depression and conditioned place preference and aversion in mice. The findings validate a novel structure-inspired paradigm for achieving beneficial in vivo profiles for analgesia through different mechanisms that include bias, partial agonism, and dual MOR/KOR agonism.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Rajendra Uprety

    Neurology, Memorial Sloan Kettering Cancer Center, New York, United States
    Competing interests
    Rajendra Uprety, RU have filed a provisional patent on MP1207 and related molecules..
  2. Tao Che

    Pharmacology, University of North Carolina, Chapel Hill, United States
    Competing interests
    No competing interests declared.
  3. Saheem A Zaidi

    Department of Biological Sciences, Molecular & Computational Biology, The Bridge Institute, University of Southern California, Los Angeles, United States
    Competing interests
    Saheem A Zaidi, SZ has filed a provisional patent on MP1207 and related molecules..
  4. Steven G Grinnell

    Molecular Therapeutics, Columbia University, New York, United States
    Competing interests
    No competing interests declared.
  5. Balázs R Varga

    Center for Clinical Pharmacology, St. Louis College of Pharmacy and Washington University School of Medicine, St Louis, United States
    Competing interests
    No competing interests declared.
  6. Abdelfattah Faouzi

    Clinical Pharmacology, Washington University in St. Louis, St Louis, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9059-4791
  7. Samuel T Slocum

    Pharmacology, University of North Carolina, Chapel Hill, United States
    Competing interests
    No competing interests declared.
  8. Abdullah Allaoa

    Neurology, Memorial Sloan Kettering Cancer Center, New York, United States
    Competing interests
    No competing interests declared.
  9. András Varadi

    Neurology, Memorial Sloan Kettering Cancer Center, New York, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5591-377X
  10. Melissa Nelson

    Molecular Therapeutics, Columbia University, New York, United States
    Competing interests
    No competing interests declared.
  11. Sarah M Bernhard

    Center for Clinical Pharmacology, Washington University, St Louis, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8549-0413
  12. Elizaveta Kulko

    Molecular Therapeutics, Columbia University, New York, United States
    Competing interests
    No competing interests declared.
  13. Valerie LeRouzic

    Neurology, Memorial Sloan Kettering Cancer Center, New York, United States
    Competing interests
    No competing interests declared.
  14. Shainnel O Eans

    Pharmacodyanamics, University of Florida, Gainesville, United States
    Competing interests
    No competing interests declared.
  15. Chloe A Simons

    Pharmacodyanamics, University of Florida, Gainesville, United States
    Competing interests
    No competing interests declared.
  16. Amanda Hunkele

    Neurology, Memorial Sloan Kettering Cancer Center, New York, United States
    Competing interests
    No competing interests declared.
  17. Joan Subrath

    Neurology, Memorial Sloan Kettering Cancer Center, New York, United States
    Competing interests
    No competing interests declared.
  18. Ying Xian Pan

    Neurology, Memorial Sloan Kettering Cancer Center, New York, United States
    Competing interests
    Ying Xian Pan, YXPis a co-founder of Sparian biosciences..
  19. Jonathan A Javitch

    Department of Psychiatry, Columbia University, New York, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7395-2967
  20. Jay P McLaughlin

    Pharmacodyanamics, University of Florida, Gainesville, United States
    Competing interests
    Jay P McLaughlin, JM has filed a provisional patent on MP1207 and related molecules..
  21. Bryan L Roth

    Department of Pharmacology, University of North Carolina, Chapel Hill, United States
    For correspondence
    bryan_roth@med.unc.edu
    Competing interests
    Bryan L Roth, BLR has filed a provisional patent on MP1207 and related molecules..
  22. Gavril W Pasternak

    Neurology, Memorial Sloan Kettering Cancer Center, New York, United States
    Competing interests
    Gavril W Pasternak, GWP is a co-founder of Sparian biosciences. GWP have filed a provisional patent on MP1207 and related molecules..
  23. Vsevolod Katritch

    Department of Biological Sciences, University of Southern California, Los Angeles, United States
    For correspondence
    katritch@usc.edu
    Competing interests
    Vsevolod Katritch, VK has filed a provisional patent on MP1207 and related molecules..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3883-4505
  24. Susruta Majumdar

    Center for Clinical Pharmacology, Washington University, st louis, United States
    For correspondence
    susrutam@email.wustl.edu
    Competing interests
    Susruta Majumdar, SM, is a co-founder of Sparian biosciences. SM have filed a provisional patent on MP1207 and related molecules..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2931-3823

Funding

National Institute on Drug Abuse (DA045884)

  • Susruta Majumdar

National Institute on Drug Abuse (DA045657)

  • Jonathan A Javitch

National Institute on Drug Abuse (DA046487)

  • Susruta Majumdar

National Institute on Alcohol Abuse and Alcoholism (AA026949)

  • Susruta Majumdar

National Institute on Drug Abuse (DA038858)

  • Vsevolod Katritch

National Institute on Drug Abuse (DA035764)

  • Bryan L Roth
  • Vsevolod Katritch

National Institute on Drug Abuse (DA007242,DA006241)

  • Ying Xian Pan
  • Gavril W Pasternak

National Institute on Drug Abuse (DA042888,DA046714)

  • Ying Xian Pan

National Institute of Mental Health (MH018870)

  • Steven G Grinnell

National Institute of Mental Health (MH112205)

  • Jonathan A Javitch

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Olga Boudker, Weill Cornell Medicine, United States

Ethics

Animal experimentation: All animal studies were preapproved by the Institutional Animal Care and Use Committees of University of Florida in accordance with the 2002 National Institutes of Health Guide for the Care and Use of Laboratory Animals. protocols 201808990 and 202011105.

Version history

  1. Received: March 1, 2020
  2. Accepted: February 7, 2021
  3. Accepted Manuscript published: February 8, 2021 (version 1)
  4. Version of Record published: February 26, 2021 (version 2)

Copyright

© 2021, Uprety et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 5,478
    views
  • 731
    downloads
  • 42
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  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 LeRouzic
  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

Share this article

https://doi.org/10.7554/eLife.56519

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Marian Brenner, Christoph Zink ... Antje Gohla
    Research Article

    Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal 5’-phosphate phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5’-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small-molecule screening, protein crystallography, and biolayer interferometry, we discover, visualize, and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Thomas RM Germe, Natassja G Bush ... Anthony Maxwell
    Research Article

    DNA gyrase, a ubiquitous bacterial enzyme, is a type IIA topoisomerase formed by heterotetramerisation of 2 GyrA subunits and 2 GyrB subunits, to form the active complex. DNA gyrase can loop DNA around the C-terminal domains (CTDs) of GyrA and pass one DNA duplex through a transient double-strand break (DSB) established in another duplex. This results in the conversion from a positive (+1) to a negative (–1) supercoil, thereby introducing negative supercoiling into the bacterial genome by steps of 2, an activity essential for DNA replication and transcription. The strong protein interface in the GyrA dimer must be broken to allow passage of the transported DNA segment and it is generally assumed that the interface is usually stable and only opens when DNA is transported, to prevent the introduction of deleterious DSBs in the genome. In this paper, we show that DNA gyrase can exchange its DNA-cleaving interfaces between two active heterotetramers. This so-called interface ‘swapping’ (IS) can occur within a few minutes in solution. We also show that bending of DNA by gyrase is essential for cleavage but not for DNA binding per se and favors IS. Interface swapping is also favored by DNA wrapping and an excess of GyrB. We suggest that proximity, promoted by GyrB oligomerization and binding and wrapping along a length of DNA, between two heterotetramers favors rapid interface swapping. This swapping does not require ATP, occurs in the presence of fluoroquinolones, and raises the possibility of non-homologous recombination solely through gyrase activity. The ability of gyrase to undergo interface swapping explains how gyrase heterodimers, containing a single active-site tyrosine, can carry out double-strand passage reactions and therefore suggests an alternative explanation to the recently proposed ‘swivelling’ mechanism for DNA gyrase (Gubaev et al., 2016).