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Research Article
DRUG DESIGN

Structure-based discovery of nonopioid analgesics acting through the α2A-adrenergic receptor

Science
30 Sep 2022
Vol 377, Issue 6614

A path to pain relief

The serious problems associated with opioid addiction have motivated the search for non-opioid pain-relief drugs. The α2A-adrenergic receptor (α2AAR) is a validated pain receptor and is targeted by dexmetadomine, a drug used in hospitals but unsuitable for broader use because it causes sedation and is not orally bioavailable. Fink et al. screened more than 300 million virtual molecules and identified agonists that bind α2AAR with reasonable affinity and are structurally unrelated to known agonists. Experimental structures of two of the compounds bound to α2AAR allowed optimization to improve potency. The optimized compounds were effective in a neuropathic pain model without causing sedation, making them promising leads for further development. —VV

Structured Abstract

INTRODUCTION

Epidemics of pain and opioid abuse underscore the need for new nonopioid therapeutics to treat pain. Many nonopioid receptors are involved in pain processing (nociception), but only a few have been validated therapeutically. Of particular interest is the α2A-adrenergic receptor (α2AAR), a G protein–coupled receptor (GPCR) whose activation in the central nervous system has pain-relieving effects. The known therapeutics targeting the α2AAR, like clonidine and dexmedetomidine, are known to be analgesic. They are also strongly sedating, which is important for the primary indication of dexmedetomidine. This, however, has restricted the use of these drugs to hospital settings and kept them from being used in broader patient populations.

RATIONALE

Because GPCRs, like α2AAR, can signal into the cell through multiple downstream effectors, we reasoned that agonists that were chemically dissimilar to the highly related dexmedetomidine, clonidine, and brimonidine might have different signaling and might be able to separate sedation from analgesia. We sought these chemotypes among a virtual library of more than 301 million diverse, readily accessible molecules in the ZINC15 library (http://zinc15.docking.org), few of which have been previously synthesized. We computationally docked each virtual molecule into the highly similar α2BAR binding site, prioritizing those that physically fit and that were chemically unrelated to the known drugs.

RESULTS

From the high-ranking docked compounds, we selected 48 for de novo synthesis and testing. Against the α2BAR used in the virtual docking screens, 30 molecules bound for a 63% hit rate, among the highest to date for docking campaigns. Seventeen further bound to α2AAR with binding constants in the low-nanomolar to low-micromolar concentration range. Several acted as full or partial agonists of α2AAR, activating the receptor. Among these was ‘9087 [mean effective concentration (EC50) of 52 nM]. Notably, the docking-derived agonists preferentially activated Gi, Go, and Gz G protein subtypes, which contrasts with known drugs, like dexmedetomidine and brimonidine, that activate a much broader set of G proteins and recruit β-arrestins. Thus, the new agonists activate a more selective set of cellular pathways than the known α2AAR drugs, something we had hoped for when prioritizing new chemotypes.
The structures of two of these agonists were experimentally determined in complex with the activated state of α2AAR. These experimental ligand geometries closely corresponded to computational predictions. They also templated the optimization of the initial docking hits and led to more potent analogs, including PS75 (EC50 4.8 nM). The physical features of these agonists allowed them to reach high brain concentrations after systemic dosing. In animal behavioral assays, six of these previously uncharacterized agonists relieved pain behaviors in neuropathic, inflammatory, and acute thermal nociception assays. Gene mutation and reversal of receptor binding with an α2AR antagonist confirmed that analgesia occurred primarily through α2AAR. Crucially, when compared with dexmedetomidine, none of the new compounds caused sedation, even at substantially higher doses than required for pain relief.

CONCLUSION

The separation of analgesic properties from sedation of the new agonists is important for further α2AAR drug development. The newly identified agonists, especially ‘9087 and PS75, overcome the sedation liability of the previously known drugs, and several are orally bioavailable. This makes them lead molecules for the development of nonopioid pain therapeutics.
Newly identified α2AAR agonists are analgesic without sedation.
More than 301 million molecules were docked against the activated α2BAR. Experimental testing identified α2AAR agonists with diverse chemical scaffolds. The experimental structure of the ‘9087-α2AAR complex superposed closely to the computational prediction. The newly discovered agonists had efficacy in an in vivo neuropathic pain model (top right) without sedation, unlike dexmedetomidine (DEX) (bottom right). Gi1-activation EC50 (nanomolar) and Emax (percentage) values are shown. Single-letter abbreviations for the amino acid residues are as follows: D, Asp; F, Phe; I, Ile; S, Ser; V, Val; and Y, Tyr. ns, not significant; *P < 0.05; ****P < 0.0001; SNI, spared nerve injury.

Abstract

Because nonopioid analgesics are much sought after, we computationally docked more than 301 million virtual molecules against a validated pain target, the α2A-adrenergic receptor (α2AAR), seeking new α2AAR agonists chemotypes that lack the sedation conferred by known α2AAR drugs, such as dexmedetomidine. We identified 17 ligands with potencies as low as 12 nanomolar, many with partial agonism and preferential Gi and Go signaling. Experimental structures of α2AAR complexed with two of these agonists confirmed the docking predictions and templated further optimization. Several compounds, including the initial docking hit ‘9087 [mean effective concentration (EC50) of 52 nanomolar] and two analogs, ‘7075 and PS75 (EC50 4.1 and 4.8 nanomolar), exerted on-target analgesic activity in multiple in vivo pain models without sedation. These newly discovered agonists are interesting as therapeutic leads that lack the liabilities of opioids and the sedation of dexmedetomidine.

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Supplementary Materials

This PDF file includes:

Figs. S1 to S20
Tables S1 to S4, S6 to S11

Other Supplementary Material for this manuscript includes the following:

Table S5
MDAR Reproducibility Checklist

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Published In

Science
Volume 377 | Issue 6614
30 September 2022

Submission history

Received: 14 December 2021
Accepted: 30 August 2022
Published in print: 30 September 2022

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Acknowledgments

We thank B. Kobilka for sharing the structure of PDB 6K41 before publication. We thank T. Tummino and J. Lyu for reading this manuscript. We also thank WuXi AppTec for providing information on compounds sourced from the WuXi GalaXi virtual library. We gratefully acknowledge OpenEye Software for Omega and related tools and Schrödinger, Inc. for the Maestro package.
Funding: This work is supported by DARPA grant HR0011-19-2-0020 (B.K.S., A.I.B., J.J.I., and M.P.J.), DFG grant GRK 1910 (P.G.), US NIH grant R35GM122481 (B.K.S.), US NIH grant R01GM133836 (J.J.I.), US R35 NS097306 (A.I.B.), Open Philanthropy (A.I.B.), the Facial Pain Research Foundation (A.I.B.), and CIHR Foundation grant FN-148431 (M.B.). C.M.W. is supported by an NSF Graduate Research Fellowship. Y.D. is supported by a grant from the Science, Technology and Innovation Commission of Shenzhen Municipality (project JCYJ20200109150019113) and in part by the Kobilka Institute of Innovative Drug Discovery and Shenzhen Key Lab (ZDSYS20190902093417963). X.-P.H. is supported by NIMH PDSP (HHSN-271-2018-00023-C), directed by B. Roth. M.B. holds the Canada Research Chair in Signal Transduction and Molecular Pharmacology.
Author contributions: E.A.F. conducted the docking screens with input from B.K.S. Ligand optimization was performed by E.A.F. and P.S. with input from M.F.S., H.H., B.K.S., and P.G. H.H. performed all binding and functional assays and analyses for adrenergic and dopamine receptors with input from D.W. J.X. determined the ‘9087-α2AAR-GoA and ‘4622-α2AAR-GoA structures and made α2AAR mutations assisted by G.C. and Z.L., with supervision from Y.D. J.M.B. performed and analyzed the in vivo pharmacology experiments assisted by V.C., supervised and coanalyzed by A.I.B. C.A. tested select compounds in the panel of G protein and β-arrestin subtypes and receptor internalization with supervision from M.B. S.G. modeled compound ‘7075 and PS75. X.-P.H. tested compounds in GPCRome and hERG assays. M.F.S. performed contact area calculation. P.S. synthesized bespoke compounds and performed pKa and analytical testing with supervision of P.G. D.W. performed ELISA experiments. C.M.W. tested compounds for μOR activity. C.K. performed permeability calculations with supervision of M.P.J. Y.S.M. supervised compound synthesis of Enamine compounds purchased from the ZINC15 database and 12 billion catalog, assisted by N.A.T. J.J.I. built the ZINC15 ultralarge libraries. B.K.S., P.G., A.I.B., and Y.D. supervised the project. E.A.F. wrote the paper with contributions from J.M.B. and J.X., input from all other authors, and primary editing from B.K.S. and P.G. B.K.S., P.G., and A.I.B. conceived the project.
Competing interests: B.K.S. and P.G. are founders of Epiodyne. B.K.S. is a founder of BlueDolphin and of Deep Apple Therapeutics and consults in docking and in the GPCR space. J.J.I. is a cofounder of BlueDolphin and Deep Apple Therapeutics. M.P.J. is a consultant to Deep Apple Therapeutics and to Schrödinger, Inc. M.B. is the chair of the scientific advisory board of Domain Therapeutics, to which some of the BRET-based biosensors used are licensed for commercial use. Y.S.M. is a CEO of Chemspace LLC and a scientific advisor at Enamine, Ltd. B.K.S., E.A.F., P.G., H.H., P.S., A.I.B., J.M.B., Y.D., and J.X. are authors of patents on the discovery of new pain modulators acting through the α2AAR. The authors declare no other competing interests.
Data and materials availability: All data are available in the main text, the supplementary materials, the listed Protein Data Bank (PDB) files, the Electron Microscopy Data Bank (EMDB) files, or at https://github.com/efink14/ADRA2AR_docking_results. The 3D cryo-EM density maps of 9087-α2AAR-GoA and ‘4622-α2AAR-GoA generated in this study have been deposited with accession codes EMD-32331 and EMD-32342, respectively. The coordinates of ‘9087-α2AAR-GoA and ‘4622-α2AAR-GoA have been deposited with PDB accession codes 7W6P and 7W7E, respectively. The identities of compounds docked in this study are freely available from the ZINC15 and ZINC20 databases (https://zinc15.docking.org/ and https://zinc20.docking.org/), and active compounds may be purchased from Enamine and WuXi AppTec or are available from the authors. The docking results, including ZINC number, SMILES, and docking score, are located at https://github.com/efink14/ADRA2AR_docking_results. DOCK3.7 is freely available for noncommercial research (https://dock.compbio.ucsf.edu/DOCK3.7/). A web-based version is freely available to all (https://blaster.docking.org/). The biosensors used for generating the data in tables S3 and S4 and figures S6 to S8 are protected by a patent but are available from M.B. for noncommercial research without restrictions under a regular academic Material Transfer Agreement with the Université de Montréal.
License information: Copyright © 2022 the authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original US government works. https://www.science.org/about/science-licenses-journal-article-reuse

Authors

Affiliations

Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, USA.
Graduate Program in Biophysics, University of California, San Francisco, San Francisco, CA, USA.
Roles: Conceptualization, Data curation, Formal analysis, Investigation, Software, Visualization, Writing - original draft, and Writing - review & editing.
Kobilka Institute of Innovative Drug Discovery, School of Life and Health Sciences, Chinese University of Hong Kong, Shenzhen, Guangdong 518172, China.
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA.
Roles: Investigation, Methodology, Validation, Visualization, Writing - original draft, and Writing - review & editing.
Department of Chemistry and Pharmacy, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91058 Erlangen, Germany.
Roles: Formal analysis, Investigation, Resources, Validation, Visualization, and Writing - review & editing.
Department of Anatomy, University of California, San Francisco, San Francisco, CA, USA.
Roles: Formal analysis, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, and Writing - original draft.
Department of Chemistry and Pharmacy, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91058 Erlangen, Germany.
Roles: Formal analysis and Investigation.
Charlotte Avet
Department of Biochemistry and Molecular Medicine, Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, QC, Canada.
Roles: Formal analysis, Investigation, Visualization, and Writing - review & editing.
Department of Anatomy, University of California, San Francisco, San Francisco, CA, USA.
Roles: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing - original draft, and Writing - review & editing.
Department of Chemistry and Pharmacy, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91058 Erlangen, Germany.
Roles: Formal analysis, Investigation, and Writing - review & editing.
Department of Chemistry and Pharmacy, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91058 Erlangen, Germany.
Roles: Formal analysis, Investigation, Methodology, Writing - original draft, and Writing - review & editing.
Chase M. Webb
Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, USA.
Graduate Program in Pharmaceutical Sciences and Pharmacogenomics, University of California, San Francisco, San Francisco, CA, USA.
Roles: Formal analysis, Investigation, Methodology, Validation, Visualization, and Writing - review & editing.
Nataliya A. Tolmachova
Enamine Ltd., 02094 Kyiv, Ukraine.
Institute of Bioorganic Chemistry and Petrochemistry, National Ukrainian Academy of Science, 02660 Kyiv, Ukraine.
Roles: Project administration and Resources.
National Taras Shevchenko University of Kyiv, 01601 Kyiv, Ukraine.
Chemspace, Riga LV-1082, Latvia.
Roles: Formal analysis, Resources, and Writing - original draft.
National Institute of Mental Health Psychoactive Drug Screening Program (NIMH PDSP), School of Medicine, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA.
Roles: Formal analysis and Investigation.
Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, USA.
Roles: Formal analysis, Investigation, Software, and Writing - original draft.
Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, USA.
Roles: Formal analysis and Investigation.
Geng Chen
Kobilka Institute of Innovative Drug Discovery, School of Life and Health Sciences, Chinese University of Hong Kong, Shenzhen, Guangdong 518172, China.
Roles: Investigation and Resources.
Kobilka Institute of Innovative Drug Discovery, School of Life and Health Sciences, Chinese University of Hong Kong, Shenzhen, Guangdong 518172, China.
Roles: Data curation, Investigation, Methodology, Project administration, and Validation.
Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, USA.
Roles: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Visualization, and Writing - original draft.
Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, USA.
Roles: Funding acquisition, Resources, and Software.
Department of Biochemistry and Molecular Medicine, Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, QC, Canada.
Roles: Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, and Writing - review & editing.
Kobilka Institute of Innovative Drug Discovery, School of Life and Health Sciences, Chinese University of Hong Kong, Shenzhen, Guangdong 518172, China.
Roles: Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, and Writing - review & editing.
Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, USA.
Roles: Conceptualization, Data curation, Funding acquisition, Project administration, Supervision, and Writing - review & editing.
Department of Anatomy, University of California, San Francisco, San Francisco, CA, USA.
Roles: Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, and Writing - review & editing.
Department of Chemistry and Pharmacy, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91058 Erlangen, Germany.
Roles: Conceptualization, Funding acquisition, Supervision, Visualization, and Writing - review & editing.

Funding Information

Science, Technology and Innovation Commission of Shenzhen Municipality: JCYJ20200109150019113
Science, Technology, and Innovation Commission of Shenzhen Municipality: JCYJ20200109150019113
Shenzhen Key Lab: ZDSYS20190902093417963
Shenzhen Key Lab Project: ZDSYS20190902093417963

Notes

These authors contributed equally to this work.
*
Corresponding author. Email: [email protected] (P.G.); [email protected] (A.I.B.); [email protected] (B.K.S.); [email protected] (Y.D.); [email protected] (M.B.)

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