Received: 14 December 2021
Accepted: 30 August 2022
Published in print: 30 September 2022
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-α2A
generated in this study have been deposited with accession codes EMD-32331 and EMD-32342, respectively. The coordinates of ‘9087-α2A
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 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.