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An allosteric modulator binds to a conformational hub in the β2 adrenergic receptor

Abstract

Most drugs acting on G-protein-coupled receptors target the orthosteric binding pocket where the native hormone or neurotransmitter binds. There is much interest in finding allosteric ligands for these targets because they modulate physiologic signaling and promise to be more selective than orthosteric ligands. Here we describe a newly developed allosteric modulator of the β2-adrenergic receptor (β2AR), AS408, that binds to the membrane-facing surface of transmembrane segments 3 and 5, as revealed by X-ray crystallography. AS408 disrupts a water-mediated polar network involving E1223.41 and the backbone carbonyls of V2065.45 and S2075.46. The AS408 binding site is adjacent to a previously identified molecular switch for β2AR activation formed by I3.40, P5.50 and F6.44. The structure reveals how AS408 stabilizes the inactive conformation of this switch, thereby acting as a negative allosteric modulator for agonists and positive allosteric modulator for inverse agonists.

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Fig. 1: Hit-to-lead optimization, pharmacological characterization and structure of allosteric modulator AS408 bound to β2AR.
Fig. 2: Structural basis of the negative allosteric activity of AS408 on agonist binding to β2AR.
Fig. 3: Structure–activity relationships of AS408 analogs.
Fig. 4: AS408 helps to stabilize the inactive conformation.
Fig. 5: AS408 uses E1223.41 of β2AR, a residue that participates in an allosteric network.

Data availability

Atomic coordinates and structure factors have been deposited in the PDB under accession code 6OBA.

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Acknowledgements

We acknowledge support from the US NIH grant nos. GM106990 (B.K.S., P.G. and R.K.S.), and the DFG Grants Gm 13/10 and GRK 1910 (P.G.), and Beijing Advanced Innovation Center for Structural Biology, Tsinghua University (X.L.), as well as the compute resources provided by the RRZE. B.K.K. is a Chan Zuckerberg Biohub Investigator. We thank D. Steffen and M. Gilardi for help with confocal microscopy. We also thank A. Christopoulos for his critical insight with the analysis of cooperativity.

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X.L. expressed and purified the receptor, crystallized the receptor–ligand complex and solved the crystal structure. K.H. performed the automatic data collection and processing. A.S., D.D. and M.S. synthesized and analytically characterized the chemical compounds. H.H., M.J.C., R.A.M., J.M., X.L., D.D. and X.X. performed ligand binding and signaling experiments. M.K. performed docking studies. J.K. performed MD simulations. The manuscript was written by B.K.K., X.L. and P.G. with editing and suggestions from R.K.S. and input from J.K. and H.H., P.G. supervised chemical synthesis of compounds, B.K.K., R.K.S. and P.G. supervised binding and signaling experiments. B.K.S. supervised docking. X.L. supervised structure determination. The project was conceived by B.K.K., P.G., R.K.S. and B.K.S.

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Correspondence to Roger K. Sunahara or Brian K. Kobilka or Peter Gmeiner.

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B.K.K. is a cofounder of and consultant for ConfometRx, Inc.

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Liu, X., Kaindl, J., Korczynska, M. et al. An allosteric modulator binds to a conformational hub in the β2 adrenergic receptor. Nat Chem Biol 16, 749–755 (2020). https://doi.org/10.1038/s41589-020-0549-2

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