Following insights from recent crystal structures of the muscarinic acetylcholine receptor, binding modes of Positive Allosteric Modulators (PAMs) were predicted under the assumption that PAMs should bind to the extracellular surface of the active state. A series of well-characterized PAMs for adenosine (A1R, A2AR, A3R) and muscarinic acetylcholine (M1R, M5R) receptors were modeled using both rigid and flexible receptor CHARMM-based molecular docking. Studies of adenosine receptors investigated the molecular basis of the probe-dependence of PAM activity by modeling in complex with specific agonist radioligands. Consensus binding modes map common pharmacophore features of several chemical series to specific binding interactions. These models provide a rationalization of how PAM binding slows agonist radioligand dissociation kinetics. M1R PAMs were predicted to bind in the analogous M2R PAM LY2119620 binding site. The M5R NAM (ML-375) was predicted to bind in the PAM (ML-380) binding site with a unique induced-fit receptor conformation.
Catecholamine-stimulated β 2 -adrenergic receptor (β 2 AR) signaling via the canonical G s –adenylyl cyclase–cAMP–PKA pathway regulates numerous physiological functions, including the therapeutic effects of exogenous β-agonists in the treatment of airway disease. β 2 AR signaling is tightly regulated by GRKs and β-arrestins, which together promote β 2 AR desensitization and internalization as well as downstream signaling, often antithetical to the canonical pathway. Thus, the ability to bias β 2 AR signaling toward the G s pathway while avoiding β-arrestin-mediated effects may provide a strategy to improve the functional consequences of β 2 AR activation. Since attempts to develop G s -biased agonists and allosteric modulators for the β 2 AR have been largely unsuccessful, here we screened small molecule libraries for allosteric modulators that selectively inhibit β-arrestin recruitment to the receptor. This screen identified several compounds that met this profile, and, of these, a difluorophenyl quinazoline (DFPQ) derivative was found to be a selective negative allosteric modulator of β-arrestin recruitment to the β 2 AR while having no effect on β 2 AR coupling to G s . DFPQ effectively inhibits agonist-promoted phosphorylation and internalization of the β 2 AR and protects against the functional desensitization of β-agonist mediated regulation in cell and tissue models. The effects of DFPQ were also specific to the β 2 AR with minimal effects on the β 1 AR. Modeling, mutagenesis, and medicinal chemistry studies support DFPQ derivatives binding to an intracellular membrane-facing region of the β 2 AR, including residues within transmembrane domains 3 and 4 and intracellular loop 2. DFPQ thus represents a class of biased allosteric modulators that targets an allosteric site of the β 2 AR.
The β2‐adrenergic receptor (β2AR) is a Gs‐coupled receptor that promotes an agonist‐dependent cyclic AMP (cAMP)‐mediated reversal of bronchoconstriction that is therapeutic for airway diseases like asthma. Long‐term use of β‐agonists is associated with arrestin mediated desensitization of response to treatment. Gs‐biased signaling through the β2AR would have clinical utility in promoting airway relaxation while avoiding desensitization of response to β‐agonists. Using bioluminescence resonance energy transfer (BRET) for arrestin recruitment, cAMP ELISAs, radioligand assays, mutagenesis, X‐ray crystallography, and ex vivo airway tissue models, we characterized compounds that inhibit arrestin recruitment to the β2AR without inhibiting cAMP production in the presence of β‐agonists. Our data reveal a druggable allosteric site in the extracellular vestibule of the β2AR that can be targeted to modulate arrestin recruitment to the receptor without affecting Gs coupling. Here we characterize a small molecule that targets this site to promote β2AR specific, negative allosteric modulation of arrestin recruitment that is protective against agonist‐induced desensitization in cells and airway tissue.Support or Funding InformationResearch funded by NIH training grant GM100836, P01 HL114471, and F31 HL139104.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.