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In Silico Identification of a β2 Adrenergic Receptor Allosteric Site that Selectively Augments Canonical β 2 AR‐Gs Signaling and Function
Author(s) -
Shah Sushrut D.,
Lind Christoffer,
De Pascali Francesco,
Penn Raymond B.,
MacKerell Alexander D.,
Deshpande Deepak A.
Publication year - 2022
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2022.36.s1.r3986
Subject(s) - allosteric regulation , in silico , chemistry , agonist , g protein coupled receptor , receptor , signal transduction , allosteric modulator , binding site , biophysics , microbiology and biotechnology , biochemistry , biology , gene
Objective & Hypothesis Activation of β 2 ‐adrenergic receptors (β 2 ARs) leads to airway smooth muscle (ASM) relaxation and bronchodilation. While this facilitates β 2 ARs agonists (β‐agonists) as the front‐line treatments for asthma and related obstructive airway diseases, their therapeutic efficacy of β‐agonists is limited by agonist‐induced β 2 AR desensitization and activation of non‐canonical β 2 AR signaling involving β‐arrestin. Accordingly, we undertook the identification of an allosteric site on β 2 AR and potential ligands, and ascertain their effects in‐vitro . We hypothesize that activation of specific allosteric site on β 2 AR could selectively modulate the activity of β‐agonists to overcome these limitations. Methods We employed the site identification by ligand competitive saturation (SILCS) computational method to map the entire 3D structure of an in silico ‐generated β 2 AR intermediate conformation. Further, in‐silico database was screened to identify potential ligands using SILCS‐PHARM approach. Levels of cyclic‐AMP were used to assess activation of β 2 AR‐Gs activation using ELISA. Activation of downstream effector of cAMP, protein kinase A (PKA) was assessed by western blotting and luciferase assay. β‐arrestin recruitment to β 2 AR was assessed using Bioluminescence Resonance Energy Transfer (BRET) assay. ELISA and immunofluorescence were used to assess cell surface expression. Mutagenesis analysis was used to investigate the potential binding site for the allosteric modulators. Lastly, functional effects of the allosteric modulators were determined by assessing relaxation of human ASM cells embedded in 3D‐collagen gels and murine airways. Results Database screening using SILCS identified not only putative allosteric site but also potential drug‐like compounds that can bind to the site. Experimental assays in HEK293 cells and human ASM cells identified positive and negative allosteric modulators (PAM and NAM respectively) of β 2 AR as assessed by generation of cAMP. Co‐stimulation of cells with PAM or NAM modulated β‐agonist‐induced PKA activation. Allosteric modulators had no effect on β‐agonist‐induced recruitment of β‐arrestin to β 2 AR or β‐agonist‐induced loss of cell surface expression in HEK293 cells expressing β 2 AR. Mutagenesis analysis of β 2 AR confirmed the SILCS identified allosteric modulator binding site that comprises of F282, Y219, and R131. Finally, functional studies revealed augmentation of β‐agonist‐induced relaxation of contracted human ASM cells and bronchodilation of contracted murine airways by PAM. Conclusion These findings identify a novel allosteric binding site on the β 2 AR, whose activation selectively augments β‐agonist‐induced Gs signaling and increases relaxation of ASM cells, the principal therapeutic effect of β‐agonists in obstructive lung disease such as asthma and COPD.