• In Silico Identification of a β2 Adrenergic Receptor Allosteric Site that Selectively Augments Canonical β<inf>2</inf> AR-Gs Signaling and Function

      Shah, Sushrut D.; Lind, Christoffer; De Pascali, Francesco; Penn, Raymond B.; MacKerell, Alexander D.; Deshpande, Deepak A. (Wiley-Blackwell, 2022-05-01)
      OBJECTIVE & HYPOTHESIS: Activation of β2 -adrenergic receptors (β2 ARs) leads to airway smooth muscle (ASM) relaxation and bronchodilation. While this facilitates β2 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 β2 AR desensitization and activation of non-canonical β2 AR signaling involving β-arrestin. Accordingly, we undertook the identification of an allosteric site on β2 AR and potential ligands, and ascertain their effects in-vitro. We hypothesize that activation of specific allosteric site on β2 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 β2 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 β2 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 β2 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 β2 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 β2 AR or β-agonist-induced loss of cell surface expression in HEK293 cells expressing β2 AR. Mutagenesis analysis of β2 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 β2 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. © FASEB.