In silico identification of a β <sub>2</sub> -adrenoceptor allosteric site that selectively augments canonical β <sub>2</sub> AR-Gs signaling and function
Sushrut D. Shah, Christoffer Lind, Francesco De Pascali, Raymond B. Penn, Alexander D. MacKerell, Deepak A. Deshpande
Abstract
Activation of β 2 -adrenoceptors (β 2 ARs) causes airway smooth muscle (ASM) relaxation and bronchodilation, and β 2 AR agonists (β-agonists) are front-line treatments for asthma and other obstructive lung diseases. However, the therapeutic efficacy of β-agonists is limited by agonist-induced β 2 AR desensitization and noncanonical β 2 AR signaling involving β-arrestin that is shown to promote asthma pathophysiology. Accordingly, we undertook the identification of an allosteric site on β 2 AR that could modulate the activity of β-agonists to overcome these limitations. We employed the site identification by ligand competitive saturation (SILCS) computational method to comprehensively map the entire 3D structure of in silico-generated β 2 AR intermediate conformations and identified a putative allosteric binding site. Subsequent database screening using SILCS identified drug-like molecules with the potential to bind to the site. Experimental assays in HEK293 cells (expressing recombinant wild-type human β 2 AR) and human ASM cells (expressing endogenous β 2 AR) identified positive and negative allosteric modulators (PAMs and NAMs) of β 2 AR as assessed by regulation of β-agonist-stimulation of cyclic AMP generation. PAMs/NAMs 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 site based on mutants of amino acids R131, Y219, and F282. Finally, functional studies revealed augmentation of β-agonist-induced relaxation of contracted human ASM cells and bronchodilation of contracted airways. These findings identify a 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.