Activation of β2-adrenoceptors (β2ARs) causes airway smooth muscle (ASM) relaxation and bronchodilation, and β2AR 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 β2AR desensitization and noncanonical β2AR signaling involving β-arrestin that is shown to promote asthma pathophysiology. Accordingly, we undertook the identification of an allosteric site on β2AR 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 β2AR 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 β2AR) and human ASM cells (expressing endogenous β2AR) identified positive and negative allosteric modulators (PAMs and NAMs) of β2AR 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 β2AR- or β-agonist-induced loss of cell surface expression in HEK293 cells expressing β2AR. Mutagenesis analysis of β2AR 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 β2AR, whose activation selectively augments β-agonist-induced Gs signaling, and increases relaxation of ASM cells, the principal therapeutic effect of β-agonists.
Keywords: SILCS; adrenergic receptor; allosteric modulator; asthma; biased signaling.