In silico identification of a β ₂ -adrenoceptor allosteric site that selectively augments canonical β ₂ AR-Gs signaling and function

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.

Biased Signaling in Mutated Variants of β2-Adrenergic Receptor: Insights from Molecular Dynamics Simulations

“…In particular, simulation results show the role of critical residues and structural motifs responsible for transmitting allosteric signals from the agonist to the transducer-binding regions; TM3 and TM7 residues act as allosteric communication hubs in GRK/β-arrestin selective signaling, whereas TM5 residues favor G protein signaling. The residues and allosteric functional regions in β 2 AR, identified in the present study, can be targeted to design novel biased drugs, as information about functional hotspots in β-adrenergic receptors has been demonstrated to assist in delineating the mechanism of agonist-induced activation and biased signaling. ,,,,− …”

Allosteric Regulation of G-Protein-Coupled Receptors: From Diversity of Molecular Mechanisms to Multiple Allosteric Sites and Their Ligands

“…As in the structure of most GPCRs, allosteric sites in β-AR are located in all main subdomains, which is confirmed by the influence of autoantibodies to ECLs, synthetic ICL-derived pepducins, as well as the endogenous and synthetic small compounds capable of interacting with sites located in different loci of the TMD and its interfaces with ICLs. The multiplicity of these sites was demonstrated using the ligand competitive saturation (SILCS) computational method, which made it possible not only to predict the possible localization and functional activity of allosteric sites in the β 2 -AR but also to suggest potential candidates as regulators of these sites [ 417 ]. Below, we will briefly discuss some synthetic small molecular weight allosteric regulators since they, like small endogenous regulators (cholesterol, metal ions, etc.…”

“…Below, we will briefly discuss some synthetic small molecular weight allosteric regulators since they, like small endogenous regulators (cholesterol, metal ions, etc. ), are described in detail in a number of reviews and experimental works [ 417 , 418 , 419 , 420 , 421 , 422 ], and we will pay more attention to autoantibodies and pepducins.…”

Colabind: A Cloud-Based Approach for Prediction of Binding Sites Using Coarse-Grained Simulations with Molecular Probes

“…20−22 The approach was further developed, adapted, and demonstrated flexibility across various classes of targets and specific applications in a number of succeeding studies, 23,24 including membrane proteins. 25,26 Notably, Bahar et al 27 introduced a unique algorithm to combine detected interaction hotspots and assess pocket druggability.…”