Saturday, September 7, 2024
9/7/2024
PROJECT SUMMARY: Beta 2 adrenergic receptor (AR) agonists (-agonists) are prominent prophylaxis and rescue medicines in asthma. However, the therapeutic efficacy of -agonists is limited by multiple clinical problems associated with the chronic use of -agonists, including tachyphylaxis and worsening asthma symptoms. Therefore, there is a clinical need to develop strategies to improve the efficacy of β-agonists. At a molecular level, β2AR binds to multiple proteins such as Gs, -arrestins, G-protein receptor kinase, and protein kinase A, leading to intricate, multidimensional signaling and functional effects. The canonical β2AR-Gs signaling relaxes airway smooth muscle (ASM), whereas β2AR-arrestin signaling promotes receptor desensitization, cell proliferation, and mucus production in the airways. Leveraging the concepts of “Biased agonism” and “Allosteric modulation” in G protein-coupled receptor pharmacology, in this application, we propose to employ advanced computational approaches and identify novel β2AR conformations and allosteric sites, and develop ligands that bind to those sites and promote specific (beneficial) signaling and functional effect in airways. Our studies identified unique -agonist-induced conformations and a novel allosteric site on the β2AR. Database screening identified small-molecule ligands that can bind to this site and modulate β2AR-Gs signaling and relaxation of human ASM cells and bronchodilation of human and murine airways. Motivated by these data, we hypothesize that selective enhancement of the β2AR-Gs signaling by positive allosteric modulators (AMs) of β2AR developed by computational approaches will provide therapeutic benefits in asthma. In Aim 1, we will employ advanced computational approaches, including atomistic molecular dynamics simulations at a superior spatial and temporal resolution in the presence of interacting proteins and different clinically relevant -agonists. Further, we will develop functional group affinity patterns, FragMaps, for different conformations of β2AR and identify allosteric binding sites and screen for ligands binding to those sites. These studies will also establish the structural basis for the modulation of specific signaling of β2AR by the AMs. Aim 2 studies will elucidate the signaling (Gs: Gs activation, cyclic AMP generation, protein kinase A activation; -arrestin: arrestin recruitment, phosphorylation of p42/p44, loss of cell surface expression of β2AR) mechanisms and functional effects (ASM relaxation, bronchodilation, ASM cell proliferation, and mucus/cytokine secretion by human airway epithelial cells) of - agonists modulated by AMs using HEK293 cells expressing human β2AR and human ASM/airway epithelial cells, murine and human lung slices, and transgenic mice expressing human β2AR in smooth muscle. Previous studies have demonstrated that -agonists promote asthma pathology in a β2AR-arrestin-dependent manner in murine models. Enhancing canonical β2AR-Gs signaling may overcome the harmful effects of -agonists. We will assess the therapeutic benefit of AMs in a murine model of asthma and human lung slices (Aim 3). We anticipate that the PAMs of 2AR will act in a biased fashion to augment desirable Gs signaling and functional effects of -agonists, providing a basis for exploring a biased AM-based therapeutic approach to improve the efficacy of current -agonists.
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