Project Summary Asthma is a complex disease that results in airway smooth muscle (ASM) contraction and subsequent airway constriction. The major drugs used to treat asthma include β-agonists that promote ASM relaxation, Gq- coupled receptor antagonists that inhibit bronchoconstriction, and corticosteroids that reduce inflammation. Recent studies suggest that long acting β-agonists increase the risk of having a severe asthmatic attack that can result in death. While the mechanisms whereby β-agonists cause such severe side effects are poorly understood, β2-adrenergic receptor (β2AR) desensitization and β-arrestin-mediated signaling appear to contribute to this process. We hypothesize that biased agonists that selectively promote β2AR interaction with Gs will serve as an effective way of treating asthma. To test this hypothesis, we will characterize lead compounds that we have developed as well as additional compounds, for their ability to provide superior inhibition of ASM contraction by virtue of their ability to promote Gs-biased β2AR signaling. In aim 1, we will develop compounds that mediate Gs-biased signaling through the β2AR. We have identified two broad classes of compounds that bias Gs signaling through the β2AR, arrestin-biased negative allosteric modulators (NAMs), that effectively inhibit β2AR interaction with arrestins, and Gs-biased agonists. The lead compounds will be further characterized and then optimized using molecular modeling, structure activity analysis, and medicinal chemistry to improve the potency, bias and drug-like properties. In aim 2, we will identify the molecular basis of Gs-biased signaling using structural and biophysical approaches to study the interaction of arrestin-biased NAMs and Gs-biased agonists with the β2AR. In aim 3, we will characterize the ability of Gs-biased agonists and arrestin-biased NAMs to promote human airway smooth muscle relaxation as compared to β-agonists currently used to treat asthma. Overall, our development of Gs-biased β2AR agonists and allosteric modulators will provide a structural framework for better understanding the mechanistic basis of β2AR biased signaling, which should ultimately lead to novel drugs for a wide range of airway diseases.