?-agonists acting at the ?2-adrenergic receptor (?2AR) on human airway smooth muscle (HASM) relax the muscle and dilate the airways, and are used for acute (rescue) and chronic (maintenance) therapy for asthma. With many asthmatics not achieving control, there is a need to understand the basis for efficacy observed with acute ?-agonists and the development of tolerance, or tachyphylaxis, during chronic therapy. These issues are due in part to a lack of understanding of fundamental aspects of ?2AR signaling in HASM. This proposal has three aims to close this gap, with the broad, long-term objective of developing optimal ?-agonist treatment for asthma to reduce morbidity.
In Aim 1, we will screen a 40 million compound library to discover ?-agonists that stabilize a specific ?2AR conformation that is favorable for asthma. This ?biasing? would be towards Gs/cAMP coupling (improves bronchodilation) and away from ?-arrestin recruitment (thus minimizing tolerance). This will be accomplished through a sequential screening approach that measures cAMP, ?-arrestin recruitment, and then physiologic function in transgenic mouse and human airways under asthmatic phenotypes. Agonists that approach optimal biasing will be modeled to the crystal structure of the ?2AR and refined via medicinal chemistry. A high resolution ?2AR crystal structure bound to the biased ligand will be generated.
In Aim 2, the interactions between ?2AR and another airway receptor that bronchodilates, the bitter taste receptor (TAS2R), will be ascertained. Agonists for TAS2Rs are in development for treating asthma, and it is envisioned that TAS2R and ?2AR agonists will be administered concomitantly. Yet, the two receptors appear to be intertwined at the cell surface in heterodimers, where activation of one receptor alters function of the other. Heterodimers and their in vitro function will be studied using cellular techniques, and function will be ascertained by measuring second messengers from each monomeric component and isolated cell mechanics in HASM from asthmatic and nonasthmatic donors.
In Aim 3, the mechanism of translational repression of the ?2AR gene by the miRNA let-7f will define how the resting level of ?2AR protein (and thus responsiveness) is established, and is regulated by agonist, in asthmatic and nonasthmatic HASM. Physiologic function will be established with isolated HASM mechanics, and in sensitized transgenic mice with and without the let-7f binding domain. The in vitro, ex vivo, and in vivo studies will merge cell signaling and biochemistry with physiologic function, providing a cohesive analysis of these new concepts in receptor biology relative to asthma. Collectively, they will define mechanisms that point to new therapeutics for improved therapy of asthma.
Many of the 25 million asthmatics in the US do not achieve adequate control of their disease. It is proposed to understand certain aspects of ?2-adrenergic function that will provide for novel ?-agonists with superior efficacy and lack of tolerance compared to those currently available.
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