Project Summary Effective asthma management requires regulating airway smooth muscle (ASM) contractile state to avoid or reverse bronchoconstriction. Whether this is attempted by use of direct bronchodilators (e.g., β-agonists), by anti-inflammatory agents (e.g., corticosteroids), or some combination of both, too often management is lacking, as an estimated 55% of all asthmatics have suboptimal control. All current bronchodilator drugs have limitations which respect to efficacy, and safety issues still persist with the most frequently used class of bronchodilator drugs- long-acting β-agonists (LABAs). We submit that the limitations of bronchodilator drugs can be overcome by an approach that targets the 2 most powerful regulators of ASM contractile state: pharmacomechanical coupling and the actin cytoskeleton. We hypothesize that combinations of beta-agonists (that primarily target pharmacomechanical coupling) and drugs that specifically target the actin cytoskeleton can be highly efficacious bronchodilators, with a functional cooperatively that enables lower drug doses and therefore a better safety profile. Three aims are proposed to test this hypothesis. In Aim 1, we will establish, using cell, tissue, and in vivo models of ASM contraction, the cooperative nature of combined targeting of pharmacomechanical coupling and actin polymerization, and identify optimal combinations of beta-agonists and cytoskeleton-targeting drugs that relax ASM. In Aim 2, we will determine the mechanistic basis for this functional cooperativity by characterizing the effects of these drugs on signaling intermediates and outcomes that control cross bridge cycle (myosin light chain kinase and phosphatase phosphorylation) or actin polymerization state (F/G actin ratio), and on the upstream signals that regulate these outcomes. Lastly, in Aim 3 we will assess the effect of asthma pathobiology on the efficacy of combining β-agonists and actin cytoskeleton-targeting drugs in inhibiting ASM contraction and airway resistance, by employing cell and tissue model systems derived from cells/tissues from human asthmatics, or in which asthma pathobiology is imposed either in vitro/ex vivo (to cells, tissue), or in vivo (2 differrent in vivo murine models). The proposed studies performed by 3 established PIs with complementary expertise represent an innovative approach to establish an asthma management strategy that overcomes the current limitations of efficacy and safety. Moreover, the proposed mechanistic studies will provide new insight into how to optimally disrupt the cooperation between cross bridge cycling and cytoskeleton stiffening that generates tension in the ASM cell.