Strategies for biasing and augmenting b2-adrenoceptor signaling and function in ASM

Abstract Project 1 (“Strategies for biasing and augmenting 2-adrenoceptor signaling and function in ASM” Penn, PI) In the last 3 years, our investigative team has achieved what the field of GPCR biology has intensely and unsuccessfully pursued over the prior 2 decades- the discovery of biased 2AR (small molecule) ligands that effectively and selectively activate the (beneficial) Gs/cAMP/PKA pathway in artificial systems and primary human ASM (HASM) cells, while not activating the (deleterious) arrestin-dependent pathway. This PPG project proposes to leverage the strengths of Core A to advance our discovery of biased 2AR ligands, building on new insight gained from our recent modeling and synthesis successes. In addition, we will test the effect of our lead biased 2AR ligands, in combination with inhibitors of cAMP transporter ABCC1 (Project 2), to further bias and augment ASM Gs/cAMP/PKA signaling. We will test the central hypothesis that small molecules that stabilize a dimeric complex of the 2AR that is resistant to −arrestin recruitment function as Gs-biased ligands and enable superior inhibition of ASM contraction and the asthma phenotype. In addition, signaling and functional bias of these ligands can be further enhanced by ABCC1 inhibition. Aim 1 will identify the structural basis of 2AR/GPCR allosteric modulation, and develop new 2AR allosteric modulators (AMs) that bias 2AR signaling. Recent studies by our team have determined critical structural features of the 2AR that affect their interactions with AMs. Working closely with Core A, we will employ CHARMM-based receptor modeling to identify and validate new small molecule ligands that will ultimately feed into Aims 2 and 3. Aim 2 will establish that Gs-biased 2AR signaling, via biased ligands and combination strategies, translates into greater anti-contractile effects in multiple models of HASM function, and in a murine model of allergic lung inflammation. Employing multiple cell, tissue, and in vivo models of ASM contraction, we will demonstrate that Gs-biased 2AR ligands will provide superior relaxation/prevention of contracted ASM, and ABCC1 will further augment this effect. Then we will explore the therapeutic efficacy of these treatments on the asthma phenotype (airway hyperresponsiveness (AHR), airway inflammation, and airway remodeling) in a murine model of allergic lung inflammation. Aim 3 will establish the mechanisms by which biased 2AR ligands inhibit pro-contractile signaling, and that concomitant treatment with ABCC1 inhibitors further biases 2AR signaling to enhance such inhibition. We hypothesize that increased and prolonged PKA activity stimulated by biased 2AR ligands will result in greater inhibition of multiple effectors of pro-contractile Gq-signaling. Concomitant ABCC1 inhibition will further bias 2AR signaling and increase these inhibitory effects.