Biased Allosteric Modulation of β₂-adrenergic Receptor Signaling and Bronchodilation by different β-agonists

Abstract ID 94144 Poster Board 004 Objective & Hypothesis: β-agonist serves as the frontline treatment of obstructive airway diseases like asthma and COPD to induce very essential functions - airway smooth muscle (ASM) relaxation and bronchodilation. However, due to β-arrestin mediated β₂-adrenergic receptor (β₂AR) signaling leads to receptor desensitization and loss of β-agonist efficacy. Therefore, new research for developing anti-asthma therapeutics is aimed at discovering biased ligands that augment beneficial Gs-mediated canonical signaling. We recently discovered allosteric modulators (AMs) of the β₂AR that augment Gs signaling and function in ASM cells using a prototypic β-agonist, isoproterenol (ISO). However, different endogenous and clinically used β-agonists induce different β₂AR conformational changes and thus potentially restricting the allosteric site on the β₂AR. The goal of the study is to establish the efficacy of these AMs on different B-agonists induced signaling and function. We hypothesize that AMs differentially modulate β₂AR signaling and bronchodilation induced by clinically distinct β-agonists. Methods: HEK-293 cells transfected with human β₂AR and human ASM (HASM) cells were stimulated with different β-agonists at varying concentrations in the presence of vehicle/AMs, and cAMP levels were assessed using ELISA. β-arrestin recruitment was assessed by BRET assay in β₂AR transfected HEK-293 cells. Additionally, bronchodilation, a functional effect, was examined using murine Precision Cut Lung Slices (PCLS). Airway lumen area was measured before and 10 minutes after treatment with β-agonists in the presence of vehicle or AMs. Results: Positive AM (PAM; cmpd 37) augmented ISO-induced cAMP generation. Interestingly, cmpd 37 had varying effects on different β-agonists-induced cAMP generation. Cmpd 37 augmented epinephrine-, formoterol-, and salmeterol-, but not albuterol- induced increase in cAMP levels. Further, in HASM cells – a cell model expressing endogenous β₂AR, cmpd 37 failed to augment salmeterol-mediated cAMP generation. Conversely, negative AMs (NAMs – cmpds 36 and 42), had variable effect on cAMP generation induced by different β-agonists. In HEK-293 cells, NAMs cmpd 36 and 42 inhibited ISO-, albuterol-, epinephrine-, and formoterol-, but not salmeterol- induced cAMP generation. In murine PCLS, cmpd 37 augmented ISO-, epinephrine-, formoterol-, salmeterol-, and albuterol- induced bronchodilation, albeit to a varying degree. NAMs cmpd 36 and 42 inhibited ISO-, epinephrine-, and formoterol-induced relaxation of murine airways. Further, cmpd 42, and not cmpd 36, inhibited albuterol- and salmeterol-induced relaxation of murine airways. Conclusion: Our findings suggest that the newly discovered AMs of the β₂AR modulate β₂AR-Gs signaling and bronchodilation by multiple b-agonists and could serve as a new therapeutic option for obstructive lung diseases by enhancing the bronchodilator effect of b-agonists. Further, these findings demonstrate the differential effect of biased AMs on different endogenous and clinically used β-agonists, possibly due to unique degree of the allosteric site accessibility induced by each of the agonists, positing the complexity of biased signaling. On-going computational studies are aimed at examining the mechanistic basis for differential effects of the AMs on different β-agonists.