Identification and characterization of an atypical Gαs-biased β 2 AR agonist that fails to evoke airway smooth muscle cell tachyphylaxis.

G protein-coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For β 2 -adrenergic receptors (β 2 AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and β-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S -stereoisomer of compound C1 shows no detectable β-arrestin engagement/signaling by four methods. However, C1- S retained Gαs signaling-a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: β 2 AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1- S , and desensitization of HASM cell relaxation was observed with albuterol but not with C1- S These HASM results indicate biologically pertinent biasing of C1- S , in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1- S was apparently strongly biased away from β - arrestin, in contrast to albuterol and C5- S C1- S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1- S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5- S (R2 = benzene group), which appears to contribute to C1- S biasing away from β-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of β 2 AR actions favorable for treating obstructive lung disease.