Identification of a β-arrestin- biased negative allosteric modulator for the β2-adrenergic receptor

Catecholamine-stimulated beta(2)-adrenergic receptor (beta(2)AR) signaling via the canonical Gs-adenylyl cyclase-cAMP-PKA pathway regulates numerous physiological functions, including the therapeutic effects of exogenous beta-agonists in the treatment of airway disease. beta(2)AR signaling is tightly regulated by GRKs and beta-arrestins, which together promote beta(2)AR desensitization and internalization as well as downstream signaling, often antithetical to the canonical pathway. Thus, the ability to bias beta(2)AR signaling toward the Gs pathway while avoiding beta-arrestin- mediated effects may provide a strategy to improve the functional consequences of beta(2)AR activation. Since attempts to develop Gs-biased agonists and allosteric modulators for the beta(2)AR have been largely unsuccessful, here we screened small molecule libraries for allosteric modulators that selectively inhibit beta-arrestin recruitment to the receptor. This screen identified several compounds that met this profile, and, of these, a difluorophenyl quinazoline (DFPQ) derivative was found to be a selective negative allosteric modulator of beta-arrestin recruitment to the beta(2)AR while having no effect on beta(2)AR coupling to Gs. DFPQ effectively inhibits agonist-promoted phosphorylation and internalization of the beta(2)AR and protects against the functional desensitization of beta-agonist mediated regulation in cell and tissue models. The effects of DFPQ were also specific to the beta(2)AR with minimal effects on the beta(1)AR. Modeling, mutagenesis, and medicinal chemistry studies support DFPQ derivatives binding to an intracellular membrane-facing region of the beta(2)AR, including residues within transmembrane domains 3 and 4 and intracellular loop 2. DFPQ thus represents a class of biased allosteric modulators that targets an allosteric site of the beta(2)AR.