Identification of a β-arrestin-biased negative allosteric modulator for the β 2 -adrenergic receptor

Catecholamine-stimulated β -adrenergic receptor (β AR) signaling via the canonical G -adenylyl cyclase-cAMP-PKA pathway regulates numerous physiological functions, including the therapeutic effects of exogenous β-agonists in the treatment of airway disease. β AR signaling is tightly regulated by GRKs and β-arrestins, which together promote β AR desensitization and internalization as well as downstream signaling, often antithetical to the canonical pathway. Thus, the ability to β AR signaling toward the G pathway while avoiding β-arrestin-mediated effects may provide a strategy to improve the functional consequences of β AR activation. Since attempts to develop G -biased agonists and allosteric modulators for the β AR have been largely unsuccessful, here we screened small molecule libraries for allosteric modulators that selectively inhibit β-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 β-arrestin recruitment to the β AR while having no effect on β AR coupling to G . DFPQ effectively inhibits agonist-promoted phosphorylation and internalization of the β AR and protects against the functional desensitization of β-agonist mediated regulation in cell and tissue models. The effects of DFPQ were also specific to the β AR with minimal effects on the β AR. Modeling, mutagenesis, and medicinal chemistry studies support DFPQ derivatives binding to an intracellular membrane-facing region of the β 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 β AR.