Identification of a β-arrestin-biased negative allosteric modulator for the β <sub>2</sub> -adrenergic receptor
Michael Ippolito, Francesco De Pascali, Nathan Hopfinger, Konstantin E. Komolov, Daniela K. Laurinavichyute, Poli Adi Narayana Reddy, Leon A. Sakkal, Kyle Z. Rajkowski, Ajay P. Nayak, Justin B. Lee, Jordan Lee, Gaoyuan Cao, Preston S. Donover, Melvin Reichman, Steven S. An, Joseph M. Salvino, Raymond B. Penn, Roger S. Armen, Charles P. Scott, Jeffrey Benovic
Abstract
Catecholamine-stimulated β 2 -adrenergic receptor (β 2 AR) signaling via the canonical G s –adenylyl cyclase–cAMP–PKA pathway regulates numerous physiological functions, including the therapeutic effects of exogenous β-agonists in the treatment of airway disease. β 2 AR signaling is tightly regulated by GRKs and β-arrestins, which together promote β 2 AR desensitization and internalization as well as downstream signaling, often antithetical to the canonical pathway. Thus, the ability to bias β 2 AR signaling toward the G s pathway while avoiding β-arrestin-mediated effects may provide a strategy to improve the functional consequences of β 2 AR activation. Since attempts to develop G s -biased agonists and allosteric modulators for the β 2 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 2 AR while having no effect on β 2 AR coupling to G s . DFPQ effectively inhibits agonist-promoted phosphorylation and internalization of the β 2 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 2 AR with minimal effects on the β 1 AR. Modeling, mutagenesis, and medicinal chemistry studies support DFPQ derivatives binding to an intracellular membrane-facing region of the β 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 β 2 AR.