Small Molecule Modulators of β-arrestins

β-arrestins (βarrs) are key regulators of G protein-coupled receptors (GPCRs), essential for modulating signaling pathways and physiological processes. While current pharmacological strategies target GPCR orthosteric and allosteric sites, as well as G protein transducers, comparable tools for studying βarrs are lacking. Here, we present the discovery and characterization of novel small-molecule allosteric inhibitors of βarrs through comprehensive biophysical, biochemical, pharmacological, and structural analyses. These inhibitors disrupt βarr interactions with agonist-activated GPCRs, impairing receptor internalization, desensitization, and βarr-mediated physiological functions. A cryo-EM structure of βarr1 in complex with the allosteric inhibitor Cmpd-5, complemented by molecular dynamics simulations and mutagenesis studies, reveals that Cmpd-5 binds within a cryptic cleft formed by the middle, C-, and lariat loops–a critical site for βarr activation and recruitment to GPCRs. Thus, Cmpd-5 acts as a molecular lock, hindering βarr1 activation via an allosteric mechanism. These findings introduce novel strategies and tools for probing βarr functions.