Conventional drug discovery efforts at the β₂-adrenoceptor (β₂AR) have led to the development of ligands that bind almost exclusively to the receptor's hormone-binding orthosteric site. However, targeting the largely unexplored and evolutionarily unique allosteric sites has potential for developing more specific drugs with fewer side effects than orthosteric ligands. Using our recently developed approach for screening G protein-coupled receptors (GPCRs) with DNA-encoded small-molecule libraries, we have discovered and characterized the first β₂AR small-molecule positive allosteric modulators (PAMs)-compound (Cmpd)-6 [(R)-N-(4-amino-1-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-(N-isopropyl-N-methylsulfamoyl)-2-((4-methoxyphenyl)thio)benzamide] and its analogs. We used purified human β₂ARs, occupied by a high-affinity agonist, for the affinity-based screening of over 500 million distinct library compounds, which yielded Cmpd-6. It exhibits a low micro-molar affinity for the agonist-occupied β₂AR and displays positive cooperativity with orthosteric agonists, thereby enhancing their binding to the receptor and ability to stabilize its active state. Cmpd-6 is cooperative with G protein and β-arrestin1 (a.k.a. arrestin2) to stabilize high-affinity, agonist-bound active states of the β₂AR and potentiates downstream cAMP production and receptor recruitment of β-arrestin2 (a.k.a. arrestin3). Cmpd-6 is specific for the β₂AR compared with the closely related β₁AR. Structure-activity studies of select Cmpd-6 analogs defined the chemical groups that are critical for its biologic activity. We thus introduce the first small-molecule PAMs for the β₂AR, which may serve as a lead molecule for the development of novel therapeutics. The approach described in this work establishes a broadly applicable proof-of-concept strategy for affinity-based discovery of small-molecule allosteric compounds targeting unique conformational states of GPCRs.

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