Small-Molecule Positive Allosteric Modulators of the β2-Adrenoceptor Isolated from DNA-Encoded Libraries

Conventional drug discovery efforts at the beta(2)-adrenoceptor (beta(2)AR) 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 beta(2)AR 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-nnethoxyphenyl)thio)benzamide] and its analogs. We used purified human beta(2)ARs, 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 beta(2)AR 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 beta-arrestin1 (a.k.a. arresting) to stabilize high-affinity, agonist-bound active states of the beta(2)AR and potentiates downstream cAMP production and receptor recruitment of beta-arresting2 (a.k.a. arrestin3). Cmpd-6 is specific for the beta(2)AR compared with the closely related beta(1)AR. Structure-activity studies of select Cmpd-6 analogs defined the chemical groups that are critical for its biologic activity. We thus introduce the first smallmolecule PAMs for the beta(2)AR, 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.