PT - JOURNAL ARTICLE AU - Seungkirl Ahn AU - Biswaranjan Pani AU - Alem W. Kahsai AU - Eva K. Olsen AU - Gitte Husemoen AU - Mikkel Vestergaard AU - Lei Jin AU - Shuai Zhao AU - Laura M. Wingler AU - Paula K. Rambarat AU - Rishabh K. Simhal AU - Thomas T. Xu AU - Lillian D. Sun AU - Paul J. Shim AU - Dean P. Staus AU - Li-Yin Huang AU - Thomas Franch AU - Xin Chen AU - Robert J. Lefkowitz TI - Small-Molecule Positive Allosteric Modulators of the <em>β</em><sub>2</sub>-Adrenoceptor Isolated from DNA-Encoded Libraries AID - 10.1124/mol.118.111948 DP - 2018 Aug 01 TA - Molecular Pharmacology PG - 850--861 VI - 94 IP - 2 4099 - http://molpharm.aspetjournals.org/content/94/2/850.short 4100 - http://molpharm.aspetjournals.org/content/94/2/850.full SO - Mol Pharmacol2018 Aug 01; 94 AB - 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.