title: Allosteric "beta-blocker" isolated from a DNA-encoded small molecule library.
abstract: The β2-adrenergic receptor (β2AR) has been a model system for understanding regulatory mechanisms of G-protein-coupled receptor (GPCR) actions and plays a significant role in cardiovascular and pulmonary diseases. Because all known β-adrenergic receptor drugs target the orthosteric binding site of the receptor, we set out to isolate allosteric ligands for this receptor by panning DNA-encoded small-molecule libraries comprising 190 million distinct compounds against purified human β2AR. Here, we report the discovery of a small-molecule negative allosteric modulator (antagonist), compound 15 [([4-((2S)-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide], exhibiting a unique chemotype and low micromolar affinity for the β2AR. Binding of 15 to the receptor cooperatively enhances orthosteric inverse agonist binding while negatively modulating binding of orthosteric agonists. Studies with a specific antibody that binds to an intracellular region of the β2AR suggest that 15 binds in proximity to the G-protein binding site on the cytosolic surface of the β2AR. In cell-signaling studies, 15 inhibits cAMP production through the β2AR, but not that mediated by other Gs-coupled receptors. Compound 15 also similarly inhibits β-arrestin recruitment to the activated β2AR. This study presents an allosteric small-molecule ligand for the β2AR and introduces a broadly applicable method for screening DNA-encoded small-molecule libraries against purified GPCR targets. Importantly, such an approach could facilitate the discovery of GPCR drugs with tailored allosteric effects.
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, NC 27710.
authors.forename: Seungkirl
authors.initials: S
authors.lastname: Ahn
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, NC 27710.
authors.forename: Alem W
authors.initials: AW
authors.lastname: Kahsai
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, NC 27710.
authors.forename: Biswaranjan
authors.initials: B
authors.lastname: Pani
authors.affiliation: Department of Medicinal Chemistry, School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou 213164, Jiangsu, China.
authors.forename: Qin-Ting
authors.initials: QT
authors.lastname: Wang
authors.affiliation: Department of Medicinal Chemistry, School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou 213164, Jiangsu, China.
authors.forename: Shuai
authors.initials: S
authors.lastname: Zhao
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, NC 27710.
authors.forename: Alissa L
authors.initials: AL
authors.lastname: Wall
authors.affiliation: Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599.
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Strachan
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, NC 27710.|Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710.
authors.forename: Dean P
authors.initials: DP
authors.lastname: Staus
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, NC 27710.|Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710.
authors.forename: Laura M
authors.initials: LM
authors.lastname: Wingler
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, NC 27710.
authors.forename: Lillian D
authors.initials: LD
authors.lastname: Sun
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, NC 27710.
authors.forename: Justine
authors.initials: J
authors.lastname: Sinnaeve
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, NC 27710.
authors.forename: Minjung
authors.initials: M
authors.lastname: Choi
authors.affiliation: Department of Biology, Duke University Medical Center, Durham, NC 27710.
authors.forename: Ted
authors.initials: T
authors.lastname: Cho
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, NC 27710.
authors.forename: Thomas T
authors.initials: TT
authors.lastname: Xu
authors.affiliation: Lexicon Pharmaceuticals, Inc., The Woodlands, TX 77381.
authors.forename: Gwenn M
authors.initials: GM
authors.lastname: Hansen
authors.affiliation: Lexicon Pharmaceuticals, Inc., The Woodlands, TX 77381.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Burnett
authors.affiliation: DiscoverX Co., Fremont, CA 94538.
authors.forename: Jane E
authors.initials: JE
authors.lastname: Lamerdin
authors.affiliation: DiscoverX Co., Fremont, CA 94538.
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Bassoni
authors.affiliation: DiscoverX Co., Fremont, CA 94538.
authors.forename: Bryant J
authors.initials: BJ
authors.lastname: Gavino
authors.affiliation: Nuevolution A/S, 2100 Copenhagen, Denmark.
authors.forename: Gitte
authors.initials: G
authors.lastname: Husemoen
authors.affiliation: Nuevolution A/S, 2100 Copenhagen, Denmark.
authors.forename: Eva K
authors.initials: EK
authors.lastname: Olsen
authors.affiliation: Nuevolution A/S, 2100 Copenhagen, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Franch
authors.affiliation: Department of Chemistry, American University, Washington, DC 20016.
authors.forename: Stefano
authors.initials: S
authors.lastname: Costanzi
authors.affiliation: Department of Medicinal Chemistry, School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou 213164, Jiangsu, China; lefko001@receptor-biol.duke.edu xinchen@cczu.edu.cn.
authors.forename: Xin
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, NC 27710; lefko001@receptor-biol.duke.edu xinchen@cczu.edu.cn.|Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710.|Department of Biochemistry, Duke University Medical Center, Durham, NC 27710.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Lefkowitz
mesh_terms: D000319:Adrenergic beta-Antagonists / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000187:drug effects; D004247:DNA / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008024:Ligands; D015394:Molecular Structure; D009154:Mutation; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D061987:Sf9 Cells; D054852:Small Molecule Libraries / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D018411:Spodoptera
chemical_list: D000319:Adrenergic beta-Antagonists; D008024:Ligands; D018343:Receptors, Adrenergic, beta-2; D054852:Small Molecule Libraries; D004247:DNA
keywords: DNA-encoded small-molecule library; G-protein–coupled receptor; allosteric modulator; drug discovery; β2-adrenergic receptor