title: Development of a high-throughput screening paradigm for the discovery of small-molecule modulators of adenylyl cyclase: identification of an adenylyl cyclase 2 inhibitor.
abstract: Adenylyl cyclase (AC) isoforms are implicated in several physiologic processes and disease states, but advancements in the therapeutic targeting of AC isoforms have been limited by the lack of potent and isoform-selective small-molecule modulators. The discovery of AC isoform-selective small molecules is expected to facilitate the validation of AC isoforms as therapeutic targets and augment the study of AC isoform function in vivo. Identification of chemical probes for AC2 is particularly important because there are no published genetic deletion studies and few small-molecule modulators. The present report describes the development and implementation of an intact-cell, small-molecule screening approach and subsequent validation paradigm for the discovery of AC2 inhibitors. The NIH clinical collections I and II were screened for inhibitors of AC2 activity using PMA-stimulated cAMP accumulation as a functional readout. Active compounds were subsequently confirmed and validated as direct AC2 inhibitors using orthogonal and counterscreening assays. The screening effort identified SKF-83566 [8-bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hydrobromide] as a selective AC2 inhibitor with superior pharmacological properties for selective modulation of AC2 compared with currently available AC inhibitors. The utility of SKF-83566 as a small-molecule probe to study the function of endogenous ACs was demonstrated in C2C12 mouse skeletal muscle cells and human bronchial smooth muscle cells.
authors.affiliation: Medicinal Chemistry & Molecular Pharmacology, Purdue University, West Lafayette, Indiana (J.M.C., E.P.S.P., R.X., G.H.H., V.J.W.); Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, Texas (C.S.B., C.W.D.); and Department of Pharmacology, University of Tennessee Health Science Center, Memphis, Tennessee (A.S.B., R.S.O.).
authors.forename: Jason M
authors.initials: JM
authors.lastname: Conley
authors.forename: Cameron S
authors.initials: CS
authors.lastname: Brand
authors.forename: Amy S
authors.initials: AS
authors.lastname: Bogard
authors.forename: Evan P S
authors.initials: EP
authors.lastname: Pratt
authors.forename: Ruqiang
authors.initials: R
authors.lastname: Xu
authors.forename: Gregory H
authors.initials: GH
authors.lastname: Hockerman
authors.forename: Rennolds S
authors.initials: RS
authors.lastname: Ostrom
authors.forename: Carmen W
authors.initials: CW
authors.lastname: Dessauer
authors.forename: Val J
authors.initials: VJ
authors.lastname: Watts
mesh_terms: D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000235:genetics; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology / Q000276:immunology; D000242:Cyclic AMP / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D051379:Mice; D032389:Myocytes, Smooth Muscle / Q000201:enzymology / Q000276:immunology; D061987:Sf9 Cells; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D018411:Spodoptera; D014162:Transfection
chemical_list: D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases; D000242:Cyclic AMP; D004791:Enzyme Inhibitors; D054852:Small Molecule Libraries; C044846:SKF83566; C100622:adenylyl cyclase 2