title: Development of a small-molecule screening method for inhibitors of cellular response to myostatin and activin A.
abstract: Myostatin, a member of the transforming growth factor (TGF)-β family of secreted ligands, is a strong negative regulator of muscle growth. As such, therapeutic inhibitors of myostatin are actively being investigated for their potential in the treatment of muscle-wasting diseases such as muscular dystrophy and sarcopenia. Here, we sought to develop a high-throughput screening (HTS) method for small-molecule inhibitors that target myostatin. We created a HEK293 stable cell line that expresses the (CAGA)12-luciferase reporter construct and robustly responds to signaling of certain classes of TGF-β family ligands. After optimization and miniaturization of the assay to a 384-well format, we successfully screened a library of compounds for inhibition of myostatin and the closely related activin A. Selection of some of the tested compounds was directed by in silico screening against myostatin, which led to an enrichment of target hits as compared with random selection. Altogether, we present an HTS method that will be useful for screening potential inhibitors of not only myostatin but also many other ligands of the TGF-β family.
authors.affiliation: Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, OH 45267, USA.
authors.forename: Jennifer N
authors.initials: JN
authors.lastname: Cash
authors.forename: Elizabeth B
authors.initials: EB
authors.lastname: Angerman
authors.forename: R Jason
authors.initials: RJ
authors.lastname: Kirby
authors.forename: Lisa
authors.initials: L
authors.lastname: Merck
authors.forename: William L
authors.initials: WL
authors.lastname: Seibel
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Wortman
authors.forename: Ruben
authors.initials: R
authors.lastname: Papoian
authors.forename: Sandra
authors.initials: S
authors.lastname: Nelson
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Thompson
mesh_terms: D028341:Activins / Q000037:antagonists & inhibitors*; D003198:Computer Simulation; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D006133:Growth Substances / Q000494:pharmacology*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D049410:Luciferases, Renilla / Q000096:biosynthesis / Q000235:genetics; D055435:Myostatin / Q000037:antagonists & inhibitors*; D054852:Small Molecule Libraries
chemical_list: D006133:Growth Substances; D055435:Myostatin; D054852:Small Molecule Libraries; C422405:activin A; D028341:Activins; D049410:Luciferases, Renilla
keywords: cell-based assays; musculoskeletal diseases; pharmacology: ligand binding; receptor binding; reporter gene assays; signal transduction