title: Virtual High-Throughput Screening To Identify Novel Activin Antagonists.
abstract: Activin belongs to the TGFβ superfamily, which is associated with several disease conditions, including cancer-related cachexia, preterm labor with delivery, and osteoporosis. Targeting activin and its related signaling pathways holds promise as a therapeutic approach to these diseases. A small-molecule ligand-binding groove was identified in the interface between the two activin βA subunits and was used for a virtual high-throughput in silico screening of the ZINC database to identify hits. Thirty-nine compounds without significant toxicity were tested in two well-established activin assays: FSHβ transcription and HepG2 cell apoptosis. This screening workflow resulted in two lead compounds: NUCC-474 and NUCC-555. These potential activin antagonists were then shown to inhibit activin A-mediated cell proliferation in ex vivo ovary cultures. In vivo testing showed that our most potent compound (NUCC-555) caused a dose-dependent decrease in FSH levels in ovariectomized mice. The Blitz competition binding assay confirmed target binding of NUCC-555 to the activin A:ActRII that disrupts the activin A:ActRII complex's binding with ALK4-ECD-Fc in a dose-dependent manner. The NUCC-555 also specifically binds to activin A compared with other TGFβ superfamily member myostatin (GDF8). These data demonstrate a new in silico-based strategy for identifying small-molecule activin antagonists. Our approach is the first to identify a first-in-class small-molecule antagonist of activin binding to ALK4, which opens a completely new approach to inhibiting the activity of TGFβ receptor superfamily members. in addition, the lead compound can serve as a starting point for lead optimization toward the goal of a compound that may be effective in activin-mediated diseases.
authors.affiliation: †Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, 303 East Superior Street, Lurie 10-250, Chicago, Illinois 60611, United States.|‡Center for Reproductive Science, Northwestern University, Evanston, Illinois 60208, United States.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhu
authors.affiliation: §Center for Molecular Innovation and Drug Discovery, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States.
authors.forename: Rama K
authors.initials: RK
authors.lastname: Mishra
authors.affiliation: §Center for Molecular Innovation and Drug Discovery, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States.
authors.forename: Gary E
authors.initials: GE
authors.lastname: Schiltz
authors.affiliation: †Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, 303 East Superior Street, Lurie 10-250, Chicago, Illinois 60611, United States.
authors.forename: Yogeshwar
authors.initials: Y
authors.lastname: Makanji
authors.affiliation: §Center for Molecular Innovation and Drug Discovery, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States.|⊥Department of Chemistry, Northwestern University, Evanston, 60208, Illinois, United States.|∥Department of Pharmacology, Northwestern University, Chicago, Illinois 60611, United States.
authors.forename: Karl A
authors.initials: KA
authors.lastname: Scheidt
authors.affiliation: ∥Department of Pharmacology, Northwestern University, Chicago, Illinois 60611, United States.|□Chemistry of Life Processes Institute, Northwestern University, 2170 Campus Drive, Evanston, Illinois 60208, United States.
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Mazar
authors.affiliation: †Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, 303 East Superior Street, Lurie 10-250, Chicago, Illinois 60611, United States.|‡Center for Reproductive Science, Northwestern University, Evanston, Illinois 60208, United States.|□Chemistry of Life Processes Institute, Northwestern University, 2170 Campus Drive, Evanston, Illinois 60208, United States.
authors.forename: Teresa K
authors.initials: TK
authors.lastname: Woodruff
mesh_terms: D028341:Activins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D018360:Crystallography, X-Ray; D004353:Drug Evaluation, Preclinical; D005260:Female; D005640:Follicle Stimulating Hormone / Q000037:antagonists & inhibitors; D056945:Hep G2 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D051379:Mice; D062105:Molecular Docking Simulation; D010053:Ovary / Q000166:cytology / Q000187:drug effects; D011487:Protein Conformation; D015398:Signal Transduction / Q000187:drug effects; D014584:User-Computer Interface
chemical_list: C422405:activin A; D028341:Activins; D005640:Follicle Stimulating Hormone