title: A cell-based screening assay to identify pharmaceutical compounds that enhance the regenerative quality of corneal repair.
abstract: The goal of this study was to develop and validate a simple but quantitative cell-based assay to identify compounds that might be used pharmaceutically to give tissue repair a more regenerative character. The cornea was used as the model, and some specific aspects of repair in this organ were incorporated into assay design. A quantitative cell-based assay was developed based on transcriptional promoter activity of fibrotic marker genes ACT2A and TGFB2. Immortalized corneal stromal cells (HTK) or corneal epithelial cells (HCLE) were tested and compared to primary corneal stromal cells. Cells were transiently transfected with constructs containing the firefly luciferase reporter gene driven by transcriptional promoters for the selected fibrotic marker genes. A selected panel of seven chemical test compounds was used, containing three known fibrosis inhibitors: lovastatin (LOV), tyrphostin AG 1296 (6,7-dimethoxy-3-phenylquinoxaline) and SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole), and four potential fibrosis inhibitors: 5-iodotubercidin (4-amino-5-iodo-7-(β-D-ribofuranosyl)-pyrrolo(2,3-d)pyrimidine), anisomycin, DRB (5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole) and latrunculin B. Transfected cells were treated with TGFB2 in the presence or absence of one of the test compounds. To validate the assay, compounds were tested for their direct effects on gene expression in the immortalized cell lines and primary human corneal keratocytes using RT-PCR and immunohistochemistry. Three "hits" were validated LOV, SB203580 and anisomycin. This assay, which can be applied in a high throughput format to screen large libraries of uncharacterized compounds, or known compounds that might be repurposed, offers a valuable tool for identifying new treatments to address a major unmet medical need. Anisomycin has not previously been characterized as antifibrotic, thus, this is a novel finding of the study.
authors.affiliation: USC Institute for Genetic Medicine, Keck School of Medicine of USC, University of Southern California, Los Angeles, California.|Department of Ophthalmology and Graduate Program in Molecular Cell and Developmental Biology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida.
authors.forename: Gabriel M
authors.initials: GM
authors.lastname: Gordon
authors.affiliation: Department of Biology, Grand View University, Des Moines, Iowa.|Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida.
authors.forename: Adriana J
authors.initials: AJ
authors.lastname: LaGier
authors.affiliation: Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida.
authors.forename: Corinne
authors.initials: C
authors.lastname: Ponchel
authors.affiliation: USC Institute for Genetic Medicine and Graduate Program in Integrative Biology of Disease, Keck School of Medicine, University of Southern California, Los Angeles, California.
authors.forename: Aditi
authors.initials: A
authors.lastname: Bauskar
authors.affiliation: USC Institute for Genetic Medicine, Keck School of Medicine of USC, University of Southern California, Los Angeles, California.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Itakura
authors.affiliation: Department of Ophthalmology, USC Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California.
authors.forename: Shinwu
authors.initials: S
authors.lastname: Jeong
authors.affiliation: USC Institute for Genetic Medicine, Keck School of Medicine of USC, University of Southern California, Los Angeles, California.
authors.forename: Nitin
authors.initials: N
authors.lastname: Patel
authors.affiliation: Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida.|Department of Cell and Neurobiology and Department of Ophthalmology, USC Institute for Genetic Medicine, USC Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California.
authors.forename: M Elizabeth
authors.initials: ME
authors.lastname: Fini
mesh_terms: D000199:Actins / Q000187:drug effects / Q000235:genetics; D000818:Animals; D000841:Anisomycin / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002460:Cell Line; D003315:Cornea / Q000166:cytology / Q000187:drug effects; D060527:Corneal Keratocytes / Q000166:cytology / Q000187:drug effects*; D003584:Cytological Techniques; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D008148:Lovastatin / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011817:Rabbits; D012038:Regeneration / Q000187:drug effects*; D053778:Thiazolidines / Q000494:pharmacology; D053781:Transforming Growth Factor beta2 / Q000187:drug effects / Q000235:genetics; D014372:Tubercidin / Q000031:analogs & derivatives / Q000494:pharmacology; D020032:Tyrphostins / Q000494:pharmacology; D014945:Wound Healing / Q000187:drug effects*
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D004791:Enzyme Inhibitors; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007093:Imidazoles; D011500:Protein Synthesis Inhibitors; D011725:Pyridines; C509041:TGFB2 protein, human; D053778:Thiazolidines; D053781:Transforming Growth Factor beta2; D020032:Tyrphostins; C106250:6,7-dimethoxy-3-phenylquinoxaline; C034214:5-iodotubercidin; D004004:Dichlororibofuranosylbenzimidazole; D000841:Anisomycin; D008148:Lovastatin; C037068:latrunculin B; D014372:Tubercidin; C093642:SB 203580