title: High-Throughput Screening for Drugs that Modulate Intermediate Filament Proteins.
abstract: Intermediate filament (IF) proteins have unique and complex cell and tissue distribution. Importantly, IF gene mutations cause or predispose to more than 80 human tissue-specific diseases (IF-pathies), with the most severe disease phenotypes being due to mutations at conserved residues that result in a disrupted IF network. A critical need for the entire IF-pathy field is the identification of drugs that can ameliorate or cure these diseases, particularly since all current therapies target the IF-pathy complication, such as diabetes or cardiovascular disease, rather than the mutant IF protein or gene. We describe a high-throughput approach to identify drugs that can normalize disrupted IF proteins. This approach utilizes transduction of lentivirus that expresses green fluorescent protein-tagged keratin 18 (K18) R90C in A549 cells. The readout is drug "hits" that convert the dot-like keratin filament distribution, due to the R90C mutation, to a wild-type-like filamentous array. A similar strategy can be used to screen thousands of compounds and can be utilized for practically any IF protein with a filament-disrupting mutation, and could therefore potentially target many IF-pathies. "Hits" of interest require validation in cell culture then using in vivo experimental models. Approaches to study the mechanism of mutant IF normalization by potential drugs of interest are also described. The ultimate goal of this drug screening approach is to identify effective and safe compounds that can potentially be tested for clinical efficacy in patients.
authors.affiliation: Department of Molecular & Integrative Physiology, University of Michigan, Ann Arbor, Michigan, USA; Department of Medicine, University of Michigan, Ann Arbor, Michigan, USA; VA Ann Arbor Healthcare System, Ann Arbor, Michigan, USA; Hepatology Unit, Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, PR China; Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, PR China.
authors.forename: Jingyuan
authors.initials: J
authors.lastname: Sun
authors.affiliation: Department of Pharmacology, The Center for Chemical Genomics, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Vincent E
authors.initials: VE
authors.lastname: Groppi
authors.affiliation: Department of Molecular & Integrative Physiology, University of Michigan, Ann Arbor, Michigan, USA; Department of Medicine, University of Michigan, Ann Arbor, Michigan, USA; VA Ann Arbor Healthcare System, Ann Arbor, Michigan, USA; Department of Infectious Diseases, Ruijin Hospital, Jiaotong University School of Medicine, Shanghai, PR China.
authors.forename: Honglian
authors.initials: H
authors.lastname: Gui
authors.affiliation: Department of Infectious Diseases, Ruijin Hospital, Jiaotong University School of Medicine, Shanghai, PR China.
authors.forename: Lu
authors.initials: L
authors.lastname: Chen
authors.affiliation: Department of Infectious Diseases, Ruijin Hospital, Jiaotong University School of Medicine, Shanghai, PR China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Xie
authors.affiliation: Hepatology Unit, Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, PR China; Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, PR China.
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.affiliation: Department of Molecular & Integrative Physiology, University of Michigan, Ann Arbor, Michigan, USA; Department of Medicine, University of Michigan, Ann Arbor, Michigan, USA; VA Ann Arbor Healthcare System, Ann Arbor, Michigan, USA. Electronic address: mbishr@umich.edu.
authors.forename: M Bishr
authors.initials: MB
authors.lastname: Omary
mesh_terms: D000818:Animals; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006801:Humans; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000378:metabolism*; D007382:Intermediate Filaments / Q000235:genetics / Q000378:metabolism*; D016086:Lentivirus / Q000235:genetics; D009154:Mutation / Q000235:genetics
chemical_list: D007381:Intermediate Filament Proteins
keywords: Diseases; Drug screening; High-throughput screening; IF-pathies; Intermediate filaments; Mutations; PKC412