title: Chemogenomic profiling of endogenous PARK2 expression using a genome-edited coincidence reporter.
abstract: Parkin, an E3 ubiquitin ligase, is a central mediator of mitochondrial quality control and is linked to familial forms of Parkinson's disease (PD). Removal of dysfunctional mitochondria from the cell by Parkin is thought to be neuroprotective, and pharmacologically increasing Parkin levels may be a novel therapeutic approach. We used genome-editing to integrate a coincidence reporter into the PARK2 gene locus of a neuroblastoma-derived cell line and developed a quantitative high-throughput screening (qHTS) assay capable of accurately detecting subtle compound-mediated increases in endogenous PARK2 expression. Interrogation of a chemogenomic library revealed diverse chemical classes that up-regulate the PARK2 transcript, including epigenetic agents, drugs controlling cholesterol biosynthesis, and JNK inhibitors. Use of the coincidence reporter eliminated wasted time pursuing reporter-biased false positives accounting for ∼2/3 of the actives and, coupled with titration-based screening, greatly improves the efficiency of compound selection. This approach represents a strategy to revitalize reporter-gene assays for drug discovery.
authors.affiliation: †National Institute of Neurological Disorders and Stroke, Bethesda, Maryland 20892, United States.
authors.forename: Samuel A
authors.initials: SA
authors.lastname: Hasson
authors.affiliation: †National Institute of Neurological Disorders and Stroke, Bethesda, Maryland 20892, United States.
authors.forename: Adam I
authors.initials: AI
authors.lastname: Fogel
authors.affiliation: †National Institute of Neurological Disorders and Stroke, Bethesda, Maryland 20892, United States.
authors.forename: Chunxin
authors.initials: C
authors.lastname: Wang
authors.affiliation: ‡National Center for Advancing Translational Sciences, Rockville, Maryland 20850, United States.
authors.forename: Ryan
authors.initials: R
authors.lastname: MacArthur
authors.affiliation: ‡National Center for Advancing Translational Sciences, Rockville, Maryland 20850, United States.
authors.forename: Rajarshi
authors.initials: R
authors.lastname: Guha
authors.affiliation: §NIH Center for Regenerative Medicine, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland 20892, United States.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Heman-Ackah
authors.affiliation: ‡National Center for Advancing Translational Sciences, Rockville, Maryland 20850, United States.
authors.forename: Scott
authors.initials: S
authors.lastname: Martin
authors.affiliation: †National Institute of Neurological Disorders and Stroke, Bethesda, Maryland 20892, United States.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Youle
authors.affiliation: ‡National Center for Advancing Translational Sciences, Rockville, Maryland 20850, United States.|∥National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, United States.
authors.forename: James
authors.initials: J
authors.lastname: Inglese
mesh_terms: D045744:Cell Line, Tumor; D002784:Cholesterol / Q000096:biosynthesis; D044127:Epigenesis, Genetic; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D023281:Genomics; D057166:High-Throughput Screening Assays; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000037:antagonists & inhibitors; D044767:Ubiquitin-Protein Ligases / Q000235:genetics*
chemical_list: D002784:Cholesterol; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein; D048670:MAP Kinase Kinase 4