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ACS Chem Biol. 2015 May 15;10(5):1188-97. doi: 10.1021/cb5010417. Epub 2015 Feb 26.

Chemogenomic profiling of endogenous PARK2 expression using a genome-edited coincidence reporter.

Author information

1
†National Institute of Neurological Disorders and Stroke, Bethesda, Maryland 20892, United States.
2
‡National Center for Advancing Translational Sciences, Rockville, Maryland 20850, United States.
3
§NIH Center for Regenerative Medicine, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland 20892, United States.
4
∥National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, United States.

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.

PMID:
25689131
DOI:
10.1021/cb5010417
[Indexed for MEDLINE]

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