title: Development of a cell-based high throughput luciferase enzyme fragment complementation assay to identify nuclear-factor-e2-related transcription factor 2 activators.
abstract: Nuclear-factor-E2-related transcription factor 2 (Nrf2) regulates a large panel of Phase II genes and plays an important role in cell survival. Nrf2 activation has been shown as preventing cigarette smoke-induced alveolar enlargement in mice. Therefore, activation of the Nrf2 protein by small-molecule activators represents an attractive therapeutic strategy that is used for chronic obstructive pulmonary disease. In this article, we describe a cell-based luciferase enzyme fragment complementation assay that identifies Nrf2 activators. This assay is based on the interaction of Nrf2 with its nuclear partner MafK or runt-related transcription factor 2 (RunX2) and is dependent on the reconstitution of a "split" luciferase. Firefly luciferase is split into two fragments, which are genetically fused to Nrf2 and MafK or RunX2, respectively. BacMam technology was used to deliver the fusion constructs into cells for expression of the tagged proteins. When the BacMam-transduced cells were treated with Nrf2 activators, the Nrf2 protein was stabilized and translocated into the nucleus where it interacted with MafK or RunX2. The interaction of Nrf2 and MafK or RunX2 brought together the two luciferase fragments that form an active luciferase. The assay was developed in a 384-well format and was optimized by titrating the BacMam concentration, transduction time, cell density, and fetal bovine serum concentration. It was further validated with known Nrf2 activators. Our data show that this assay is robust, sensitive, and amenable to high throughput screening of a large compound collection for the identification of novel Nrf2 activators.
authors.affiliation: Department of Biological Reagents and Assay Development, Platform Technology and Science, GlaxoSmithKline Pharmaceuticals, Collegeville, Pennsylvania 19426, USA.
authors.forename: Wensheng
authors.initials: W
authors.lastname: Xie
authors.forename: Christina
authors.initials: C
authors.lastname: Pao
authors.forename: Taylor
authors.initials: T
authors.lastname: Graham
authors.forename: Ed
authors.initials: E
authors.lastname: Dul
authors.forename: Quinn
authors.initials: Q
authors.lastname: Lu
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Sweitzer
authors.forename: Robert S
authors.initials: RS
authors.lastname: Ames
authors.forename: Hu
authors.initials: H
authors.lastname: Li
mesh_terms: D000465:Algorithms; D001331:Automation; D002452:Cell Count; D003001:Cloning, Molecular; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D003470:Culture Media; D003587:Cytomegalovirus / Q000235:genetics; D003627:Data Interpretation, Statistical; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005816:Genetic Complementation Test / Q000379:methods*; D005822:Genetic Vectors; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008156:Luciferases / Q000378:metabolism*; D051267:NF-E2-Related Factor 2 / Q000819:agonists / Q000187:drug effects*; D060888:Real-Time Polymerase Chain Reaction; D054852:Small Molecule Libraries; D014161:Transduction, Genetic
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D003470:Culture Media; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; C493709:RUNX2 protein, human; D054852:Small Molecule Libraries; D008156:Luciferases; D004121:Dimethyl Sulfoxide