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.