Optimization and validation of a high throughput screen for identifying Nrf2
inhibitors
Tiffany Nguyen1, David R Lamson1, Xiaoxin L Chen, Ph.D.2, Ben Major, Ph.D.3, Kevin P. Williams, Ph.D.1
1
Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise (BRITE),
North Carolina Central University, Durham, NC
2
Department of Biological & Biomedical Sciences, Julius L. Chambers Biomedical/Biotechnology Research Institute
(BBRI), North Carolina Central University, Durham, NC
3
Department of Cell Biology and Physiology, Lineberger Comprehensive Cancer Center, University of North
Carolina at Chapel Hill, Chapel Hill, NC
The Nrf2-Keap1 signaling pathway is a primary cell defense and survival pathway. Keap1 (Kelch-like ECHassociated protein 1) regulates the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2,)
which, in turn, activates a wide range of cytoprotective genes including NQO1 to stimulate an
antioxidant response and protect cells from a number of toxins and carcinogens.
Though the Nrf2-Keap1 pathway is an essential defense mechanism for normal cell survival, recent
evidence shows that Nrf2 also promotes cancer cell survival. In particular, esophageal cancer exhibits
high expression of Nrf2. Nrf2 overexpression is also associated with chemotherapeutic drug resistance.
Several studies have shown that cancer cells with elevated levels of Nrf2 are less sensitive to common
chemotherapeutic agents such as etoposide, carboplatin, cisplatin, 5-fluorouracil, and doxorubicin.
Additionally, cells that have developed resistance to chemotherapeutic agents have been shown to
express high levels of Nrf2.
The purpose of this study is to screen for compounds which inhibit the activation of Nrf2 to potentially
reduce both its cancer cell protective role and drug resistance role. A GFP reporter human non-small cell
lung carcinoma cell line (H1299-GFP NQO1) was used. This cell line contained GFP under control of the
NQO1 promoter. In a previous study, these cells were treated with known Nrf2 agonists CDDO [2-cyano3, 12-dioxo-oleana-1,9(11)-dien-28-oic acid, methyl ester] and tBHQ (tert-butylhydroquinone) in a 12well assay to overexpress Nrf2. This resulted in increased GFP levels due to activation of the NQO1
promoter controlling GFP expression. This study optimizes the assay to a 384-well format to validate
reproducibility. When it is evident that variability is minimal, a compound library will be screened to
find potential inhibitors against Nrf2. In the presence of an Nrf2 inhibitor, decreased Nrf2 expression
would be detected as decreased GFP expression.