Development of Neh2-luciferase reporter and its application for high throughput screening and real-time monitoring of Nrf2 activators

Natalya A. Smirnova, Renee E. Haskew-Layton, Manuela Basso, Dmitry M. Hushpulian, Jimmy B. Payappilly, Rachel E. Speer, Young Hoon Ahn, Ilay Rakhman, Philip A. Cole, John T. Pinto, Rajiv R. Ratan, Irina G. Gazaryan

Research output: Contribution to journalArticle

Abstract

The NF-E2-related factor 2 (Nrf2) is a key transcriptional regulator of antioxidant defense and detoxification. To directly monitor stabilization of Nrf2, we fused its Neh2 domain, responsible for the interaction with its nucleocytoplasmic regulator, Keap1, to firefly luciferase (Neh2-luciferase). We show that Neh2 domain is sufficient for recognition, ubiquitination, and proteasomal degradation of Neh2-luciferase fusion protein. The Neh2-luc reporter system allows direct monitoring of the adaptive response to redox stress and classification of drugs based on the time course of reporter activation. The reporter was used to screen the Spectrum library of 2000 biologically active compounds to identify activators of Nrf2. The most robust and yet nontoxic Nrf2 activators found - nordihydroguaiaretic acid, fisetin, and gedunin - induced astrocyte-dependent neuroprotection from oxidative stress via an Nrf2-dependent mechanism.

Original languageEnglish (US)
Pages (from-to)752-765
Number of pages14
JournalChemistry and Biology
Volume18
Issue number6
DOIs
StatePublished - Jun 24 2011

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

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  • Cite this

    Smirnova, N. A., Haskew-Layton, R. E., Basso, M., Hushpulian, D. M., Payappilly, J. B., Speer, R. E., Ahn, Y. H., Rakhman, I., Cole, P. A., Pinto, J. T., Ratan, R. R., & Gazaryan, I. G. (2011). Development of Neh2-luciferase reporter and its application for high throughput screening and real-time monitoring of Nrf2 activators. Chemistry and Biology, 18(6), 752-765. https://doi.org/10.1016/j.chembiol.2011.03.013