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

Fingerprint

NF-E2-Related Factor 2
Luciferases
Screening
Throughput
Monitoring
Masoprocol
Firefly Luciferases
Detoxification
Oxidative stress
Ubiquitination
Astrocytes
Oxidation-Reduction
Oxidative Stress
Fusion reactions
Stabilization
Antioxidants
Chemical activation
Degradation
Pharmaceutical Preparations
Proteins

ASJC Scopus subject areas

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

Cite this

Smirnova, N. A., Haskew-Layton, R. E., Basso, M., Hushpulian, D. M., Payappilly, J. B., Speer, R. E., ... 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

Development of Neh2-luciferase reporter and its application for high throughput screening and real-time monitoring of Nrf2 activators. / Smirnova, Natalya A.; Haskew-Layton, Renee E.; Basso, Manuela; Hushpulian, Dmitry M.; Payappilly, Jimmy B.; Speer, Rachel E.; Ahn, Young Hoon; Rakhman, Ilay; Cole, Philip A.; Pinto, John T.; Ratan, Rajiv R.; Gazaryan, Irina G.

In: Chemistry and Biology, Vol. 18, No. 6, 24.06.2011, p. 752-765.

Research output: Contribution to journalArticle

Smirnova, NA, Haskew-Layton, RE, Basso, M, Hushpulian, DM, Payappilly, JB, Speer, RE, Ahn, YH, Rakhman, I, Cole, PA, Pinto, JT, Ratan, RR & Gazaryan, IG 2011, 'Development of Neh2-luciferase reporter and its application for high throughput screening and real-time monitoring of Nrf2 activators', Chemistry and Biology, vol. 18, no. 6, pp. 752-765. https://doi.org/10.1016/j.chembiol.2011.03.013
Smirnova, Natalya A. ; Haskew-Layton, Renee E. ; Basso, Manuela ; Hushpulian, Dmitry M. ; Payappilly, Jimmy B. ; Speer, Rachel E. ; Ahn, Young Hoon ; Rakhman, Ilay ; Cole, Philip A. ; Pinto, John T. ; Ratan, Rajiv R. ; Gazaryan, Irina G. / Development of Neh2-luciferase reporter and its application for high throughput screening and real-time monitoring of Nrf2 activators. In: Chemistry and Biology. 2011 ; Vol. 18, No. 6. pp. 752-765.
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