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

Chem Biol. 2011 Jun 24;18(6):752-65. doi: 10.1016/j.chembiol.2011.03.013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Cell Line, Tumor
  • Computer Simulation
  • Flavonoids / chemistry
  • Flavonoids / pharmacology
  • Genes, Reporter*
  • High-Throughput Screening Assays*
  • Humans
  • Intracellular Signaling Peptides and Proteins / antagonists & inhibitors
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Kelch-Like ECH-Associated Protein 1
  • Kinetics
  • Limonins / chemistry
  • Limonins / pharmacology
  • Luciferases / genetics
  • Luciferases / metabolism
  • Masoprocol / chemistry
  • Masoprocol / pharmacology
  • NF-E2-Related Factor 2 / agonists
  • NF-E2-Related Factor 2 / genetics
  • NF-E2-Related Factor 2 / metabolism*
  • Oxidative Stress
  • Protein Structure, Tertiary
  • RNA Interference
  • RNA, Small Interfering / metabolism
  • Recombinant Fusion Proteins / agonists
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Structure-Activity Relationship
  • Ubiquitination

Substances

  • Flavonoids
  • Intracellular Signaling Peptides and Proteins
  • KEAP1 protein, human
  • Kelch-Like ECH-Associated Protein 1
  • Limonins
  • NF-E2-Related Factor 2
  • RNA, Small Interfering
  • Recombinant Fusion Proteins
  • gedunin
  • Masoprocol
  • Luciferases
  • fisetin