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Development of a High-Throughput Cul3-Keap1 Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay for Identifying Nrf2 Activators.
Poore, Derek D; Hofmann, Glenn; Wolfe, Lawrence A; Qi, Hongwei; Jiang, Ming; Fischer, Michael; Wu, Zining; Sweitzer, Thomas D; Chakravorty, Subhas; Donovan, Brian; Li, Hu.
Affiliation
  • Poore DD; 1 Screening, Profiling and Mechanistic Biology, Platform Technology and Science, GlaxoSmithKline, Collegeville, PA, USA.
  • Hofmann G; 1 Screening, Profiling and Mechanistic Biology, Platform Technology and Science, GlaxoSmithKline, Collegeville, PA, USA.
  • Wolfe LA; 1 Screening, Profiling and Mechanistic Biology, Platform Technology and Science, GlaxoSmithKline, Collegeville, PA, USA.
  • Qi H; 2 Protein, Cellular and Structural Sciences, Platform Technology and Science, GlaxoSmithKline, Collegeville, PA, USA.
  • Jiang M; 1 Screening, Profiling and Mechanistic Biology, Platform Technology and Science, GlaxoSmithKline, Collegeville, PA, USA.
  • Fischer M; 1 Screening, Profiling and Mechanistic Biology, Platform Technology and Science, GlaxoSmithKline, Collegeville, PA, USA.
  • Wu Z; 1 Screening, Profiling and Mechanistic Biology, Platform Technology and Science, GlaxoSmithKline, Collegeville, PA, USA.
  • Sweitzer TD; 2 Protein, Cellular and Structural Sciences, Platform Technology and Science, GlaxoSmithKline, Collegeville, PA, USA.
  • Chakravorty S; 3 Computational Biology, Target Sciences, GlaxoSmithKline, Collegeville, PA, USA.
  • Donovan B; 1 Screening, Profiling and Mechanistic Biology, Platform Technology and Science, GlaxoSmithKline, Collegeville, PA, USA.
  • Li H; 1 Screening, Profiling and Mechanistic Biology, Platform Technology and Science, GlaxoSmithKline, Collegeville, PA, USA.
SLAS Discov ; 24(2): 175-189, 2019 02.
Article in En | MEDLINE | ID: mdl-30383469
ABSTRACT
Nrf2, a master regulator of the phase II gene response to stress, is kept at low concentrations in the cell through binding to Keap1, an adaptor protein for the Cul3 ubiquitin ligase complex. To identify Nrf2 activators, two separate time-resolved fluorescence resonance energy transfer (TR-FRET) assays were developed to monitor the binding of Nrf2-Keap1 and Cul3-Keap1, respectively. The triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl] imidazole (CDDO-Im) and its analogs, exhibited approximately 100-fold better potency in the Cul3-Keap1 assay than in the Nrf2-Keap1 assay, and this difference was more profound at 37 °C than at room temperature in the Nrf2-Keap1 assay, but this phenomenon was not observed in the Cul3-Keap1 assay. A full diversity screen of approximately 2,200,000 GSK compounds was run with the Cul3-Keap1 TR-FRET assay and multiple chemical series were identified and characterized.
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Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Fluorescence Resonance Energy Transfer / Cullin Proteins / NF-E2-Related Factor 2 / High-Throughput Screening Assays / Kelch-Like ECH-Associated Protein 1 Type of study: Prognostic_studies Limits: Humans Language: En Journal: SLAS Discov Year: 2019 Type: Article Affiliation country: United States
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Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Fluorescence Resonance Energy Transfer / Cullin Proteins / NF-E2-Related Factor 2 / High-Throughput Screening Assays / Kelch-Like ECH-Associated Protein 1 Type of study: Prognostic_studies Limits: Humans Language: En Journal: SLAS Discov Year: 2019 Type: Article Affiliation country: United States