Development of a High-Throughput Cul3-Keap1 Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay for Identifying Nrf2 Activators

被引：5
|
作者：
Poore, Derek D. [1 ]
Hofmann, Glenn [1 ]
Wolfe, Lawrence A., III [1 ]
Qi, Hongwei [2 ]
Jiang, Ming [1 ]
Fischer, Michael [1 ]
Wu, Zining [1 ]
Sweitzer, Thomas D. [2 ]
Chakravorty, Subhas [3 ]
Donovan, Brian [1 ]
Li, Hu [1 ]
机构：
[1] GlaxoSmithKline, Screening Profiling & Mechanist Biol, Platform Technol & Sci, 1250 South Collegeville Rd, Collegeville, PA 19426 USA
[2] GlaxoSmithKline, Prot Cellular & Struct Sci, Platform Technol & Sci, Collegeville, PA 19426 USA
[3] GlaxoSmithKline, Computat Biol, Target Sci, Collegeville, PA 19426 USA
来源：
SLAS DISCOVERY | 2019年 / 24卷 / 02期
基金：
美国国家卫生研究院;
关键词：
high-throughput screening; TR-FRET; assay development; compound screening; ANTIOXIDANT RESPONSE ELEMENT; CUL3-BASED E3 LIGASE; OXIDATIVE STRESS; PROTEASOMAL DEGRADATION; PHASE-2; RESPONSE; SENSOR KEAP1; DLG MOTIFS; PROTEIN; INDUCTION; ELECTROPHILE;
D O I：
10.1177/2472555218807698
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
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 degrees 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.
引用
收藏
页码：175 / 189
页数：15
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