Development of a Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay for the Inhibition of Keap1-Nrf2 Protein-Protein Interaction.
Development of a Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay for the Inhibition of Keap1-Nrf2 Protein-Protein Interaction.
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开发均匀的时间分辨荧光共振能量转移（TR-FRET）测定，以抑制Keap1-NRF2蛋白 - 蛋白质相互作用。
DOI：
10.1177/2472555220935816
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发表时间：
2021-01
期刊：
影响因子：
3.1
通讯作者：
Hu, Longqin
中科院分区：
文献类型：
--
作者：
Lee, Sumi;Abed, Dhulfiqar Ali;Beamer, Lesa J.;Hu, Longqin
转录因子Nrf2通过Keap1-Nrf2-ARE途径在调节抗氧化防御系统中发挥重要作用。针对Keap1-Nrf2蛋白-蛋白相互作用(PPI)的小分子抑制剂降低了26S蛋白酶体对Nrf2的降解速度，从而增加了Nrf2的细胞内水平，Nrf2移位到细胞核中，导致细胞保护酶和抗氧化酶的表达上调。这些抑制剂可以被开发成潜在的氧化损伤引起的疾病的预防和治疗药物。为了更有效地通过抑制Keap1-Nrf2 PPI来识别潜在的Nrf2激活剂，本工作建立了一种均相时间分辨荧光共振能量转移(TRRET)分析方法，该方法以Tb-抗His抗体间接标记Keap1 Kelch结构域蛋白为供体，以FITC标记的9聚Nrf2肽胺为受体，该荧光探针与早期荧光偏振分析(FP)中使用的荧光探针相同。在与已知的Keap1-Nrf2 PPI小分子抑制剂的tr-fret竞争试验中，优化了包括各组分的浓度、缓冲液类型和孵育时间在内的检测条件。在优化的条件下，Keap1-Nrf2 TrFRET法具有较高的灵敏度、较高的动态范围和较高的稳定性，其Z‘因子为0.82，适合于高通量筛选和优化Keap1-Nrf2 PPI的抑制剂。此外，TRRET分析能够将Keap1-Nrf2 PPI的有效抑制剂区分为亚纳米分子。
The transcription factor Nrf2 plays a major role in regulating the antioxidant defense system through the Keap1-Nrf2-ARE pathway. Small molecule inhibitors targeting Keap1-Nrf2 protein-protein interaction (PPI) decrease the rate of Nrf2 degradation by the 26S proteasome and thus increase the intracellular level of Nrf2, which translocates into the nucleus leading to upregulated expression of cytoprotective and antioxidant enzymes. Such inhibitors can be developed into potential preventive and therapeutic agents of diseases caused by oxidative damage. In order to more effectively identify promising Nrf2 activators through the inhibition of Keap1-Nrf2 PPI, a homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) assay was developed in this work by indirectly labeling the Keap1 Kelch domain protein with Tb-anti-His antibody as the donor and using, as the acceptor, FITC-labeled 9mer Nrf2 peptide amide, the same fluorescent probe that has been used in the earlier fluorescence polarization (FP) assay. Assay conditions including concentrations of the various components, buffer type and incubation time were optimized in the TR-FRET competition assay with known small molecule inhibitors of Keap1-Nrf2 PPI. Under the optimized conditions, the Keap1-Nrf2 TR-FRET assay exhibited great sensitivity with a high dynamic range and considerable stability up to 5 h. The Z' factor was determined to be 0.82, suggesting that the assay is suitable for high-throughput screening and lead optimization of inhibitors of Keap1-Nrf2 PPI. Furthermore, the TR-FRET assay is capable of differentiating potent inhibitors of Keap1-Nrf2 PPI down to subnanomolar .
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DOI：
10.1007/4243_2008_021
发表时间：
2008-01-01
期刊：
STANDARDIZATION AND QUALITY ASSURANCE IN FLUORESCENCE MEASUREMENTS I: TECHNIQUES
影响因子：
--
作者：
Goulko, Alevtina A.;Zhao, Qiang;Le, X. Chris
通讯作者：
Le, X. Chris
影响因子：
16
作者：
Baird, Liam;Swift, Sam;Lleres, David;Dinkova-Kostova, Albena T.
通讯作者：
Dinkova-Kostova, Albena T.
影响因子：
10.5
作者：
Itoh, K;Wakabayashi, N;Yamamoto, M
通讯作者：
Yamamoto, M
影响因子：
--
作者：
Huang, XY
通讯作者：
Huang, XY
影响因子：
6.3
作者：
Lea WA;Simeonov A
通讯作者：
Simeonov A