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BioAssay: AID 504540

Broad Institute HTS fluorescence polarization assay for inhibitors of Keap1-Nrf2 interaction Inhibitor Probe Project

The goal is to identify small molecules that directly disrupt the Keap1-Nrf2 interaction at the protein-protein interface. The identified compounds will offer a safer alternative that can activate Nrf2 and induce the expression of oxidative stress response enzymes. These novel selective Keap1-Nrf2 inhibitors would mimic the actions of electrophiles like isothiocyanates and Michael acceptors in more ..
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AID: 504540
Data Source: Broad Institute (2119_Inhibitor_Project)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-03-23
Modify Date: 2013-08-09

Data Table ( Complete ):           Active    All
Targets
BioActive Compound: Chemical Probe: 1    Active: 1
Depositor Specified Assays
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AIDNameTypeComment
504523Fluorescence polarization to screen for inhibitors that disrupt the protein-protein interaction between Keap1 and Nrf2 Measured in Biochemical System Using Plate Reader - 2119-01_Inhibitor_SinglePoint_HTS_ActivityscreeningPrimary HTS
651798Fluorescence polarization to screen for inhibitors that disrupt the protein-protein interaction between Keap1 and Nrf2 Measured in Biochemical System Using Plate Reader - 2119-01_Inhibitor_Dose_DryPowder_Activityconfirmatory
651801Fluorescence polarization to screen for inhibitors that disrupt the protein-protein interaction between Keap1 and Nrf2 Measured in Biochemical System Using Plate Reader - 2119-01_Inhibitor_Dose_DryPowder_Activity_Set2confirmatory
651807The inhibition of Keap1-Nrf2 interaction in cells by beta-lactamase reporter assay Measured in Cell-Based System Using Plate Reader - 2119-03_Inhibitor_Dose_DryPowder_Activity_Set4confirmatory
651833Nrf2 PathHunter Nuclear Translocation assay in U2OS cells Measured in Cell-Based System Using Plate Reader - 2119-04_Inhibitor_Dose_DryPowder_Activityconfirmatory
651860HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set2confirmatory
651829Thermal shift assay to identify compound binding to Kelch domain of Keap1 protein Measured in Biochemical System Using RT-PCR - 2119-02_Inhibitor_Dose_DryPowder_Activityconfirmatory
651858Counterscreen to eliminate false positive from thermal shift assay Measured in Biochemical System Using Plate Reader - 2119-07_Inhibitor_Dose_CherryPick_Activityconfirmatory
651834Fluorescence polarization to screen for inhibitors that disrupt the protein-protein interaction between Keap1 and Nrf2 Measured in Biochemical System Using Plate Reader - 2119-01_Inhibitor_Dose_DryPowder_Activity_Set4confirmatory
651863HEK293 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-01_Inhibitor_Dose_DryPowder_Activity_Set4confirmatory
651806Fluorescence polarization to screen for inhibitors that disrupt the protein-protein interaction between Keap1 and Nrf2 Measured in Biochemical System Using Plate Reader - 2119-01_Inhibitor_Dose_DryPowder_Activity_Set3confirmatory
651876Competitive SPR to confirm the inhibition of the compound to keap1-Nrf2 interaction Measured in Biochemical System Using Plate Reader - 2119-08_Inhibitor_Dose_DryPowder_Activityother
588683Fluorescence polarization to screen for inhibitors that disrupt the protein-protein interaction between Keap1 and Nrf2 Measured in Biochemical System Using Plate Reader - 2119-01_Inhibitor_Dose_CherryPick_Activityconfirmatory
651823Thermal shift assay to identify compound binding to Kelch domain of Keap1 protein Measured in Biochemical System Using RT-PCR - 2119-02_Inhibitor_Dose_CherryPick_Activityconfirmatory
Description:
Primary Collaborators:
Longqin Hu, Rutgers University, Piscataway, NJ, LongHu@rutgers.edu,(732) 445-5291

Project Goal:

The goal is to identify small molecules that directly disrupt the Keap1-Nrf2 interaction at the protein-protein interface. The identified compounds will offer a safer alternative that can activate Nrf2 and induce the expression of oxidative stress response enzymes. These novel selective Keap1-Nrf2 inhibitors would mimic the actions of electrophiles like isothiocyanates and Michael acceptors in the induction of cytoprotective enzymes but without the associated side effects. Such inhibitors will be useful as important pharmacological probes for the elucidation of cytoprotective pathways and as potential chemopreventive agents for cancer and other diseases in high risk populations.

Transcriptional factor Nrf2, Keap1-Nrf2 protein complex, Inhibitor, Oxidative stress, antioxidant response element (ARE)
Biological Relevance:

Keap1-Nrf2-antioxidant response element (ARE) system represents a key signaling pathway of cytoprotective gene expression against oxidative stress. The Keap1 is a 69-kDa oxidative stress "sensor" protein that forms a complex with the transcription factor Nrf2 and keeps Nrf2 in the cytosol for ubiquitination and subsequent proteosomal degradation. However, under oxidative stress conditions, Keap1 liberates Nrf2 from its repression, resulting in increased steady-state levels of Nrf2 and increased transcription of cytoprotective ARE-dependent genes. The cytoprotective gene products controlled by Nrf2 include glutathione S-transferases (GST), UDP-glucuronyl transferase, NAD(P)H:quinone oxidoreductase 1 (NQO1), epoxide hydrolase, glutamate cysteine ligase, glutathione reductase, thioredoxin, catalase, superoxide dismutase, and heme oxygenase 1 (HO1), that are important in the deactivation of reactive oxygen species and electrophiles.
Many natural products like sulforaphane, curcumin, and epigallocatechin gallate from natural sources such as fruits, vegetables, and tea products are known inducers/activators of Nrf2. All these inducers share a common chemical property: they are either chemically reactive or can be metabolized to become chemically reactive toward sulfhydryl groups. It is now believed that these inducers activate ARE through chemical modification of the cellular "sensor" protein Keap1. Treatment of cells with these inducers results in the dissociation of the Keap1-Nrf2 complex and the inhibition of proteosomal degradation of Nrf2. Multiple cysteine residues in Keap1, especially Cys257, Cys273, Cys288, and Cys297, are modified by the chemically reactive isothiocyanates and Michael acceptors, leading to conformational changes in Keap1 that are responsible for the dissociation and/or inhibition of degradation. The increase in cytosolic levels of Nrf2 would allow more Nrf2 to translocate to the nucleus, heterodimerize with small Maf, and bind to ARE, resulting in transcriptional activation of the ARE genes. Although additional factors and regulatory mechanisms exist, Keap1-Nrf2-ARE is clearly the main signaling pathway by the cysteine-reactive chemical inducers. Some of these natural and synthetic ARE inducers are being evaluated as chemopreventive agents, indicating that inhibition of Keap1-Nrf2 interaction is a sound strategy for chemoprevention. However, their chemical reactivity raises safety concerns over their long-term use as chemopreventive agents. New nonreactive direct inhibitors of Keap1-Nrf2 interaction discovered through the screening of chemical libraries could be more promising as chemopreventive agents.
In addition to carcinogenesis, the Nrf2-mediated response has also been shown to alter acute chemical toxicity, asthma, acute inflammation, septic shock and neurodegenerative diseases such as Alzheimer's and Parkinson's. Therefore, molecules that interfere with the Keap1-Nrf2 interaction could potentially be useful as chemopreventive agents for a variety of diseases and conditions including cancer, Parkinson's, and Alzheimer's. Furthermore, these novel agents could be used as pharmacological probes for the elucidation of biological processes and mechanisms involving Keap1-Nrf2.
Result Definitions
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OutcomeThe BioAssay activity outcomeOutcome
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Additional Information
Grant Number: R03 MH093197-01

Data Table (Concise)
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