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 ..
Longqin Hu, Rutgers University, Piscataway, NJ, LongHu@rutgers.edu,(732) 445-5291
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)
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.
Categorized Comment - additional comments and annotations
From MLP Probe Report:
Probe count: 1
MLP Probe ML# for probe 1: ML334
PubChem Substance ID (SID) for probe 1: 137276180
PubChem Compound ID (CID) for probe 1: 56840728
Probe type for probe 1: Inhibitor
IC50/EC50 (nM) for probe 1: 1600
Target for probe 1: Keap1 (gi: 45269145,224028257)
Disease relevance for probe 1: Cancer, inflamation
Anti-target for probe 1: Cytotoxicity
Fold selectivity for probe 1: >16
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK169452/ (ID: 3060620)
Grant number for probe 1: MH093197
PubMed Publication ID (PMID) for probe 1: 23562243
NCBI Book chapter title for probe 1: The identification and characterization of non-reactive inhibitor of Keap1-Nrf2 interaction through HTS using a fluorescence polarization assay
Data Table (Concise)