Screening for natural chemoprevention agents that modify human Keap1

Upregulation of cytoprotective enzymes by therapeutic agents to prevent damage by reactive oxygen species and xenobiotic electrophiles is a strategy for cancer chemoprevention. The Kelch-like ECH-associated protein 1 (Keap1) and its binding partner, transcription factor NF-E2-related factor-2 (NRF2)...

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Bibliographic Details
Published in:Analytical biochemistry 2012-02, Vol.421 (1), p.108-114
Main Authors: Hu, Chenqi, Nikolic, Dejan, Eggler, Aimee L., Mesecar, Andrew D., van Breemen, Richard B.
Format: Article
Language:English
Subjects:
Anticarcinogenic Agents - pharmacology
antioxidants
Antioxidants - pharmacology
beta-mercaptoethanol
Biological Products - pharmacology
Cacao - chemistry
Chemoprevention
Chromatography, Liquid
desorption
Drug Evaluation, Preclinical - methods
enzymes
Humans
In Vitro Techniques
Intracellular Signaling Peptides and Proteins - chemistry
Intracellular Signaling Peptides and Proteins - drug effects
Isoliquiritigenin
Keap1
Kelch-Like ECH-Associated Protein 1
Mass spectrometry
matrix-assisted laser desorption-ionization mass spectrometry
Mercaptoethanol
Nrf2
oxidative stress
Plant Extracts - pharmacology
reactive oxygen species
Recombinant Proteins - chemistry
Recombinant Proteins - drug effects
screening
Spectrometry, Mass, Electrospray Ionization
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Sulforaphane
Tandem Mass Spectrometry
transcription factors
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Summary:Upregulation of cytoprotective enzymes by therapeutic agents to prevent damage by reactive oxygen species and xenobiotic electrophiles is a strategy for cancer chemoprevention. The Kelch-like ECH-associated protein 1 (Keap1) and its binding partner, transcription factor NF-E2-related factor-2 (NRF2), are chemoprevention targets because of their role in regulating the antioxidant response element (ARE) in response to oxidative stress and exposure to electrophiles. Modification of the sensor protein Keap1 by electrophiles such as the isothiocyanate sulforaphane can direct Nrf2 accumulation in the nucleus and subsequent ARE activation. Since our previous matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS)-based screening method to discover natural products that modify Keap1 does not detect covalent modification of Keap1 by some highly reversible agents such as sulforaphane, a more sensitive screening assay was developed. In this new assay, electrophiles that have reversibly modified Keap1 can be released, trapped, and detected as β-mercaptoethanol adducts by mass spectrometry. Isoliquiritigenin and sulforaphane, known ARE activators that target Keap1, were used to validate the assay. To determine the ability of the assay to identify electrophiles in complex matrixes that modify Keap1, sulforaphane was spiked into a cocoa extract, and LC–MS/MS using high resolution mass spectrometry with accurate mass measurement was used to identify β-mercaptoethanol adducts of sulforaphane that had been released from Keap1. This screening assay permits identification of potential chemoprevention agents in complex natural product mixtures that reversibly modify Keap1 but cannot be detected using MALDI-TOF MS.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2011.10.028