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Title: The biological evaluation of inhibitors of the Keap1-Nrf2 protein-protein interaction
Author: Schaap, M. C. A.
ISNI:       0000 0004 7229 5266
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2015
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The Keap1-Nrf2 pathway has been identified as a key regulator of the cytoprotective response of cells when exposed to oxidative stress. The induction of detoxification gene products by increasing the activity of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) can be used as a chemopreventive approach to cancer treatment. Natural and synthetic agents can disrupt the interaction between the substrate adaptor protein Kelch-like ECH-associated protein 1 (Keap1) and Nrf2, which allows nuclear Nrf2 translocation and accumulation. The aim of this project was to characterize the behaviour of direct and indirect Nrf2 inducer molecules by applying several biological methods that were specifically optimised or developed for the Keap1-Nrf2 pathway. Potential inhibitors of the Keap1-Nrf2 complex were screened using a cellular NQO1 induction assay and an in vitro fluorescence polarisation (FP) assay for direct inhibitors. A novel in vitro Förster resonance energy transfer (FRET) assay was developed as an additional screening tool to identify Keap1 binding partners. Further biological evaluation was conducted with the most promising molecules using western blotting analysis and a new intracellular Nrf2 staining method for flow cytometry. The natural product and indirect Nrf2 inducer sulforaphane was used as a reference compound (NQO1 CD = 0.4 μM). From a library of Michael acceptor-containing cyclohexadienone analogues, a 3-chlorophenyl compound (6.49) was identified as a potent inducer (NQO1 CD = 1 μM). Stearoyl-capped Nrf2-derived peptides were potent inhibitors of the direct Keap1-Nrf2 protein-protein interaction and showed promising cell penetrating properties. Direct inhibitor small molecules included two bissulphonamide derivatives (7A4, NQO1 CD = 8 μM and 7B1, NQO1 CD = 4 μM) and a 1,2,3-triazole derivative (7C55, NQO1 CD = 0.6 μM) with FP IC50 values in the nano- to micromolar range. The selectivity of the compounds was assessed by revealing potential aryl hydrocarbon receptor (AhR) transcription factor cross-talk.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available