Design and Synthesis of Inhibitors of the Nrf2/MafG Protein-Protein Interaction as a Research Probe.

Hyde, Ellen Elizabeth (2025) Design and Synthesis of Inhibitors of the Nrf2/MafG Protein-Protein Interaction as a Research Probe. Doctoral thesis, University of East Anglia.

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Abstract

Nuclear erythroid factor 2 (Nrf2) and v-maf musculoaponeurotic fibrosarcoma oncogene family protein G (MafG) are basic leucine zipper (bZIP) transcription factors. A coiled-coil protein-protein interaction (PPI) forms between the leucine zipper domains of Nrf2 and MafG, stabilising Nrf2/DNA binding for activation of gene transcription. Nrf2 is responsible for regulating oxidative stress, controlling gene expression of antioxidant and cytoprotective activity in cells. The overexpression of Nrf2 is linked to the development of chemoresistance in non-small cell lung cancer (NSCLC). Consequently, there is a need for developing inhibitors of Nrf2 as an anti-cancer therapeutic.

In Chapter 1 the discovery of PPI inhibitors is outlined, highlighting the techniques used for different categories of interaction. A literature review of existing human PPI inhibitors is presented, demonstrating a need to apply new approaches towards the design of coiled-coil inhibitors. This thesis presents research into novel inhibitors of the coiled-coil interaction between Nrf2 and MafG.

In the absence of an experimentally resolved structure of the Nrf2/MafG PPI, AlphaFold offers a new method for studying the Nrf2/MafG PPI. Applying this approach, Chapter 2 reports the design and synthesis of peptides derived from the MafG leucine zipper, exploring the minimum sequence length required to afford inhibition. Using recombinantly expressed Nrf2 and MafG protein, described in Chapter 5, fluorescence polarisation, electrophoretic mobility shift assays and surface plasmon resonance assays were investigated, to evaluate peptide activity towards the Nrf2/MafG PPI. A lead 21-mer peptide was found capable of disrupting the PPI (IC50 of 36 μM) by fluorescence polarisation assay.

In Chapter 3, the lead peptide was applied to peptide-directed ligand design to explore small molecule fragments capable of disrupting the coiled-coil interaction. Peptide-small molecule hybrids were synthesised and screened by fluorescence polarisation assay, with two hybrid compounds capable of disrupting ternary complex formation between Nrf2/MafG/DNA (IC50 of 62 and 125 μM).

Inhibitory peptide and peptide-small molecule hybrids were evaluated for cellular efficacy in Chapter 4. A549 cells, a NSCLC cell line known to express high levels of Nrf2, were selected for the study. Inhibitors demonstrated suppression of NQO1 activity, a target gene of Nrf2. Excitingly, our lead peptide-small molecule hybrid was capable of sensitising resistant A549 cells to gemcitabine.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Chemistry, Pharmacy and Pharmacology
Depositing User:	Chris White
Date Deposited:	29 Jan 2026 11:10
Last Modified:	29 Jan 2026 11:10
URI:	https://ueaeprints.uea.ac.uk/id/eprint/101786
DOI:

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