Investigating a PROTAC approach to NRF2 activation

Robinson, William (2025) Investigating a PROTAC approach to NRF2 activation. Masters thesis, University of East Anglia.

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Abstract

Nuclear factor erythroid-2 related factor 2 (NRF2) is a transcription factor central to cellular defence against oxidative stress and inflammation. Under basal conditions, NRF2 is constitutively degraded by Kelch-like ECH-associated protein 1 (Keap1), which facilitates NRF2 ubiquitination and proteasomal degradation. Disruption of the Keap1-NRF2 protein-protein interaction (PPI) stabilises NRF2, allowing for nuclear accumulation and binding to the antioxidant response element of genes, including heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO1) amongst others. Small molecules and peptide-based protein-protein interaction (PPI) inhibitors targeting this pathway have demonstrated anti-inflammatory activity in vitro and in vivo. TAT-14, a previously reported peptide PPI inhibitor, binds Keap1 with nanomolar affinity and upregulates NRF2/ARE genes. More recently, proteolysis-targeting chimeras (PROTACs) have emerged as a novel strategy to activate NRF2 by degrading Keap1, offering enhanced potency, selectivity and duration of action compared to PPI inhibitors and small molecules.

In this project, TAT-14 was synthesised by Fmoc solid-phase peptide synthesis and confirmed to increase NRF2 protein expression and induce HO-1 and NQO1 mRNA expression with minimal cytotoxicity, consistent with previous reports. TAT-14 was therefore employed in PROTAC design to function as the Keap1-binding warhead. The VHL-recruiting ligand VH032 was successfully synthesised and linker optimisation enabled conjugation in PROTAC development. Synthesis of PROTAC1, which incorporated a PEG linker, was unsuccessful, whereas replacement with a glutaric anhydride-derived alkyl linker yielded PROTAC2 which was purified and characterised. In vitro, PROTAC2 elicited greater HO-1 and NQO1 mRNA induction than TAT-14, although with notable cytotoxicity found. These findings provide evidence that Keap1-targeting peptide PPI inhibitors can be adapted to PROTAC design to activate NRF2, although further studies are required to confirm robust NRF2 activation through Keap1 degradation.

Item Type:	Thesis (Masters)
Faculty \ School:	Faculty of Science > School of Chemistry, Pharmacy and Pharmacology
Depositing User:	Chris White
Date Deposited:	19 Feb 2026 15:03
Last Modified:	19 Feb 2026 15:03
URI:	https://ueaeprints.uea.ac.uk/id/eprint/101989
DOI:

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