TARGETING THE NRF2/KEAP1 INTERACTION
type: CreativeWork
description: The Nrf2/Keap1 protein-protein interaction (PPI) regulates activity of the Nrf2 antioxidant and anti-inflammatory pathway. The transcription factor Nrf2 has been found to be a key mediator in the resolution of inflammation and the progression of chronic diseases. Most known inducers of the Nrf2 pathway act by covalent modification of Keap1 via electrophilic functional groups. Controlled induction of the Nrf2 pathway via specific disruption of the Nrf2/Keap1 interaction is an attractive therapeutic target. This work describes a cell penetrating, TAT-Nrf2 peptide which targets the Nrf2/Keap1 interaction in vitro. Induction of downstream genes is both sequence and dose dependent. In an established model of bacterial sepsis, the peptide reduces pro-inflammatory mediators. Investigation of both cell penetrating and Keap1 binding sequences has identified the requirements for effective Nrf2 induction in cell based assays. An in vitro purified protein, fluorescence polarisation (FP) assay was established in order to rapidly characterise these peptides. Based on the secondary structure of the Keap1 binding portion of Nrf2, further peptides were designed to constrain the conformation and mimic the full protein, while reducing overall size. Synthesis of cyclic peptides has identified the minimal sequence required for efficient binding and provides significant improvement in affinity over linear sequences. Several macrocyclisation techniques were explored in an attempt to retain biological activity, without the need for cell penetration sequences. Initially, disulfide bridge formation was used to produce peptides with affinities for Keap1 similar to the TAT-Nrf2 peptides at considerably reduced size. Subsequently, both head-to-tail cyclisation and peptide stapling were examined in order to restore potency in cell based assays. Finally, an alternative method for identification of Nrf2/Keap1 disruptors was explored. In silico docking calculations were used to identify potential n…
abstract: The Nrf2/Keap1 protein-protein interaction (PPI) regulates activity of the Nrf2 antioxidant and anti-inflammatory pathway. The transcription factor Nrf2 has been found to be a key mediator in the resolution of inflammation and the progression of chronic diseases. Most known inducers of the Nrf2 pathway act by covalent modification of Keap1 via electrophilic functional groups. Controlled induction of the Nrf2 pathway via specific disruption of the Nrf2/Keap1 interaction is an attractive therapeutic target. This work describes a cell penetrating, TAT-Nrf2 peptide which targets the Nrf2/Keap1 interaction in vitro. Induction of downstream genes is both sequence and dose dependent. In an established model of bacterial sepsis, the peptide reduces pro-inflammatory mediators. Investigation of both cell penetrating and Keap1 binding sequences has identified the requirements for effective Nrf2 induction in cell based assays. An in vitro purified protein, fluorescence polarisation (FP) assay was established in order to rapidly characterise these peptides. Based on the secondary structure of the Keap1 binding portion of Nrf2, further peptides were designed to constrain the conformation and mimic the full protein, while reducing overall size. Synthesis of cyclic peptides has identified the minimal sequence required for efficient binding and provides significant improvement in affinity over linear sequences. Several macrocyclisation techniques were explored in an attempt to retain biological activity, without the need for cell penetration sequences. Initially, disulfide bridge formation was used to produce peptides with affinities for Keap1 similar to the TAT-Nrf2 peptides at considerably reduced size. Subsequently, both head-to-tail cyclisation and peptide stapling were examined in order to restore potency in cell based assays. Finally, an alternative method for identification of Nrf2/Keap1 disruptors was explored. In silico docking calculations were used to identify potential n…
author: Steel, Richard James
language: en
date published: 2014-01
date modified: 2026-07-24T02:12:01Z
provider: University of East Anglia
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of East Anglia