Nuclear factor (erythroid-derived 2)-like 2 (NRF2) drug discovery: Biochemical toolbox to develop NRF2 activators by reversible binding of Kelch-like ECH-associated protein 1 (KEAP1)

Arch Biochem Biophys. 2017 Oct 1;631:31-41. doi: 10.1016/j.abb.2017.08.003. Epub 2017 Aug 8.

Abstract

Mechanisms that activate innate antioxidant responses, as a way to mitigate oxidative stress at the site of action, hold much therapeutic potential in diseases, such as Parkinson's disease, Alzheimer's disease and Huntington's disease, where the use of antioxidants as monotherapy has not yielded positive results. The nuclear factor NRF2 is a transcription factor whose activity upregulates the expression of cell detoxifying enzymes in response to oxidative stress. NRF2 levels are modulated by KEAP1, a sensor of oxidative stress. KEAP1 binds NRF2 and facilitates its ubiquitination and subsequent degradation. Recently, compounds that reversibly disrupt the NRF2-KEAP1 interaction have been described, opening the field to a new era of safer NRF2 activators. This paper describes a set of new, robust and informative biochemical assays that enable the selection and optimization of non-covalent KEAP1 binders. These include a time-resolved fluorescence resonance energy transfer (TR-FRET) primary assay with high modularity and robustness, a surface plasmon resonance (SPR) based KEAP1 direct binding assay that enables the quantification and analysis of full kinetic binding parameters and finally a 1H-15N heteronuclear single quantum coherence (HSQC) NMR assay suited to study the interaction surface of KEAP1 with residue-specific information to validate the interaction of ligands in the KEAP1 binding site.

Keywords: Antioxidant; Biochemical methods; Drug discovery; KEAP1; NRF2; Neurodegeneration; Protein-protein interaction.

Publication types

  • Review

MeSH terms

  • Amino Acid Sequence
  • Antioxidants / chemistry
  • Antioxidants / pharmacology*
  • Binding Sites
  • Drug Discovery / methods*
  • Fluorescence Resonance Energy Transfer / methods
  • Humans
  • Kelch Repeat / drug effects
  • Kelch-Like ECH-Associated Protein 1 / chemistry
  • Kelch-Like ECH-Associated Protein 1 / metabolism*
  • Ligands
  • Magnetic Resonance Spectroscopy / methods
  • Models, Molecular
  • NF-E2-Related Factor 2 / agonists*
  • NF-E2-Related Factor 2 / metabolism*
  • Oxidative Stress / drug effects
  • Protein Interaction Maps / drug effects*
  • Small Molecule Libraries / chemistry
  • Small Molecule Libraries / pharmacology
  • Surface Plasmon Resonance / methods

Substances

  • Antioxidants
  • KEAP1 protein, human
  • Kelch-Like ECH-Associated Protein 1
  • Ligands
  • NF-E2-Related Factor 2
  • NFE2L2 protein, human
  • Small Molecule Libraries