ABSTRACT
Inhibitors of Kelch-like ECH-associated protein 1 (Keap1) increase the activity of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) by stalling its ubiquitination and degradation. This enhances the expression of genes encoding proteins involved in drug detoxification, redox homeostasis, and mitochondrial function. Nrf2 activation offers a potential therapeutic approach for conditions including Alzheimer's and Parkinson's diseases, vascular inflammation, and chronic obstructive airway disease. Non-electrophilic Keap1-Nrf2 protein-protein interaction (PPI) inhibitors may have improved toxicity profiles and different pharmacological properties to cysteine-reactive electrophilic inhibitors. Here, we describe and characterize a series of phenyl bis-sulfonamide PPI inhibitors that bind to Keap1 at submicromolar concentrations. Structural studies reveal that the compounds bind to Keap1 in a distinct "peptidomimetic" conformation that resembles the Keap1-Nrf2 ETGE peptide complex. This is different to other small molecule Keap1-Nrf2 PPI inhibitors, including bicyclic aryl bis-sulfonamides, offering a starting point for new design approaches to Keap1 inhibitors.
Subject(s)
NF-E2-Related Factor 2 , Sulfonamides , Kelch-Like ECH-Associated Protein 1/metabolism , NF-E2-Related Factor 2/metabolism , Protein Binding , Sulfonamides/pharmacologyABSTRACT
Cancer chemoprevention is an important strategy to prevent, reverse, or suppress the development of cancer. One of the target pathways that has emerged in recent years is the Keap1-Nrf2-ARE system that regulates the protection of cells against various carcinogens and their metabolites. Increased concentrations of the redox transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) induces the activation of antioxidant and phase 2 detoxifying genes. Nrf2 is regulated by substrate adaptor protein Kelch-like ECH-associated protein 1 (Keap1) that can target Nrf2 for ubiquitination and degradation by the proteasome. The interaction between Nrf2 and Keap1 can be disrupted at the protein-protein interface in order to increase Nrf2 activity for potential therapeutic purposes. This chapter describes a protocol for a steady-state fluorescence or Förster resonance energy transfer (FRET) assay to examine the Keap1-Nrf2 protein-protein interaction (PPI), to investigate the effects of Nrf2 mutations on Keap1 binding and finally to identify potential inhibitors of this PPI. In the assay system Keap1 is conjugated to an YFP protein at the N-terminus whereas an Nrf2-derived 16-mer peptide containing a high-affinity "ETGE" motif is conjugated to a CFP protein at the N-terminus.
Subject(s)
Chemoprevention , Fluorescence Resonance Energy Transfer/methods , Neoplasms/prevention & control , Amino Acid Motifs , Green Fluorescent Proteins/genetics , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Kelch-Like ECH-Associated Protein 1 , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Neoplasms/metabolism , Transformation, GeneticABSTRACT
The transcription factor Nrf2 regulates the expression of a large network of cytoprotective and metabolic enzymes and proteins. Compounds that directly and reversibly inhibit the interaction between Nrf2 and its main negative regulator Keap1 are potential pharmacological agents for a range of disease types including neurodegenerative conditions and cancer. We describe the development of a series of 1,4-diphenyl-1,2,3-triazole compounds that inhibit the Nrf2-Keap1 protein-protein interaction (PPI) in vitro and in live cells and up-regulate the expression of Nrf2-dependent gene products.
Subject(s)
Heme Oxygenase-1/biosynthesis , Intracellular Signaling Peptides and Proteins/metabolism , NAD(P)H Dehydrogenase (Quinone)/biosynthesis , NF-E2-Related Factor 2/metabolism , Triazoles/chemistry , Cell Line, Tumor , Click Chemistry , Computer Simulation , Databases, Chemical , Dose-Response Relationship, Drug , Fluorescence Polarization , HEK293 Cells , Heme Oxygenase-1/genetics , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Isothiocyanates/chemistry , Isothiocyanates/pharmacology , Kelch-Like ECH-Associated Protein 1 , Molecular Docking Simulation , NAD(P)H Dehydrogenase (Quinone)/genetics , NF-E2-Related Factor 2/chemistry , Protein Binding , Structure-Activity Relationship , Sulfoxides , Triazoles/chemical synthesis , Triazoles/pharmacologyABSTRACT
Mitophagy is central to mitochondrial and cellular homeostasis and operates via the PINK1/Parkin pathway targeting mitochondria devoid of membrane potential (ΔΨm) to autophagosomes. Although mitophagy is recognized as a fundamental cellular process, selective pharmacologic modulators of mitophagy are almost nonexistent. We developed a compound that increases the expression and signaling of the autophagic adaptor molecule P62/SQSTM1 and forces mitochondria into autophagy. The compound, P62-mediated mitophagy inducer (PMI), activates mitophagy without recruiting Parkin or collapsing ΔΨm and retains activity in cells devoid of a fully functional PINK1/Parkin pathway. PMI drives mitochondria to a process of quality control without compromising the bio-energetic competence of the whole network while exposing just those organelles to be recycled. Thus, PMI circumvents the toxicity and some of the nonspecific effects associated with the abrupt dissipation of ΔΨm by ionophores routinely used to induce mitophagy and represents a prototype pharmacological tool to investigate the molecular mechanisms of mitophagy.
Subject(s)
Mitochondria/metabolism , Mitophagy/drug effects , Triazoles/pharmacology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Antioxidant Response Elements , Cell Line , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Membrane Potential, Mitochondrial/drug effects , Mice , Microtubule-Associated Proteins/metabolism , NF-E2-Related Factor 2/metabolism , Protein Kinases/deficiency , Protein Kinases/genetics , Protein Kinases/metabolism , RNA Interference , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Sequestosome-1 Protein , Signal Transduction/drug effects , Triazoles/chemistry , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitination/drug effects , Up-Regulation/drug effectsABSTRACT
One of the strategies proposed for the chemoprevention of degenerative diseases and cancer involves upregulation of antioxidant and free radical detoxification gene products by increasing the intracellular concentration of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). This can be achieved by disrupting the interaction between Nrf2 and Kelch-like ECH associated protein 1 (Keap1), a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Here, we describe the development of a high-throughput fluorescence (or Förster) resonance energy transfer assay for the identification of inhibitors of the Keap1-Nrf2 protein-protein interaction (PPI). The basis of this assay is the binding of a YFP-conjugated Keap1 Kelch binding domain to a CFP-conjugated Nrf2-derived 16-mer peptide containing a highly conserved "ETGE" motif. The competition aspect of the assay was validated using unlabeled Nrf2-derived 7-mer and 16-mer peptides and has potential as a screening tool for small molecule inhibitors of the PPI. We discuss the development of this assay in the context of other methods used to evaluate this PPI.
Subject(s)
Fluorescence Resonance Energy Transfer/methods , Intracellular Signaling Peptides and Proteins/metabolism , NF-E2-Related Factor 2/metabolism , Peptides/chemistry , Peptides/metabolism , Protein Interaction Domains and Motifs , Amino Acid Motifs , Binding, Competitive , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Kelch-Like ECH-Associated Protein 1 , NF-E2-Related Factor 2/chemistry , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Staining and LabelingABSTRACT
Inhibitors of the Keap1-Nrf2 protein-protein interaction (PPI) have been proposed as potential anti-inflammatory and cancer chemopreventive agents. Such compounds have the potential to increase the intracellular concentrations of Nrf2 in a reversible manner and consequently increase the expression of a battery of gene products with antioxidant response elements (AREs) in their promoter region. In this manuscript we describe the development of peptide inhibitors with modified C- and N-termini and reduced overall charge. The activity of the compounds in inhibiting the PPI and in cellular assays of Nrf2 function are described. Compound 10 has potent activity (IC50 = 22 nM) in a cell-free fluorescence polarisation assay and induced the expression of Nrf2 dependent gene products in cells, suggesting that it has potential as a lead molecule for the development of peptidomimetic inhibitors.
