SummaryMitophagy 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.

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.

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.

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.

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.