Development of a steady-state FRET-based assay to identify inhibitors of the Keap1-Nrf2 protein-protein interaction.

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.

Peptide inhibitors of the Keap1-Nrf2 protein-protein interaction with improved binding and cellular activity.

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.

Peptide inhibitors of the Keap1-Nrf2 protein-protein interaction.

Disruption of the interaction between the ubiquitination facilitator protein Keap1 and the cap'n'collar basic-region leucine-zipper transcription factor Nrf2 is a potential strategy to enhance expression of antioxidant and free radical detoxification gene products regulated by Nrf2. Agents that disrupt this protein-protein interaction may be useful pharmacological probes and future cancer-chemopreventive agents. We describe the structure-activity relationships for a series of peptides based upon regions of the Nrf2 Neh2 domain, of varying length and sequence, that interact with the Keap1 Kelch domain and disrupt the interaction with Nrf2. We have also investigated sequestosome-1 (p62) and prothymosin-α sequences that have been reported to interact with Keap1. To achieve this we have developed a high-throughput fluorescence polarization (FP) assay to screen inhibitors. In addition to screening synthetic peptides, we have used a phage display library approach to identify putative peptide ligands with non-native sequence motifs. Candidate peptides from the phage display library screening protocol were evaluated in the FP assay to quantify their binding activity. Hybrid peptides based upon the Nrf2 "ETGE" motif and the sequestosome-1 "Keap1-interaction region" have superior binding activity compared to either native peptide alone.