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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.

Background: Compounds that block the cellular effects of carcinogens have the potential to be cancer chemopreventive agents. A number of dietary constituents such as sulforaphane found in broccoli and curcumin found in turmeric increase the expression of antioxidant response element (ARE) genes involved in detoxifying reactive primary metabolites. The mode of action of these chemopreventive agents has been determined as an interaction with cysteine residues in the protein Keap1, this interaction causes Keap1 to release its binding partner, the transcription factor Nrf2. The free Nrf2 then translocates to the nucleus and up-regulates ARE dependent genes resulting in cellular protection against reactive species (McMahon et al., 2003). Aims: Our aim is to develop compounds that directly inhibit the Keap1-Nrf2 protein-protein interaction and thereby enhance Nrf2 activity by a mechanism different to that of existing chemopreventive agents. Such compounds will have the potential to be useful molecular probes for studying the Keap1-Nrf2 interaction and the Nrf2 signaling pathway in detail and also as candidate preventive agents for cancer. Methods: A series of linear peptides based on the high affinity binding motif of Nrf2 (the ETGE sequence) were designed, synthesized and screened to determine the optimum chain length for β-turn formation and interaction with the Keap1 protein. A second generation of peptides with changes to the sequence were synthesized to improve the binding profile. The peptides were synthesized using standard Fmoc chemistry and were purified by preparative HPLC, then lyophilized prior to use. The peptides were characterized by 1H-NMR, HPLC, LC-MS and high resolution MS. Results: The peptides were synthesized successfully and were shown to cause inhibition of the Keap1-Nrf2 protein-protein interaction, determined using a fluorescence polarization assay developed in our laboratory. A number of the sequence change peptides have equivalent or superior activity to the native Nrf2 sequence. Conclusion: In vitro tests have shown that the peptide sequence, structure and length are important for inhibition of the protein-protein interaction and the data have allowed us to propose an initial structure activity relationship for the Keap1-peptide interaction. The results and their implications for future drug design will be presented. Reference: McMahon, M., Itoh, K., Yamamoto, M., Hayes, J. (2003) J. Biol. Chem. 278, 21592-21600 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 828. doi:10.1158/1538-7445.AM2011-828