Optimization of Fluorescently Labeled Nrf2 Peptide Probes and the Development of a Fluorescence Polarization Assay for the Discovery of Inhibitors of Keap1-Nrf2 Interaction

Abstract

Activation of the antioxidant response element (ARE) up-regulates enzymes involved in detoxification of electrophiles and reactive oxygen species. The induction of ARE genes is regulated by the interaction between redox sensor protein, Keap1, and the transcription factor, Nrf2. Fluorescently labeled Nrf2 peptides containing the ETGE motif were synthesized and optimized as tracers in the development of a fluorescence polarization (FP) assay to identify small molecule inhibitors of Keap1-Nrf2 interaction. The tracers were optimized to increase the dynamic range of the assay and their binding affinities to the Keap1 Kelch domain. The binding affinities of Nrf2 peptide inhibitors obtained in our FP assay using FITC-9mer Nrf2 peptide amide as the probe were in good agreement with those obtained previously by a surface plasmon resonance (SPR) assay. The FP assay exhibits considerable tolerance towards DMSO and produced a Z'-factor greater than 0.6 in a 384-well format. Further optimization of the probe led to cyanine-labeled 9mer Nrf2 peptide amide, which can be used along with the FITC-9mer Nrf2 peptide amide in a high throughput screening (HTS) assay to discover small molecule inhibitors of Keap1-Nrf2 interaction.

Transcription Factor NRF2 as a Therapeutic Target for Chronic Diseases: A Systems Medicine Approach

“…Briefly, a series of truncated NRF2 peptides was initially evaluated as direct inhibitors of PPI using surface plasmon resonance and fluorescence polarization assays (Hu et al, 2013). The minimal peptide sequence with inhibitory capacity was the 9-mer sequence of LDE-ETGE-FL (Chen et al, 2011;Inoyama et al, 2012). In parallel, Wells and collaborators (Hancock et al, 2013) searched for new putative peptide ligands using a phage display library combined with high-throughput fluorescence polarization assay.…”

Non-electrophilic modulators of the canonical Keap1/Nrf2 pathway

“…The 14mer peptide 74 LQLDEETGEFLPIQ 87 experiences a drop in potency ( K D = 61.9 nM). 39 The 10mer peptide 76 LDEETGEFLP 85 , was less effective than the 16mer at inhibiting the complex ( K D = 30.1 nM) but still managed to displace Nrf2 from the complex. 39 Upon further deletion of residues from the 16mer peptide to increase its dynamic range, it was found that the 9mer 76 LDEETGEFL 84 ( K D = 352 nM) is the minimal Nrf2 peptide sequence required for the complex to be inhibited, as the 8mer peptide no longer bound to Keap1 with any affinity ( K D = ~1000 nM).…”

“…38 The ETGE motif has a higher affinity ( K D = 5 nM) than the DLG motif ( K D = 1 µM). 39,40 This difference in affinities stems from the motifs’ interactions with the pocket, detailed below.…”

“…These small peptides are truncated versions of the Nrf2 Neh2 domain, often used for competition fluorescence polarization assays, and contain the ETGE binding motif along with several amino acid residues on either side of the motif. 39 The 16mer Nrf2 peptide 69 AFFAQLQLDEETGEFL 84 is the most potent of the peptides ( K D = 28.7 nM); however, because the size of the labeled peptide is directly proportional to the rotational correlation time, 59 the larger peptide does not provide as large a dynamic range for fluorescence polarization assays as a smaller peptide would. The 14mer peptide 74 LQLDEETGEFLPIQ 87 experiences a drop in potency ( K D = 61.9 nM).…”

Peptide and small molecule inhibitors of the Keap1–Nrf2 protein–protein interaction

“…FITC-β-EETGE E did not bind (K D >> 1000 nM), indicating a minimal recognition sequence of 6-7 amino acids [27]. Complementary assays based upon surface plasmon resonance [28,29], FRET [30], thermal denaturation [31], MS, NMR [32] and isothermal calorimetry [21] have also been applied to confirm ligand binding activity.…”