Design and synthesis of Nrf2-derived hydrocarbon stapled peptides for the disruption of protein-DNA-interactions

Abstract

Misregulation and mutations of the transcription factor Nrf2 are involved in the development of a variety of human diseases. In this study, we employed the technology of stapled peptides to address a protein-DNA-complex and designed a set of Nrf2-based derivatives. Varying the length and position of the hydrocarbon staple, we chose the best peptide for further evaluation in both fixed and living cells. Peptide 4 revealed significant enrichment within the nucleus compared to its linear counterpart 5, indicating potent binding to DNA. Our studies suggest that these molecules offer an interesting strategy to target activated Nrf2 in cancer cells.

Double Stranded DNA Binding Stapled Peptides: An Emerging Tool for Transcriptional Regulation

“…In 2022, stapled peptides were used to inhibit the DNA-protein interaction of the TF Nrf2 (nuclear factor erythroid 2-related factor 2). [90] Nrf-2 binds DNA using a leucine zipper motif and is responsible for mediating the transcription of genes involved in protein degradation and in the regulation of inflammatory responses. [91,92] As it is over activated in tumour cells, leading to tumour progression and metastasis, it is an attractive therapeutic target.…”

“…Using a homology model of Nrf2-DNA binding, Wiedemann et al designed and synthesized four stapled peptides based on the major-groove binding helix with varying all-hydrocarbon stapling at the i and i + 3, i and i + 4, i and i + 7 positions. [90] The peptides were evaluated for their helical content using CD, which showed them to be helical, and the DNA binding affinities were probed using fluorescence polarization with the FAM-labelled Nrf2 dsDNA sequence. Further evidence of DNA binding was evaluated with EMSA.…”

“…This is born out in the Nrf2-based stapled peptides where there is a five-fold difference in affinity between the stapled and unstapled peptide control. [90] HTHs on the other hand are well folded prior to binding. As such peptides derived from these motifs show a significant increase in affinity upon stapling.…”

“…Both the GCN4 and Nrf2 leucine zipper derived stapled peptides show binding affinities, where reported, in the low μM range. [75,90] The HTH derive stapled peptides typically show low nM dissociation constants, such as with the HNF-3γ-derived stapled peptide (K D =4 nM) and the Q50Kderived stapled peptide (K D = 7.5 nM), [87] though the RpoN-derived stapled peptide is an outlier with a high nM affinity (K D = 366 nM). [96] A particular need in the field is rigorous evaluation of sequence specific DNA binding.…”

Stapled Peptides as Direct Inhibitors of Nrf2-sMAF Transcription Factors

“…This heterodimeric complex is responsible for the transcription of Nrf2-dependent genes like antioxidant proteins and oncogenes. , A few Nrf2 inhibitors have been reported, including the small molecules brusatol, AEM1, and ML385 . While these molecules suppress gene expression in cell culture, they do not directly inhibit the Nrf2-sMAF interaction. − Two peptide-based inhibitors, an Nrf2 stapled peptide for inhibiting the Nrf2/DNA interaction and a heterodimeric peptide inhibitor of the NRF2-MAFG-DNA ternary complex, have been developed. These have either not been tested for activity in cells or are not cell permeable.…”