Ramlan, S. R. | bioRxiv Science

bioRxiv2022

Stitched peptides as potential cell permeable inhibitors of oncogenic DAXX protein

The death domain associated protein 6 (DAXX) is frequently upregulated in a number of common cancers where its suppression has been linked to reduced tumour progression. As a master regulator protein, with >70 reported protein interaction partners, the role of DAXX in its oncogenecity remains unclear. We designed and developed a set of novel stapled/stitched peptides that target a surface on the N-terminal helical bundle domain of DAXX which is the anchor-point for binding to multiple interaction partners (including Rassf1C, P53, Mdm2 and ATRX) and also for the auto regulation of the DAXX N-terminal SUMO interaction motif (SIM). We demonstrate that these peptides bind to and inhibit DAXX with an affinity higher than those reported for the known interaction partners and release the auto-inhibited SIM for interaction with SUMO-1. NanoBret assays show that the peptides enter cells and that their intracellular concentrations remain at nanomolar levels even after 24 hours, without causing membrane perturbation. Together our data suggest that these peptides are both tools for probing the molecular interactions of DAXX and potential precursors to the development of therapeutics.

biochemistry↗

bioRxiv2022

Development of a Novel Peptide Aptamer that Interacts with the eIF4E Capped-mRNA Binding Site using Peptide Epitope Linker Evolution (PELE).

Identifying new binding sites and poses that modify biological function are an important step towards drug discovery. We have identified a novel disulphide constrained peptide that interacts with the cap-binding site of eIF4E, an attractive therapeutic target that is commonly overexpressed in many cancers and plays a significant role in initiating a cancer specific protein synthesis program though binding the 5cap (7methyl-guanoisine) moiety found on mammalian mRNAs. The use of disulphide constrained peptides to explore intracellular biological targets is limited by their lack of cell permeability and the instability of the disulphide bond in the reducing environment of the cell, loss of which results in abrogation of binding. To overcome these challenges, the cap-binding site interaction motif was placed in a hypervariable loop on an VH domain, and then selections performed to select a molecule that could recapitulate the interaction of the peptide with the target of interest in a process termed Peptide Epitope Linker Evolution (PELE). A novel VH domain was identified that interacted with the eIF4E cap binding site with a nanomolar affinity and that could be intracellularly expressed in mammalian cells. Additionally, it was demonstrated to specifically modulate eIF4E function by decreasing cap-dependent translation and cyclin D1 expression, common effects of eIF4F complex disruption.

biochemistry↗