Binding Dynamics of a Stapled Peptide Targeting the Transcription Factor NF-Y.

Transcription factors (TFs) play a central role in gene regulation, and their malfunction can result in a plethora of severe diseases. TFs are therefore interesting therapeutic targets, but their involvement in protein-protein interaction networks and the frequent lack of well-defined binding pockets render them challenging targets for classical small molecules. As an alternative, peptide-based scaffolds have proven useful, in particular with an α-helical active conformation. Peptide-based strategies often require extensive structural optimization efforts, which could benefit from a more detailed understanding of the dynamics in inhibitor/protein interactions. In this study, we investigate how truncated stapled α-helical peptides interact with the transcription factor Nuclear Factor-Y (NF-Y). We identified a 13-mer minimal binding core region, for which two crystal structures with an altered C-terminal peptide conformation when bound to NF-Y were obtained. Subsequent molecular dynamics simulations confirmed that the C-terminal part of the stapled peptide is indeed relatively flexible while still showing defined interactions with NF-Y. Our findings highlight the importance of flexibility in the bound state of peptides, which can contribute to overall binding affinity.

Structure-Based Design of Bicyclic Helical Peptides That Target the Oncogene ß-Catenin.

The inhibition of intracellular protein-protein interactions is challenging, in particular, when involved interfaces lack pronounced cavities. The transcriptional co-activator protein and oncogene ß­catenin is a prime example of such a challenging target. Despite extensive targeting efforts, available high-affinity binders comprise only large molecular weight Inhibitors. This hampers the further development of therapeutically useful compounds. Herein, we report the design of a considerably smaller peptidomimetic scaffold derived from the α-helical ß­catenin-binding motif of Axin. Sequence maturation and bicyclization provided a stitched peptide with an unprecedented crosslink architecture. The binding mode and site were confirmed by a crystal structure. Further derivatization yielded a ß-catenin inhibitor with single-digit micromolar activity in a cell-based assay. This study sheds a light on how to design helix mimetics with reduced molecular weight thereby improving their biological activity.