Orthogonal ring-closing alkyne and olefin metathesis for the synthesis of small GTPase-targeting bicyclic peptides.

Bicyclic peptides are promising scaffolds for the development of inhibitors of biological targets that proved intractable by typical small molecules. So far, access to bioactive bicyclic peptide architectures is limited due to a lack of appropriate orthogonal ring-closing reactions. Here, we report chemically orthogonal ring-closing olefin (RCM) and alkyne metathesis (RCAM), which enable an efficient chemo- and regioselective synthesis of complex bicyclic peptide scaffolds with variable macrocycle geometries. We also demonstrate that the formed alkyne macrocycle can be functionalized subsequently. The orthogonal RCM/RCAM system was successfully used to evolve a monocyclic peptide inhibitor of the small GTPase Rab8 into a bicyclic ligand. This modified peptide shows the highest affinity for an activated Rab GTPase that has been reported so far. The RCM/RCAM-based formation of bicyclic peptides provides novel opportunities for the design of bioactive scaffolds suitable for the modulation of challenging protein targets

Constraining an irregular peptide secondary structure through ring-closing alkyne metathesis

Macrocyclization can be used to constrain peptides in their bioactive conformations, thereby supporting target affinity and bioactivity. In particular, for the targeting of challenging protein–protein interactions, macrocyclic peptides have proven to be very useful. Available approaches focus on the stabilization of α-helices, which limits their general applicability. Here we report for the first time on the use of ring-closing alkyne metathesis for the stabilization of an irregular peptide secondary structure. A small library of alkyne-crosslinked peptides provided a number of derivatives with improved target affinity relative to the linear parent peptide. In addition, we report the crystal structure of the highest-affinity derivative in a complex with its protein target 14-3-3ζ. It can be expected that the alkyne-based macrocyclization of irregular binding epitopes should give rise to new scaffolds suitable for targeting of currently intractable proteins

Protease-resistant and cell-permeable double-stapled peptides targeting the Rab8a GTPase.

Small GTPases comprise a family of highly relevant targets in chemical biology and medicinal chemistry research and have been considered “undruggable” due to the persisting lack of effective synthetic modulators and suitable binding pockets. As molecular switches, small GTPases control a multitude of pivotal cellular functions, and their dysregulation is associated with many human diseases such as various forms of cancer. Rab-GTPases represent the largest subfamily of small GTPases and are master regulators of vesicular transport interacting with various proteins <i>via</i> flat and extensive protein–protein interactions (PPIs). The only reported synthetic inhibitor of a PPI involving an activated Rab GTPase is the hydrocarbon stapled peptide <b>StRIP3</b>. However, this macrocyclic peptide shows low proteolytic stability and cell permeability. Here, we report the design of a bioavailable <b>StRIP3</b> analogue that harbors two hydrophobic cross-links and exhibits increased binding affinity, combined with robust cellular uptake and extremely high proteolytic stability. Localization experiments reveal that this double-stapled peptide and its target protein Rab8a accumulate in the same cellular compartments. The reported approach offers a strategy for the implementation of biostability into conformationally constrained peptides while supporting cellular uptake and target affinity, thereby conveying drug-like properties