Macrocyclic Peptides Closed by a Thioether-Bipyridyl Unit That Grants Cell Membrane Permeability

Membrane permeabilityis an important factor that determines thevirtue of peptides targeting intracellular molecules. By introducinga membrane penetration motif, some peptides exhibit better membranepermeabilities. Previous choices for such motifs have usually beenpolycationic sequences, but their protease vulnerabilities and modestendosome escapability remain challenging. Here, we report a strategyfor macrocyclization of peptides closed by a hydrophobic bipyridyl(BPy) unit, which grants an improvement of their membrane permeabilityand proteolytic stability compared with the conventional polycationicpeptides. We chemically prepared model macrocyclic peptides closedby a thioether-BPy unit and determined their cell membranepermeability, giving 200 nM CP50 (an indicative value ofmembrane permeability), which is 40-fold better than that of the ordinarythioether macrocycle consisting of the same sequence composition.To discover potent target binders consisting of the BPy unit, we reprogrammedthe initiator with chloromethyl-BPy ((BPy)-B-ClMe) forthe peptide library synthesis with a downstream Cys residue(s) andexecuted RaPID (Random nonstandard Peptide Integrated Discovery) againstthe bromodomains of BRD4. One of the obtained sequences exhibiteda single-digit nanomolar dissociation constant against BRD4 in vitroand showed approximately 2-fold and 10-fold better membrane permeabilitythan positive controls, R9 and Tat peptides, respectively. Moreover,we observed an intracellular activity of the BPy macrocycle taggedwith a proteasome target peptide motif (RRRG), resulting in modestbut detectable degradation of BRD4. The present demonstration indicatesthat the combination of the RaPID system with an appropriate hydrophobicunit, such as BPy, would provide a potential approach for devisingcell penetrating macrocycles targeting various intracellular proteins.

Automated summary

By introducing a membrane penetration motif, peptides exhibit improved membrane permeability. Macrocyclization of peptides closed by a hydrophobic bipyridyl unit improves their membrane permeability and proteolytic stability. The combination of the RaPID system with a hydrophobic unit like BPy provides a potential approach for designing cell penetrating macrocycles.