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

Area of Science:

• Biochemistry
• Molecular Biology
• Medicinal Chemistry

Background:

• Peptide therapeutics targeting intracellular molecules require efficient membrane permeability.
• Conventional polycationic membrane penetration motifs face challenges with protease vulnerability and endosome escape.

Purpose of the Study:

• To develop a novel strategy for macrocyclization of peptides using a hydrophobic bipyridyl (BPy) unit to improve membrane permeability and proteolytic stability.
• To discover potent intracellular binders using the BPy-macrocycle system via the Random nonstandard Peptide Integrated Discovery (RaPID) platform.

Main Methods:

• Chemical synthesis of model macrocyclic peptides closed by a thioether-BPy unit.
• Application of the RaPID system with chloromethyl-BPy (ClMeBPy) initiator for peptide library synthesis against BRD4 bromodomains.
• In vitro characterization of dissociation constants and cell membrane permeability of identified BPy macrocycles.

Main Results:

• Macrocyclic peptides closed by a thioether-BPy unit showed 40-fold better membrane permeability than non-macrocyclic counterparts.
• A BPy macrocycle identified via RaPID exhibited single-digit nanomolar affinity for BRD4 and superior membrane permeability compared to R9 and Tat peptides.
• BPy macrocycles tagged with a proteasome target motif demonstrated modest BRD4 degradation within cells.

Conclusions:

• Peptide macrocyclization with hydrophobic units like BPy significantly enhances cell membrane permeability and proteolytic stability.
• The combination of the RaPID system and BPy macrocycles offers a promising strategy for developing cell-penetrating peptides targeting intracellular proteins.
• This approach holds potential for the discovery of novel therapeutics for various intracellular targets.