Macrocyclic Peptides Containing an Imidazopyridinium (IP⁺) Unit Display Enhanced Passive Cell Permeability

Area of Science:

• Medicinal Chemistry
• Molecular Biology
• Drug Discovery

Background:

• Macrocyclic peptides (MPs) show promise for targeting difficult proteins but struggle with intracellular delivery due to poor membrane permeability.
• Previous work demonstrated that incorporating an imidazopyridinium (IP+) moiety enhances passive membrane permeability (PAMPA).

Purpose of the Study:

• To investigate the cellular entry mechanisms of IP+-containing MPs.
• To assess the potential of IP+-modified MPs for targeting intracellular proteins.

Main Methods:

• Chloroalkane penetration assay (CAPA) to measure cytoplasmic entry rates.
• Live-cell imaging, ATP-depletion assays, and organelle co-localization studies.
• Assessing the biological activity of an IP+-modified p53-MDM2 inhibitor in MCF-7 cells.

Main Results:

• IP+-containing MPs exhibit rapid cytoplasmic entry, comparable to small molecules.
• Cellular uptake occurs primarily through passive diffusion, bypassing endosomal pathways.
• Mitochondrial co-localization was not observed for IP+-MPs.
• An IP+-modified MP successfully inhibited MCF-7 cell proliferation by targeting the p53-MDM2 interaction.

Conclusions:

• Imidazopyridinium incorporation is a viable strategy to enhance the cell permeability of macrocyclic peptides.
• This approach facilitates the development of MPs targeting intracellular proteins for therapeutic applications.