Peptides face an energy barrier when crossing the apolar region of a membrane due to the need for desolvation of their polar groups, such as the hydrogen (H)-bond donors found in peptide bonds. Nature has evolved a solution to this challenge through cyclization, which promotes the formation of intramolecular H-bonds within peptides. This process reduces solvation and simultaneously buries some of the polar surface, thereby enhancing the potential for membrane permeability. While linear peptides can also form intramolecular H-bonds, this is less favorable from an entropic perspective.
A well-studied example of this phenomenon is cyclosporine, an 11-amino-acid cyclic peptide drug. Despite its large size, cyclosporine is cell-permeable and can be administered orally. To efficiently cross membranes, cyclosporine undergoes Nα-methylation on 7 out of its 11 amide bonds, while the remaining amide bonds form intramolecular H-bonds to shield the donor and acceptor groups from the apolar environment of the lipid bilayer. Interestingly, when cyclosporine binds to its target, it alters its conformation and utilizes some of its polar groups for H-bond interactions. The importance of its cyclic structure is underscored by the observation that linearized cyclosporine exhibits reduced membrane permeability, highlighting the necessity of the cyclic shape for effective intramolecular hydrogen bonding. This behavior is not unique to cyclosporine; similar mechanisms have been observed in other natural cyclic peptides, where intramolecular H-bonding has been strategically leveraged to enhance membrane permeability and oral availability.
While cyclization can enhance membrane permeability, it is not a guaranteed solution, as the permeability of cyclic versus linear peptides depends on their specific structural and physicochemical properties. For example, the Kodadek group demonstrated that cyclization does not universally result in membrane permeability. Recently, computational methods have been employed to design cyclic peptide structures that conceal amide H-bond donors and acceptors through intramolecular hydrogen bonds. These peptides, designed specifically for membrane permeation, have shown promising results in parallel artificial membrane permeability assays (PAMPA), in live cells, and in rodent models, demonstrating good membrane permeability and oral bioavailability.
Cell-Permeable Peptides
Small cyclic peptides capable of crossing cell membranes are gaining significant attention due to their potential to target challenging intracellular sites that are currently considered “undruggable” by conventional small molecules. These targets, such as protein-protein interactions, often present flat, featureless surfaces without defined pockets for small molecule binding. Cyclic peptides, with their strong binding properties in such interactions, offer a promising approach. However, for these peptides to exert their effects on intracellular targets, they must first cross the plasma membrane—a challenging feat given their size and polar surface.
To date, only a few cyclic peptides have been approved for clinical use against intracellular targets. These include the immunosuppressant cyclosporine, the anti-cancer agent romidepsin, and the hepatitis C virus (HCV) drugs paritaprevir, grazoprevir, voxilaprevir, and glecaprevir. These HCV protease inhibitors are often classified as peptidomimetics or macrocycle drugs, as they contain only three amino acids along with several non-amino acid components.
Cyclic peptides can enter cells through various mechanisms, with passive diffusion being the most efficient. For effective diffusion across the nonpolar region of membranes, these peptides need to maintain a limited polar surface area, ideally less than 200 Ų, while also ensuring they are not too apolar to remain soluble in the aqueous environment required for diffusion and target binding. The factors influencing cell permeability have been extensively studied by comparing the physicochemical properties of membrane-permeable and non-permeable cyclic peptides and macrocyclic compounds. Key parameters include molecular weight, partition coefficient for n-octanol/water (logP), polar surface area, and the number of H-bond donors and acceptors. To develop membrane-permeable cyclic peptide drugs, charged and polar side chains are often excluded, and the number of peptide bonds is minimized. One common strategy to maintain the peptide’s size for effective target binding while reducing polar surface area involves using N-methylated or N-alkylated amino acids to eliminate H-bond donors. Another strategy involves engineering chameleonic cyclic peptides that can adopt different shapes and surfaces suitable for both aqueous solubility and membrane permeability.
New methods have been developed to reliably quantify the cell permeability of cyclic peptides. One notable example is the chloroalkane penetration assay (CAPA) developed by the Kritzer group. This method incorporates a small chloroalkane tag into molecules of interest, which is then detected in the cell cytosol by a HaloTag protein. Unlike microscopy-based techniques, which may struggle to distinguish between peptides in the cytosol and those in compartments like endosomes, CAPA provides a more precise estimation of peptide concentrations in the cell cytosol.
Source:
Cyclic Peptides for Drug Development
Xinjian Ji, Alexander L. Nielsen, Christian Heinis
First published: 23 October 2023 https://doi10.1002/anie.202308251