Breaking Barriers: Engineering Cyclic Peptides for Enhanced Cell Entry and Targeted Cancer Therapy

Cyclic peptides have long been recognized for their potential as modulators of intracellular protein-protein interactions (PPIs), a class of biological targets that have been notoriously difficult to drug due to their large and often flat interaction surfaces. Unlike small molecules, cyclic peptides can offer a larger surface area for interaction with their protein targets, potentially leading to more specific and high-affinity binding. However, a significant barrier to their clinical utility development has been their generally low permeability across cell membranes, which limits their ability to reach intracellular targets.

To address this longstanding challenge in the field of medicinal chemistry and achieve   cell membrane permeability enhancement for cyclic peptides without compromising their ability to inhibit specific intracellular PPIs, scientists from FUJIFILM’s Synthetic Organic Chemistry Laboratories and Analysis Technology Center, namely Mai Mizuno-Kaneko, Dr. Ichihiko Hashimoto, Dr. Kenta Miyahara, Masahiro Kochi, Noriyuki Ohashi, Dr. Kyosuke Tsumura, Koo Suzuki, and Dr. Takashi Tamura designed cyclic peptides capable of inhibiting the MDMX-p53 interaction, a critical target in cancer therapy. The new study is now published in ACS Medical Chemistry Letters. The team initiated their study by analyzing the conformation of cyclic peptides within the cell membrane to understand better how these molecules could be structurally optimized to traverse the lipid bilayer more effectively.  A novel aspect of their approach was combining NMR structure analysis and molecular dynamics simulations, to assess the conformations of cyclic peptides in membrane-mimicking environments. This led to the introduction of the “r-value,” a metric derived from the ellipsoid approximation of peptide conformations, which serves as a predictor for membrane permeability. Peptides with lower r-values, indicating more elongated conformations, were found to have higher permeability, likely due to reduced polar surface area and enhanced ability to form intramolecular hydrogen bonds, thus minimizing unfavorable interactions with the hydrophobic core of the lipid bilayer. Building on this foundation, the researchers applied computational chemistry techniques to design cyclic peptides with high affinity for MDMX, a negative regulator of the tumor suppressor protein p53. By inhibiting MDMX, the activation of p53 can be restored, leading to the induction of apoptosis in cancer cells, making this pathway a promising target for anticancer therapy. The team’s lead compound, cyclic peptide 19, demonstrated not only high affinity for MDMX (IC50 = 0.07 μM) but also favorable cell membrane permeability (Papp = 0.80 × 10^-6 cm s^-1), a significant achievement in the context of cyclic peptide drug development.

The researchers employed strategic modifications, such as N-methylation of amide bonds and the replacement of certain amino acids, to optimize the peptides’ properties. These modifications successfully reduced the polar surface area (EPSA) of the peptides, thereby enhancing their membrane permeability without diminishing their affinity for MDMX.  The study showcased the power of combining computational design with experimental validation. The computational models accurately predicted the conformations and properties of the cyclic peptides, which were subsequently confirmed through synthesis and biological testing. Additionally, the strategic replacement of certain amino acid residues with those having less polar side chains further optimized the peptide’s hydrophobicity without compromising its binding conformation or affinity for MDMX.

The new study is an excellent showcase of the strength of integrating computational and experimental methodologies in drug design research, particularly for challenging targets like PPIs. The knowledge gained from the conformational analysis of cyclic peptides in membrane-like environments have broad implications, potentially designing the development of a wide range of cyclic peptide-based therapeutics with enhanced cell membrane permeability. Moreover, targeting the MDMX-p53 interaction opens new avenues for cancer therapy, and may offer hope for potential effective treatments that can reactivate the p53 pathway in tumors where it is inactivated by overexpression of MDMX. In conclusion, the work by FUJIFILM’s scientists represents a significant step forward in the field of medicinal chemistry, particularly in the design of cyclic peptides as intracellular PPI inhibitors. The study not only provides a novel framework for understanding and improving the membrane permeability of these molecules but also demonstrates the feasibility of developing potent, cell-permeable inhibitors targeting critical cancer pathways. As such, this research holds promise for the future development of new therapeutic agents that can effectively modulate intracellular targets previously considered “undruggable,” potentially leading to advancing cancer treatment. The Fujifilm scientists also hope that, through this paper, their membrane-permeable peptide development technology will be extended to the development of inhibitors of PPI targets other than MDMX.