Scientists at the Ecole Polytechnique Fédérale de Lausanne (EPFL) have addressed a long-standing challenge in the pharmaceutical industry, with the development of a two-step, one-pot method for generating orally available cyclic peptide-based drug candidates. The achievement represents a significant milestone in drug development that could provide new opportunities for creating peptide therapeutics against challenging disease targets.

“We have now succeeded in generating cyclic peptides that bind to a disease target of our choice and can also be administered orally,” said research lead Christian Heinis, PhD. “To this end, we have developed a new method in which thousands of small cyclic peptides with random sequences are chemically synthesized on a nanoscale and examined in a high-throughput process.”

Heinis and colleagues reported on their development in Nature Chemical Biology, in a paper titled, “De novo development of small cyclic peptides that are orally bioavailable.” In their paper the team concluded, “This method for generating orally available peptides is general and provides a promising push toward unlocking the full potential of peptides as therapeutics.”

For decades, a substantial number of proteins, vital for treating various diseases, have remained elusive to oral drug therapy. “One of the biggest bottlenecks in therapeutic development is that a large proportion of the proteins can currently not be targeted by classical small molecules administered orally,” the investigators wrote. Traditional small molecules often struggle to bind to proteins with flat surfaces or require specificity for particular protein homologs. Typically, larger biologics that can target these proteins demand injection, limiting patient convenience and accessibility.

“There are many diseases for which the targets were identified but drugs binding and reaching them could not be developed,” said Heinis. “Most of them are types of cancer, and many targets in these cancers are protein-protein interactions that are important for the tumor growth but cannot be inhibited.”

Cyclic peptides can bind challenging disease targets with high affinity and specificity and are relatively small, so are effectively “in reach of being orally bioavailable,” the investigators suggested. And such molecules could offer up “enormous opportunities for addressing unmet medical needs.” However, the team continued, “… as with biological drugs, most cyclic peptides cannot be applied orally because they are rapidly digested and/or display low absorption in the gastrointestinal tract, hampering their development as therapeutics.”

Heinis added, “Cyclic peptides are of great interest for drug development as these molecules can bind to difficult targets for which it has been challenging to generate drugs using established methods. But the cyclic peptides cannot usually be administered orally—as a pill—which limits their application enormously.”

For their study the team focused on targeting the enzyme thrombin, which is a critical disease target because of its central role in blood coagulation. Regulating thrombin is key to preventing and treating thrombotic disorders, such as strokes and heart attacks.

To generate cyclic peptides that can target thrombin and are sufficiently stable, the scientists developed a two-step combinatorial synthesis strategy to synthesize a vast library of cyclical peptides with thioether bonds, which enhance their metabolic stability when taken orally.

“We have now succeeded in generating cyclic peptides that bind to a disease target of our choice and can also be administered orally,” said Heinis. “To this end, we have developed a new method in which thousands of small cyclic peptides with random sequences are chemically synthesized on a nanoscale and examined in a high-throughput process.”

The new method process involves two steps, and takes place in the same reactive container, a feature that chemists refer to as “one pot.” The first step is to synthesize linear peptides, which then undergo a chemical process of forming a ring-like structure—essentially, being “cyclized.” This is done by using bis-electrophilic linkers—chemical compounds used to connect two molecular groups together—to form stable thioether bonds. In the second phase, the cyclized peptides undergo acylation, a process that attaches carboxylic acids to them, further diversifying their molecular structure.

The technique eliminates the need for intermediate purification steps, allowing for high-throughput screening directly in the synthesis plates, combining synthesis and screening of thousands of peptides to identify candidates with high affinity for specific disease targets—in this case, thrombin. “In this study, we developed a combinatorial synthesis and screening approach based on sequential cyclization and one-pot peptide acylation and screening, with the possibility of simultaneously interrogating activity and permeability,” the scientists stated. “A key step in this development was the choice of a dithioether cyclic peptide format that is stable enough to resist gastrointestinal proteases, small enough to enable gastrointestinal absorption and, notably, combined the thiol-to-thiol macrocyclization and the subsequent amine acylation.”

Using the method, project leader Manuel Merz was able to generate a comprehensive library of 8,448 cyclic peptides with an average molecular mass of about 650 Da, only slightly above the maximum limit of 500 Da recommended for orally available small molecules. “The unique format of our rapidly synthesized sub-libraries allowed us to screen simultaneously for two in vitro properties—membrane permeability and target binding—to more rapidly identify de novo peptide drug candidates,” the team added.

The top cyclic peptides also showed a high affinity for thrombin, and when tested in rats, showed oral bioavailability up to 18%, which means that when taken orally, 18% of the cyclic peptide successfully enters the bloodstream and thus available to have a therapeutic effect. “In the end, this work culminated in a five-building-block cyclic peptide that displayed an oral availability of 18% in rats,” the researchers noted. Considering that orally administered cyclic peptides generally show a bioavailability below 2%, an oral bioavailability of 18% is a substantial advance for peptide-based biologic drugs.

Enabling the development of orally available cyclic peptides has opened up possibilities for treating a range of diseases that have been challenging to address with conventional oral drugs. The method’s versatility means it can be adapted to target a wide array of proteins, potentially leading to breakthroughs in areas where medical needs are currently unmet. “Our presented approach of synthesizing large numbers of small cyclic peptides with stable backbones is general and can be directly applied to other targets,” the scientists wrote. “A particularly attractive application may be the development of selective inhibitors or activators of enzymes, receptors or ion channels … Of high interest are also challenging targets, such as protein–protein interactions, many of which are intractable to classical small molecules.”

Merz added, “To apply the method to more challenging disease targets, such as protein-protein interactions, larger libraries will likely need to be synthesized and studied. By automating further steps of the methods, libraries with more than one million molecules seem to be within reach.”

In the next step of this project, the researchers will target several intracellular protein-protein interaction targets for which it has been difficult to develop inhibitors based on classical small molecules. They are confident that orally applicable cyclic peptides can be developed for at least some of them. In conclusion, Merz and colleagues wrote, “We expect that the simplicity and robustness of the presented concepts and methods, combined with the proof that they can deliver orally available cyclic peptides, will encourage broad application and hopefully lead to the development of drugs addressing unmet medical needs.”

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