Bicyclic peptide ligands pulled out of cysteine-rich peptide libraries

Shiyu Chen, Christian Heinis, Petr Leiman, Julia Morales Sanfrutos, Jeremy Touati
2013
Journal paper

Abstract

Bicyclic peptide ligands were found to have good binding affinity and target specificity. However, the method applied to generate bicyclic ligands based on phage-peptide alkylation is technically complex and limits its application to specialized laboratories. Herein, we report a method that involves a simpler and more robust procedure that additionally allows screening of structurally more diverse bicyclic peptide libraries. In brief, phage-encoded combinatorial peptide libraries of the format XmCXnCXoCXp are oxidized to connect two pairs of cysteines (C). This allows the generation of 3×(m+n+o+p) different peptide topologies because the fourth cysteine can appear in any of the (m+n+o+p) randomized amino acid positions (X). Panning of such libraries enriched strongly peptides with four cysteines and yielded tight binders to protein targets. X-ray structure analysis revealed an important structural role of the disulfide bridges. In summary, the presented approach offers facile access to bicyclic peptide ligands with good binding affinities.

Official source

https://infoscience.epfl.ch/record/185954?ln=en

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Related concepts

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Cyclic peptides have a range of properties that make them attractive as therapeutics such as high binding affinity, good target selectivity and low toxicity. While most peptides in the clinic are derived from nature, various powerful in vitro evolution techniques allow now the de novo development of cyclic peptide ligands to targets of interest. Our group is specialized in the development of bicyclic peptide ligands by phage display. A current limitation of phage-selected bicyclic peptides is their conformational flexibility limiting the binding affinity due to entropic effects or even preventing the generation of binders to challenging targets. The main goal of my thesis was the development of new bicyclic peptide formats that are conformationally more rigid or even have a stable fold in solution. The proposed bicyclic peptide formats are based on a rigid chemical structure containing multiple polar groups and peptides that are anchored to the structure and fold tightly around by forming non-covalent interactions. I also envisioned to cyclise peptide phage display libraries with several such chemical structures in parallel to generate and screen structurally highly diverse bicyclic peptide libraries. In a first project, I designed and synthesized three new compounds that have i) three thiol-reactive groups to react with peptides and ii) several polar groups. Two of these compounds efficiently and selectively cyclised linear peptides containing three cysteine residues. Functional and structural analysis of the bicyclic peptides showed that the relatively small compounds at the centre of bicyclic peptides significantly influence the activity and conformation of the peptides. These results demonstrated the prominent structural role of chemical linkers in peptide macrocycles. In a second project, I applied the compounds developed in the first project in phage selections. The sequences of isolted bicyclic peptide ligands were strongly influenced by the small chemical structures, suggesting that the peptides fold closely around them. X-ray structure analysis revealed that one of the chemical structures indeed nucleated peptides by forming H-bond interactions. This result showed that small rigid chemicals can serve as nucleating scaffolds and directs the way towards the generation of peptide ligands being folded in solution. A third project is based on an unexpected observation in the second project, namely that panning peptide phage libraries without prior alkylation with compounds yielded mostly peptides with four cysteines. The peptides bound with good affinity and characterization revealed that they contain two disulphide bridges and thus formed bicyclic peptide structures. An interesting feature of disulfide-based bicyclic peptide libraries was the high topological diversity: oxidation of peptide libraries of the form Xm CXn CXo CXp yielded 3 × (m+n+o+p) different bicyclic peptide topologies because the fourth cysteine could appear in any of the (m+n+o+p) randomized amino acid positions (X) and the four peptides in such peptides could pair in three different ways. X-ray structure analysis revealed an important structural role of the disulfide bridges. The new method offers an easy and robust way to generate bicyclic peptide ligands.

EPFL2013

Phage Selection of Cyclic Peptides for Application in Research and Drug Development

Christian Heinis, Xudong Kong

Cyclic peptides can bind to protein targets with high affinities and selectivities, which makes them an attractive modality for the development of research reagents and therapeutics. Additional properties, including low inherent toxicity, efficient chemical synthesis, and facile modification with labels or immobilization reagents, increase their attractiveness. Cyclic peptide ligands against a wide range of protein targets have been isolated from natural sources such as bacteria, fungi, plants, and animals. Many of them are currently used as research tools, and several have found application as therapeutics, such as the peptide hormones oxytocin and vasopressin and the antibiotics vancomycin and daptomycin, proving the utility of cyclic peptides in research and medicine. With the advent of phage display and other in vitro evolution techniques, it has become possible to generate cyclic peptide binders to diverse protein targets for which no natural peptides have been discovered. A highly robust and widely applied approach is based on the cyclization of peptides displayed on phage via a disulfide bridge. Disulfide-cyclized peptide ligands to more than a hundred different proteins have been reported in the literature. Technology advances achieved over the last three decades, including methods for generating larger phage display libraries, improved phage panning protocols, new cyclic peptide formats, and high-throughput sequencing, have enabled the generation of cyclic peptides with ever better binding affinities to more challenging targets. A relatively new cyclic peptide format developed using phage display involves bicyclic peptides. These molecules consist of two macrocyclic peptide rings cyclized through a chemical linker. Compared to monocyclic peptides of comparable molecular mass, bicyclic peptides are more constrained in their conformation. As a result, they can bind to their targets with a higher affinity and are more resistant to proteolytic degradation. Phage-encoded bicyclic peptides are generated by chemically cyclizing random peptide libraries on phage. Binders are identified by conventional phage panning and DNA sequencing. Next-generation sequencing and new sequence alignment tools have enabled the rapid identification of bicyclic peptides. Bicyclic peptide ligands were developed against a range of diverse target classes including enzymes, receptors, and cytokines. Most ligands bind with nanomolar affinities, with some reaching the picomolar range. To date, several bicyclic peptides have been positively evaluated in preclinical studies, and the first clinical tests are in sight. While bicyclic peptide phage display was developed with therapeutic applications in mind, these peptides are increasingly used as research tools for target evaluation or as basic research probes as well. Given the efficient development method, the ease of synthesis and handling, and the favorable binding and biophysical properties, bicyclic peptides are being developed against more and more targets, ever increasing their potential applications in research and medicine.

2017

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