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

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.

New methods for developing (bi)cyclic peptides by phage display

Camille Villequey

Cyclic peptide ligands are a promising molecular format for the development of therapeutics. They combine some of the advantages of large protein therapeutics (high affinity and specificity) and of small molecule drugs (accessibility to chemical synthesis and low immunogenicity). With phage display, large combinatorial libraries of more than one billion mono- and polycyclic peptides can be developed and screened against virtually any target. During phage affinity selections, the physical link between phenotype (encoded peptide) and genotype (phage DNA) allows the identification of enriched peptide binders by sequencing of the phage vector DNA. Based on these concepts, our laboratory has established a robust chemistry in which phage displayed peptides containing two or three cysteines are cyclized or bi-cyclized with thiol-reactive linkers. This procedure has been generally applied for the isolation of potent and selective (bi)cyclic peptide ligands with therapeutic potential. The isolation of binders with optimal properties is highly dependent on the possibility to generate (bi)cyclic peptide libraries with high structural diversity and on the ability to thoroughly decode the phage selection output. In my PhD work, I developed new methods to generate large and structurally diverse libraries and to decode more efficiently phage selected peptides. In my first project, I explore the cyclization of peptides with two chemical bridges as a method to provide rapid access to phage-encoded libraries with high scaffold diversity. To this end, a range of twelve structurally diverse chemicals were tested for thiol-reactivity and phage compatibility. Subsequently, peptide libraries of the format XCXnCXnCX (X = random amino acid, C = cysteine, n = 3 or 4) were cyclized with six of these chemical linkers and panned against the protease plasma kallikrein. The selection yielded inhibitors with remarkable affinity (sub-nM KI), target selectivity (>1000-fold) and proteolytic stability (t1/2 in plasma > 3 days) despite a relatively small molecular mass (~ 1200 Da). In the second project, I developed phage-encoded libraries of small cyclic peptides in order to identify ligands that have the potential to be orally absorbed. A major challenge for the generation of such library is the usually low diversity of compounds due to the limited number of amino acids. In this work, we addressed this limitation by taking advantage of a recently discovered peptide cyclization reaction in which the thiol of a cysteine is ligated with the N-terminal amino group using a chemical reagent. We applied this strategies to two phage-encoded peptide libraries of four and five amino acids (XXXC and XXXXC) that were cyclized with eight chemical linkers yielding a collection of more than 1.3 million compounds. Panning the libraries against the two disease targets plasma kallikrein and coagulation factor XIa yielded ligands with single-digit micromolar affinity. In a third project, I developed a strategy to bypass the bacterial infection step in phage display. Bacterial infection is usually crucial for the propagation and identification of enriched clones by DNA sequencing. However, mutations or chemical modifications of the phage coat proteins, sometimes lead to decreased infection rates and therefore compromise the entire procedure. Consequently, there is an interest for developing methods in which this step could be completely omitted.

EPFL2017

Development of New Bicyclic Peptide Formats and their Application in Directed Evolution

Shiyu Chen

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

Development of cyclic peptide inhibitors of coagulation factor XII and matrix metalloproteinase 2

Jonas Alfred Karl Wilbs

Peptides represent a promising molecular class for drug development. They combine several strengths of small molecules (e.g. efficient tissue diffusion, low immunogenicity and access to chemical synthesis) and key properties of biologics such as monoclonal antibodies (e.g. high affinity and specificity). Most of the peptide drugs are natural products or derivatives thereof, as for example human peptide hormones or antibacterial peptides. In recent years, a range of methodologies were established to develop peptides binding to therapeutic targets de novo. Our laboratory is specialized on the in vitro evolution of bicyclic peptides by phage display. Bicyclic peptides have two macrocyclic rings that allow for binding protein targets with high affinity and selectivity. The aim of my thesis was to improve the potency, selectivity and stability of phage-selected bicyclic peptide inhibitors of coagulation factor XII (FXII) and matrix metalloproteinase 2 (MMP-2), and to test their therapeutic potential in vivo. Specifically, I planned at improving the bicyclic peptides by substituting natural amino acids with unnatural ones. As described in the following, the substitutions to unnatural amino acids had led to substantial improvements in the bicyclic peptides, and this had enabled me the evaluation of the inhibitors in disease models. In my first and second project, I aimed at improving the potency and stability of a bicyclic peptide FXII inhibitor that was previously developed in our lab by phage display, and to test the therapeutic potential of the resulting peptide in vivo. FXII has recently been identified as a promising target for safe anticoagulation therapy. In the first project, I investigated if the insertion of a single carbon atom into the macrocyclic backbone of a bicyclic peptide FXII inhibitor can improve its binding affinity. Positions within the macrocycle susceptible to atom insertion were first identified using a scanning methodology where glycine mutants were compared to ¿-alanine mutants. Upon insertion of atoms in the backbone using ¿-amino acids or homologated cysteine analogues, two peptides showed 4.7- and 2.5-fold improved Ki values. The better one blocked FXII with a Ki of 1.5 ± 0.1 nM and was more potent than the lead peptide in inhibiting the activation of the intrinsic coagulation pathway. The strategy of ring size variation by one or several atoms should be generally applicable for the affinity maturation of in-vitro-evolved cyclic peptides. In my second project, I aimed at improving further the potency of the bicyclic peptide FXII inhibitor described before, and to additionally improve its proteolytic stability. I achieved both these goals by replacing further natural amino acids to unnatural ones. Sub-nanomolar activity for human and mouse FXII (370 and 450 pM respectively) as well as a high stability (t1/2 > 128 ± 8 h in plasma) permitted preclinical evaluation of the peptide. The inhibitor efficiently blocked intrinsic coagulation in blood plasma from human, mouse and rabbit. I further demonstrated that the peptide reduced experimental thrombosis induced by ferric chloride in mice and suppressed blood coagulation in artificial lungs in rabbits, all without increasing the risk of bleeding. This shows that the optimized bicyclic peptide is a promising candidate for thromboprotection in various medical conditions. In a third project, I aimed at improving the potency and stability of a bicyclic peptide MMP-2 inhibi

EPFL2018