Development of Stapling Methods by Selective Side Chain Modifications of Bioactive Peptides

Cyclic peptide therapeutics fill the gap between small molecules (5000 Da) as a separate class of drugs, combining the advantages of both in terms of high selectivity, bioavailability, synthetic accessibility and low toxicity. However, their conformational flexibility and proteolytic instability results in fast metabolism. Therefore, modification of their chemical and structural properties at the proteolytically vulnerable sites can circumvent these deficiencies. Several strategies exist to stabilise peptides through local or global constraints, side chain functionalisation or amide bond surrogates. This thesis describes the development and application of two chemical transformations to peptides with the aim of greatly enhancing their metabolic stability either via side chain to side chain cyclisation or disulfide modification. Following the functionalisation, a comparative evaluation of the pharmacological properties of the modified peptides was conducted with respect to the parent peptides. The first cyclisation strategy aimed to apply the redox-neutral chemistry developed in the burgeoning field of borrowing hydrogen technology to peptides, as a new class of tolerable substrates. These would contain the requisite amine and alcohol functional groups enabling their direct coupling to form the lactam or N-alkylated cycle. Numerous investigations into this transformation with a plethora of substrates and catalysts did not lead to the desired cyclisation. For the second approach, a simple one-pot procedure for the 'stapling' of disulfide bonds in native peptides was developed. Tris(2-carboxyethyl)phosphine mediated reduction of the disulfide bond was followed by concomitant insertion of a 'doubly electrophilic' carbon bridge to form a physiologically stable dithioacetal bond. The reaction was performed under mild, biocompatible reaction conditions, allowing for the facile conversion of native peptides into more stable analogues. The protocol was applicable to a range of bioactive peptides with a multitude of reactive functional groups demonstrating the high versatility of this approach. The importance and utility of the modified analogues were verified by testing their binding affinity and biological activity against the corresponding parent peptides. The impact of the disulfide modification on these properties for each peptide were analysed on a case-by-case basis. The SCS modified analogues of Oxytocin and Vasopressin maintained binding affinities in the same order of magnitude as their respective parent compounds across all corresponding receptors. Nevertheless, this was not the case for SCS-Octreotide and SCS-Somatostatin. Those peptides which maintained comparable binding affinities were then tested for their functional activity at the receptors of biological interest. SCS-Oxytocin retained its agonist potency displaying sub-nanomolar activity at the Oxytocin receptor, whilst SCS-Vasopressin exhibited sub micromolar functional response at the Vasopressin 1A receptor. Then the stability of the modified peptides in human serum was evaluated. In some cases, the enhancement in serum stability was vastly increased. In particular our modified SCS-Oxytocin analogue clearly demonstrated a significant improvement in serum half-life, as well as heat and pH stability over its native parent form. This disulfide stapling method could have wide-reaching application for the development of more stable therapeutic analogues.

Phage Selection of Bicyclic Peptide Ligands and Development of a New Peptide Cyclisation Method

Jeremy Touati

Bicyclic peptides binding to targets of interest can be isolated from combinatorial libraries using a phage display-based approach. In a first project of this thesis, we aimed at generating bicyclic peptide inhibitors of matrix metalloproteinases (MMPs), a family of proteases that share high structural similarities and have been difficult to target in a specific manner. From phage-encoded combinatorial libraries, we isolated a bicyclic peptide that inhibits specifically the gelatinase MMP-2 with a Ki of 2 μM. This inhibitor was less potent than the best peptidic MMP-2 inhibitor, the linear APP-derived inhibitory peptide (Ki = 13 nM). However, due to its conformational constraint, the bicyclic peptide is expected to be significantly more stable and hence more suitable for applications in biological systems. It may be used as a research tool alternatively to the broadly applied monocyclic peptide MMP-2 inhibitor CTT which is less potent (Ki of 142 μM). If affinity matured, the bicyclic peptide MMP-2 inhibitor might even be developed into an anti-cancer therapeutic. In a second project, bicyclic peptide binders to the centriolar protein SAS-6 were generated. SAS-6 plays an important structural role in centrioles and bicyclic peptide binders are currently applied in cellular assays by the laboratory of Professor Pierre Gönczy (EPFL) to study the process of centriole formation. In a third project of this thesis, we established an enzymatic method to selectively cyclise peptides with unprotected side chains using a transglutaminase (TGase). TGases catalyse the formation of stable amide bonds between the side chains of glutamine and primary amines. They have been used extensively in biotechnological applications to cross-link peptides and proteins or to attach labels to proteins. We found that the microbial transglutaminase (MTGase) of Streptomyces mobaraensis can cyclise quantitatively peptides with an N- terminal glutamine-donor sequence and a C-terminal lysine residue in an intramolecular reaction. Experiments with minimised glutamine-donor substrates revealed that peptides with only an Ala-Leu dipeptide N-terminal to the glutamine and a randomly chosen peptide between the glutamine and lysine residues are efficiently cyclised. By combining the MTGase-based cyclisation strategy with a chemical thiol-based cyclisation reaction, we were able to generate tricyclic peptide structures. It remains to be tested if this method can be applied to cyclise combinatorial peptide libraries on the surface of phage. In a fourth and last project, we developed a method to follow qualitatively and quantitatively chemical or enzymatic modifications applied to peptides displayed on phage. In this method, peptides on phage were chemically modified, cleaved off by a protease, purified, concentrated and analysed by mass spectrometry. The method will help to assess new reactions applied to phage peptides such as the MTGase-based enzymatic cyclisation reaction.

EPFL2012

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

Peptide ligands stabilized by small molecules

Alessandro Angelini, Davide Bertoldo, Shiyu Chen, Christian Heinis, Florence Pojer

Bicyclic peptides generated through directed evolution by using phage display offer an attractive ligand format for the development of therapeutics. Being nearly 100-fold smaller than antibodies, they promise advantages such as access to chemical synthesis, efficient diffusion into tissues, and needle-free application. However, unlike antibodies, they do not have a folded structure in solution and thus bind less well. We developed bicyclic peptides with hydrophilic chemical structures at their center to promote noncovalent intramolecular interactions, thereby stabilizing the peptide conformation. The sequences of the peptides isolated by phage display from large combinatorial libraries were strongly influenced by the type of small molecule used in the screen, thus suggesting that the peptides fold around the small molecules. X-ray structure analysis revealed that the small molecules indeed formed hydrogen bonds with the peptides. These noncovalent interactions stabilize the peptide-protein complexes and contribute to the high binding affinity.