Phage selection of peptide macrocycles against b-catenin to interfere with Wnt signaling

Upregulation of b-catenin, the primary mediator of the Wnt signaling pathway, plays an important role in the tumorigenesis of several types of human cancer. Targeting b-catenin to interfere with its ability to serve as a translational co-activator is considered an attractive therapeutic approach. However, the development of inhibitors has been challenging because of the lack of obvious binding pockets for ligands, and because inhibitors should not interfere with other b-catenin functions. Only two ligands with known molecular interactions with b-catenin have been developed so far, and are based on stabilized -helical peptides. In this study, we screened a large combinatorial library of bicyclic peptides by phage display. Binders to different surface regions of b-catenin were identified. The binding site of one group of ligands was mapped to the interaction region of the translational Wnt inhibitor ICAT (inhibitor of b-catenin and Tcf), which is a prime target site on b-catenin for therapeutic intervention, and to which no ligands could be developed before.

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

Phage selection of chemically stabilized alpha-helical peptide ligands

Davide Bertoldo, Pierre Vincent Dessen, Philippe Diderich, Christian Heinis, Maola Khan Mohammad Golam

Short alpha-helical peptides stabilized by linkages between constituent amino acids offer an attractive format for ligand development. In recent years, a range of excellent ligands based on stabilized alpha-helices were generated by rational design using alpha-helical peptides of natural proteins as templates. Herein, we developed a method to engineer chemically stabilized alpha-helical ligands in a combinatorial fashion. In brief, peptides containing cysteines in position i and i + 4 are genetically encoded by phage display, the cysteines are modified with chemical bridges to impose alpha-helical conformations, and binders are isolated by affinity selection. We applied the strategy to affinity mature an alpha-helical peptide binding beta-catenin. We succeeded in developing ligands with K-d's as low as 5.2 nM, having >200-fold improved affinity. The strategy is generally applicable for affinity maturation of any alpha-helical peptide. Compared to hydrocarbon stapled peptides, the herein evolved thioether-bridged peptide ligands can be synthesized more easily, unnatural amino acids are required and the cyclization reaction is more efficient and yields no stereoisomers. A further advantage of the thioether-bridged peptide ligands is that they can be expressed recombinantly as fusion proteins.

2016

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

Jeremy Touati

EPFL2012