Hélice alpha

thumb|redresse|Exemple d'hélice alpha. L’hélice alpha (hélice α) est une structure secondaire courante des protéines. Elle est formée par une chaîne polypeptidique de forme hélicoïdale à pas de rotation droit dans laquelle chaque groupe N-H de la chaîne principale d'un acide aminé forme une liaison hydrogène avec le groupe C=O de la chaîne principale du quatrième acide aminé le précédant. Cette structure secondaire est parfois appelée hélice α de Pauling-Corey-Branson. Le nom d’ est également utilisé, la valeur de 3,6 correspondant au nombre d'acides aminés dans le pas de l'hélice et 13 est le nombre d'atomes dans le cycle fermé par la liaison hydrogène. Parmi les types de structures locales présents dans les protéines, l'hélice α est la structure secondaire la plus prévisible par l'analyse de la séquence primaire, ainsi que la plus répandue. Découverte Dans les années 1930, William Astbury met en évidence des changements très importants par diffraction de rayon X de fibre

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

Development of bicyclic peptide Her2 binders and phage selection of alpha-helical peptide ligands

Philippe Diderich

Conformationally constrained peptide ligands offer an attractive format for the development of therapeutics. They can bind with high affinity and selectivity to protein targets, they are small enough to diffuse into tissues, they can be synthesized chemically, and their degradation products are not toxic. In recent years, a number of novel techniques were innovated to develop peptide-based ligands. One such technique is the generation of bicyclic peptides by phage display. This technique is well established in our laboratory and has led to the isolation of high-affinity antagonist for a range of important therapeutic targets. In a first project I aimed at developing bicyclic peptide ligands of the epidermal growth factor receptor Her2. Aberrant expression of Her2 has been implicated in various malignancies including breast cancer. In the treatment of this cancer type several Her2 specific antibodies were proved to be efficient. Due to their small size, bicyclic peptides could potentially offer advantages over antibodies such as ease of synthesis and conjugation, higher molecule-permass ratios, and better tumor penetration. I panned a large bicyclic peptide phage library against the extra-cellular domain of Her2 and obtained a range of peptide sequences binding to Her2 in a specific manner. After affinity maturation, a bicyclic peptide that bound Her2 with a Kd of 304 nM could be obtained. This peptide ligand offers a valuable starting point for further improvement and the development of high-affinity Her2 binders with potential application for tumor imaging and therapy. A second project was triggered by my observation that it is relatively difficult to generate high-affinity bicyclic peptide ligands to proteins with flat and featureless surfaces such as Her2. The major reason for the limited binding affinity of bicyclic peptides was supposed to lie in the lack of a defined secondary structure in solution and the resulting entropic penalty upon binding. To overcome this problem, I proposed to evolve ligands based on a-helices. Chemically stabilized a-helices, also named stapled peptides, were previously developed by rational design. I proposed to evolve a-helical peptides by phage display wherein the helix, is stabilized in a chemical reaction prior to phage panning. I tested this strategy by affinity maturing an alpha-helical peptide binding to beta-catenin. The project resulted with a 250-fold improved ligand of beta-catenin. In a third project, I developed a novel constrained peptide format that I dubbed âhelix-loopâ motif. A stabilized alpha-helix was expanded with a peptide loop in order to increase the number of amino acids that can formcontacts with a target. The loop was linked to either the N- or C-terminal end of the helix and via a cysteine residue to the chemical linker stabilizing the alpha-helix. Libraries were created adding randomized loops with different length to either side of the peptide. Ligands with improved affinity were isolated against the cancer target beta-catenin. In summary, I have exploited the existing bicyclic peptide format to evolve bicyclic peptide ligands of Her2. In addition, I have developed a new approach to affinity mature stabilized helical peptides by phage display as well as a new constrained peptide format. This new approach should be applicable for affinity maturation of any stabilized alpha-helix. The new peptide format promises the generation of high affinity ligands to any protein target, including Her2.

EPFL2014

Development of bicyclic peptide Her2 binders and phage selection of alpha-helical peptide ligands

Philippe Diderich

EPFL2014

Protein design: progress toward the synthesis of a 16-residue peptide designed to contain an N-terminal cystein and to form an amphiphilic alpha-helix.

Phage selection of chemically stabilized alpha-helical peptide ligands

Development of bicyclic peptide Her2 binders and phage selection of alpha-helical peptide ligands

Development of bicyclic peptide Her2 binders and phage selection of alpha-helical peptide ligands

Protein design: progress toward the synthesis of a 16-residue peptide designed to contain an N-terminal cystein and to form an amphiphilic alpha-helix.

Phage selection of chemically stabilized alpha-helical peptide ligands