Innovative chemical tools for the synthesis of disulfide-rich peptides, an emerging class of medium-sized pharmaceuticals – EasyMiniProt

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A key aspect for intensifying current processes is the realization of chemoselective ligations of the peptide segments on a solid support in order to considerably reduce the number of intermediate purification steps by HPLC. The assembly of the miniproteins in the opposite direction to the SPPS has an additional advantage of «self-purification«, allowing the use of crude peptide segments. Two distinct methods of ligation will be exploited: native ligation and peptidomimetic triazole ligation developed by partner 1. Oxidative folding to regioselectively form disulfide bridges may be a limiting factor: if naturally occurring DRPs can mostly be folded under thermodynamic control, this involves the use of very dilute conditions. We will exploit the solid support used for their synthesis for this folding step. An original approach based on fragments with preformed disulfide bridges will also be implemented.

Finally, the methods developed will be applied to the generation of a large number of diversified variants of a DRP, muscarinic toxin 7 of the mamba venom, which is particularly promising from a therapeutic point of view. In particular, we will use an innovative approach to combining fragments. We expect to identify several variants with unpublished pharmacological profiles.

Around ten second generation water-soluble NHC-Cu (I) catalysts of were synthesized and then evaluated in the framework of cycloaddition reactions of azides and model alkynes, in aqueous and diluted (<1 mM) and in Non-deoxygenated conditions. Disappointingly, these novel catalysts are no more reactive than the first-generation catalyst (Chem Comm 2012), and are therefore unsuitable for CuAAC for cysteine-rich peptides. On the other hand, a very promising system of catalysis under micellar conditions has been identified, allowing rapid reactions of small model peptides under very dilute aqueous conditions (10 µM).

We have rigorously evaluated the effectiveness of the N-Hnb-Cys device by comparing it to a conventional NCL reaction using a preformed alkyl thioester. This work has shown a higher efficiency than the existing systems based on a transfer of acyl NS (factor <10 in terms of kinetics), but remains relatively slow (kinetic about 5 times slower at pH 7.5) than NCL classic.

The development of a new N-terminal arm incorporating a cysteine ??rather than an azide group has been completed. This arm can be synthesized in a few steps with good yields, and its stability as well as its optimum cutting conditions have been rigorously evaluated.

We have explored the possibility of using conventional CuAAC catalysts for the synthesis of triazolo-DRPs, according to a strategy based on the use of cysteine ??protective groups. Serious pitfalls have been encountered concerning the compatibility of these protective groups with the conventional catalysts, but also of the very delicate deprotection of these protective groups after ligation. Pending the optimization of new catalytic systems, an efficient strategy for the synthesis of MT7 in solution via two successive NCLs has been developed.

The highly promising micellar catalysis system that has been identified will shortly be evaluated as part of the reaction of long peptides, including the models incorporating numerous cysteines cited in the project document (especially MT7). Particular attention will then be given to evaluating the compatibility of these systems with solid support reactions.

The development of a strategy to adapt our methodology for the synthesis of crypto-thioester peptides to multiple successive N-to-C ligations has been initiated and will be actively pursued in the coming months. Different cysteine ??protective groups (Acm, PhAcm, Mob, Nv) of the N-Hnb-Cys intramolecular thioesterification device resistant to NCL conditions were evaluated and very promising deprotection conditions of the Acm (PdCl2) group were able to Be highlighted.

Our experimental and theoretical efforts (partner 2 / molecular modeling) are currently focused on (1) fine understanding of the underlying molecular mechanisms and (2) reasoned design of a second generation device. This work has borne fruit because we now have a new device capable of much faster ligation reactions and provide convincing answers to the mechanistic questions about the N-Hnb-Cys device, which is at the heart of the EasyMiniProt project. The work leading to a first joint publication making full use of the synergies of the three partners is in the process of being finalized, including also the exploitation of this second generation device for the synthesis of our DRP model MT7 via the ligation of three fragments in solution by a C-to-N strategy optimized by the partner 3.

Publications:

1. M. T. Jacobsen, X. Ye, M. E. Petersen, M. Galibert, G. S. Lorimer, V. Aucagne, M. S. Kay. A helping hand to overcome solubility challenges in chemical protein synthesis. J. Am. Chem. Soc., 2016, 138, 11775-11782. [selected for a JACS Spotlight : « Helping hand dissolves tough peptides for protein synthesis » J. Am. Chem. Soc., 2016, 138, 13083-13084]
2. G. Martinez, J.-P. Hograindleur, S. Voisin, R. Abi Nahed, T. M. Abd El Aziz, J. Escoffier, J. Bessonnat, C.-M. Fovet, M. De Waard, S. Hennebicq, V. Aucagne, P. F. Ray, E. Schmitt, P. Bulet, C. Arnoult. Spermaurin, a La1-like peptide from the venom of the scorpion Maurus palmatus, improves sperm motility and fertilization in different mammalian species. Mol. Hum. Rep., 2017, 23, 116-131.
3. P. Kessler, P. Marchot, M. Silva D. Servent. The three-finger toxin fold: a multifunctional structural scaffold able to modulate the cholinergic functions J. Neurochem. (2017) 10.1111/jnc.13975

Oral communications:
1. M. T. Jacobsen, M. E. Petersen, V. Aucagne, M. S. Kay. A helping hand to overcome solubility challenges in chemical protein synthesis with application to GroES. Third Chemical Ligation Meeting (Lill’gation), 05/2016, Lille.
2. A. Casas-Mora, M. Galibert, A. F Delmas, V. Aucagne. A non-chromatographic catch-and-release purification method to simplify the synthesis of cysteine-rich peptides. 20ème réunion du Groupe Français des Peptides et Protéines (GFPP 20), 03/2017, Arcachon.
3. V. Aucagne. Simplifier la synthèse de miniprotéines riches en ponts disulfure. 7ème Symposium Français de Synthèse Totale (SFST7), 06/2017, Orsay.

+ 5 posters

Disulfide-rich peptides (DRPs) form an incredibly diverse group of bioactive natural products estimated to millions of distinct sequences. These polycyclic compounds are composed of between 10 and 90 amino acids (aa), more than 10% of which being cysteines involved in disulfide bridges. These medium-sized molecules (often referred as miniproteins), have been identified as highly potent and selective binders of various receptors difficult to target with small molecules, making them attractive new pharmacophores. A growing number of DRPs recently entered the market or are currently in clinical trials highlighting their great therapeutic potential.

If small DRPs (< 30 aa) can be synthesized by conventional solid phase peptide synthesis (SPPS), the production of longer ones represents a great synthetic challenge, and their chemical engineering towards a lead compound is even much more difficult. Most current efforts concentrate on solution-phase convergent synthesis, through chemoselective couplings of unprotected peptide segments by the native chemical ligation (NCL) reaction. But the throughput of these approaches is very low and still complicated by tedious purification and handling steps. This limits and slows down pharmacological studies and therapeutic or diagnostic applications.

This project focuses on the development of technologies for the synthesis of DRPs with much higher throughput than existing methods. One key aspect for intensification of the current processes relies on the realization of the chemoselective assembly of the peptide segments on a solid support, in order to considerably lower the number of intermediate HPLC purification steps. The assembly of the miniproteins in the opposite direction compared to SPPS benefits from an additional “self-purification” feature allowing the use of unpurified peptide segments. We will evaluate several methodologies in parallel on a set of representative DRPs:

(i) A method entirely based on NCL will be designed. This implies the use of a strategy to temporarily mask the reactivity of the thioester and prevent cyclisation or oligomerisation of the segments. This “one ligation approach” offers the advantage of using only two types of reaction conditions, for NCL and unmasking steps, which is particularly interesting for future automation of the process. A major bottleneck for applicability of NCL is the difficult synthesis of peptide thioesters. We recently developed a breakthrough methodology that will be used throughout the project.

(ii) In order to minimize the number of synthetic steps by suppressing the need of masking groups, we will exploit a smart combination of three different chemoselective reactions used in alternation: NCL, strain-promoted azide/alkyne cycloaddition (SPAAC), and copper-catalyzed azide/alkyne cycloaddition (CuAAC). These reactions will be used for grafting the first segment on a solid support then peptide elongation. In the latter case, we will use our original peptidomimetic triazole ligation (PTL) approach exploiting the amide-mimicking nature of the CuAAC product, a triazole.

(iii) Oxidative folding to regioselectively form the SS bonds can be a limiting factor for the production of DRPs: if most naturally occurring DRP can be folded under thermodynamic control, it implies the use of highly dilute conditions that complicate large scale production. We will take advantage of the solid support used for their syntheses for this folding step. An original approach based on fragments with preformed SS will also be implemented.

Finally, the methods developed during this project will be applied to the high-throughput generation of a large number of highly diverse variants of one selected DRP, the muscarinic toxin 7, which is particularly promising from a therapeutic point of view. In particular, we will use an original combinatorial approach, which we expect to lead to the identification of several variants with unprecedented pharmacological profiles.