Development of cyclic peptide inhibitors of coagulation factor XIa for safer anticoagulation

Vanessa Carle
EPFL, 2019
EPFL thesis

Abstract

Hemostasis is a complex physiological process responsible for the prevention of blood loss caused by vascular injury. The dysregulation of this delicate mechanism can lead to thromboembolic diseases, the leading cause of death worldwide. Anticoagulants are drugs applied for the treatment and prevention of this class of diseases. Their major side effect - severe bleeding, which is a life-threatening condition - strongly limits their potential and leads to events of inappropriate treatment. Thus, there is a strong unmet need for a safer class of anticoagulants. Coagulation factor XI (FXI), a serine protease from the intrinsic pathway of coagulation, has been identified as a promising target for safer anticoagulation. Studies in various animal models and epidemiological data have shown that FXI is involved in the pathogenesis of thrombosis, while not being essential for hemostasis. Various FXI-targeting molecules are currently in development, including small molecules, monoclonal antibodies, and antisense oligonucleotides. Peptide-based FXIa inhibitors have not yet been developed, despite the attractive properties of peptides as a drug format, including high affinity and selectivity, tunable pharmacokinetic properties, a fast onset of action and absence of toxic metabolic products. The aim of my thesis was the development of cyclic peptide-based FXIa inhibitors for safer anticoagulation. In a first project, I have developed a large cyclic peptide phage display library with unprecedented structural diversity. In a proof-of-concept selection performed with the library, peptides displaying long consensus sequences were enriched and potent binders were isolated, confirming the importance of having access to large and structurally diverse libraries. Next, I had applied the new phage display library for selecting cyclic peptide inhibitors of FXIa. Isolated peptides were characterized, and a particularly potent inhibitor was further optimized. The resulting peptide showed a strong anticoagulation effect ex vivo, which was comparable to the effect of heparin at therapeutic concentrations. I then modified the peptide, which improved its plasma stability and prolonged the circulation time in vivo. The modified peptide inhibited plasma coagulation after administration to rabbits and in an ex vivo model of hemodialysis using human blood. Finally, I had stumbled over a cyclic peptide FXIa inhibitor that had a moderate inhibitory constant but a particularly strong anticoagulation activity. In a structure-activity relationship analysis of the peptide, I identified the most important residue for the strong anticoagulation activity, which pointed to an electrostatic interaction and to potentially fast binding kinetics. The peptide indeed bound much faster than other FXIa inhibitors, showing that rapid engagement with the FXIa is important for efficient anticoagulation. Based on these findings, I concluded that it is worth to take into consideration the binding on-rate of inhibitors of coagulation proteases in the future development of anticoagulants.

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Vanessa Carle
EPFL, 2019
EPFL thesis

Abstract

Official source

https://infoscience.epfl.ch/record/268512?ln=en

About this result

Related concepts (15)

Related concepts

Related publications (29)

Related publications

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

Mass spectrometry (MS) has emerged over the last two decades as the analytical technique of choice in systems-level protein studies, known as proteomics. Two are the MS-based approaches generally applied to proteomics: bottom-up (BU), which relies on the proteolytic digestion of proteins into short (~10 amino acids) peptides, and top-down (TD), where proteolysis is omitted, intact proteins are detected and fragmented in gas phase. Both methods present advantages as well as drawbacks. Here, we sought to establish a complete platform to put forward a third MS-based proteomic approach; middle-down (MD). It implies protein digestion as in BU, but aims to generate large peptides which size approaches the one of small intact proteins that are readily analyzed in TD. This novel domain aims to account for the shortcoming of both classical approaches. Until now, the main reasons behind the limited use of MD proteomics have been the lack of easy-to-use cleaving agents capable of producing peptides in the desired 3-15 kDa mass range with high specificity, limitations in MS and allied instrumentation, and the absence of dedicated bioinformatics tools for processing of acquired data. The latter greatly impedes the next milestone in MD proteomics â large-scale analysis. MD can potentially combine the analysis of large portions of proteins carrying set of biologically-relevant modifications â allowing exploring proteins of molecular weight or complexity incompatible with current TD capabilities â with the high-throughput hallmark of BU proteomics. Here, we first in silico evaluated the potential target amino acid residues to produce peptides in the MD mass range within the proteomes of different organisms. This bioinformatics work was followed by an experimental study based on synthetic MD-sized peptides, aimed at determining the optimal MS and tandem MS parameters for large peptide characterization. Next, we pursued two distinct ways of performing MD proteolysis: i) the use of an enzyme and ii) the use of a chemical reagent. We selected the protease Sap9 as a target enzyme for MD, which we fully characterized and successfully applied to the study of a mixture of monoclonal antibodies, where it showed an advantage over traditional BU in terms of reduced introduction of artifacts to the sample, allowing the post-translational modification investigation and unambiguous antibody identification. The chemical cleavage way we addressed via judicious protocol optimization for hydrolysis at the N-terminal side of cysteine with NTCB reagent. We also advanced MD protocols by generation of large (~50 kDa) subunits of monoclonal antibodies through the use of papain and another more specific novel protease, GingisKHAN, combined with new MS signal processing and data analysis capabilities. The developed workflow improved mapping of the connectivity of cysteines involved in inter- and intra-molecular disulfide bridges in antibodies. To summarize, we demonstrated that MD approach to mass spectrometry and proteomics is a powerful, yet underdeveloped, complement to BU and TD. This work has benchmarked MD for targeted protein analysis. In the near future, with advancements of the field, we envision its growing use for large-scale complex proteome analysis.

EPFL2016

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