Development of cyclic peptide inhibitors of coagulation factor XII and matrix metalloproteinase 2

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 inhib

Novel inhibitors of the enzyme activated factor xii (fxiia)

The present invention relates to a bicyclic inhibitor of the coagulation enzyme activated factor XII (FXIIa) comprising or consisting of the peptide (X1)(X2)(X3)n(X4)(X5)(X6)m(X7)(X9)l(X10)(X11)(X12)(X13)(X14)k(X15)(X16), wherein (X1) is present or absent and, if present, is an amino acid; (X2) is an amino acid with a side chain; (X3) is an amino acid and n is between 0 and 3, preferably 0 or 1 and most preferably 0; (X4) is an aliphatic L-amino acid or a cyclic L- amino acid, preferably L, P or an aromatic L-amino acid, and most preferably an aromatic L-amino acid; (X5) is an amino acid; (X6) is an amino acid and m is between 0 and 3, preferably 0 or 1 and most preferably 0; (X7) is an amino acid with a side chain; (X9) is an amino acid and I is between 0 and 3, preferably 0 or 1 and most preferably 0; (X10) is an amino acid; (X11) is an amino acid, preferably Q; (X12) is a hydrophobic L-amino acid, preferably an aliphatic L-amino acid, and is most preferably L; (X13) is an amino acid; (X14) is an amino acid and k is between 0 and 3, preferably 0 or 1 and most preferably 0, (X15) is an amino acid with a side chain; and (X18) is present or absent and, if present, is an amino acid; and wherein the side chains of (X2), (X7) and (X15) are connected via a connecting molecule, said connecting molecule having at least three functional groups, each functional group forming a covalent bond with one of the side chains of (X2), (X7) and (X15)

Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides

The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a 'piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (K-d = 39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. degrees degrees degrees This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic-and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction

Peptide macrocycle inhibitor of coagulation factor XII with subnanomolar affinity and high target selectivity.

Factor XII (FXII) is a plasma protease that has emerged in recent years as a potential target to treat or prevent pathological thrombosis, to inhibit contact activation in extracorporeal circulation, and to treat the swelling disorder hereditary angioedema. While several protein based inhibitors with high affinity for activated FXII (FXIIa) were developed, the generation of small molecule inhibitors has been challenging. In this work, we have generated a potent and selective FXIIa inhibitor by optimizing a peptide macrocycle that was recently evolved by phage display (Ki = 0.84 ± 0.03 nM). A fluorine atom introduced in the para-position of phenylalanine enhanced the binding affinity as much as 10-fold. Furthermore, we improved the proteolytic stability by substituting the N-terminal arginine by norarginine. The resulting inhibitor combines high inhibitory affinity and selectivity with a good stability in plasma (Ki = 1.63 ± 0.18 nM, >27,000-fold selectivity, t1/2 plasma = 16 ± 4 h). The inhibitor efficiently blocked activation of the intrinsic coagulation pathway in human blood ex vivo

Engineered peptide macrocycles can inhibit matrix metalloproteinases with high selectivity

Matrix metalloproteinases (MMPs) are zinc‐dependent endopeptidases at the intersection of health and disease due to their involvement in processes such as tissue repair and immunity as well as cancer and inflammation. Because of the high structural conservation in the catalytic domains and shallow substrate binding sites, selective, small‐molecule inhibitors of MMPs have remained elusive. In a tour‐de‐force peptide engineering approach combining phage‐display selections, rational design of enhanced zinc chelation, and d‐amino acid screening, we succeeded in developing a first synthetic MMP‐2 inhibitor that combines high potency (Ki=1.9±0.5 nm), high target selectivity, and proteolytic stability, and thus fulfills all the required qualities for in cell culture and in vivo application. Our work suggests that selective MMP inhibition is achievable with peptide macrocycles and paves the way for developing specific inhibitors for application as chemical probes and potentially therapeutics

In Vitro-Evolved Peptides Mimic a Binding Motif of the G-Actin-Binding Protein Thymosin-B4 and Serve as Research Tools

Actin is the most abundant protein in eukaryotic cells and is key to many cellular functions. Natural products that specifically recognize the filamentous form of actin (F-actin) such as the bicyclic peptide phalloidin are important tools to study actin and are widely applied for imaging the cytoskeleton in cells. Herein, we aimed at developing peptide-based affinity reagents that selectively bind to the monomeric form of actin (G-actin), for which synthetic probes are not available. Panning a phage display library comprising more than a trillion different bicyclic peptides against G-actin yielded binders with low nanomolar affinity and greater than 1000-fold selectivity over F-actin. Sequence analysis revealed a strong similarity of the peptides\u27 sequences to a region of thymosin-b4, a protein that weakly binds G-actin, and competition binding experiments confirmed a common binding region at the cleft between the subdomains 1 and 3 of actin. We tested the G-actin peptides as probes in pull-down and imaging experiments and applied a peptide variant with improved dissociation constant (Kd = 5 ± 2 nM) to measure the affinity of G-actin-binding natural product toxins.</p

Cyclic peptide FXII inhibitor provides safe anticoagulation in a thrombosis model and in artificial lungs

Inhibiting thrombosis without generating bleeding risks is a major challenge in medicine. A promising solution may be the inhibition of coagulation factor XII (FXII), because its knock-out or inhibition in animals reduced thrombosis without causing abnormal bleeding. Herein, we have engineered a macrocyclic peptide inhibitor of activated FXII (FXIIa) with sub-nanomolar activity (Ki = 370 ± 40 pM) and a high stability (t1/2 > 5 days in plasma), allowing for the preclinical evaluation of a first synthetic FXIIa inhibitor. This 1899 Da molecule, termed FXII900, efficiently blocks FXIIa in mice, rabbits, and pigs. We found that it reduces ferric-chloride-induced experimental thrombosis in mice and suppresses blood coagulation in an extracorporeal membrane oxygenation (ECMO) setting in rabbits, all without increasing the bleeding risk. This shows that FXIIa activity is controllable in vivo with a synthetic inhibitor, and that the inhibitor FXII900 is a promising candidate for safe thromboprotection in acute medical conditions