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.

Development of an albumin-binding ligand for prolonging the plasma half-life of peptide therapeutics

Alessandro Zorzi

Peptides represent a promising format for the development of therapeutics since they combine the advantages of proteins and small molecules. Several techniques based on rational design or in vitro evolution can be used to develop peptides as therapeutics. A limitation of peptides is a rapid clearance from the blood circulation when applied intravenously, preventing their application in therapies that require prolonged drug exposure. Several methods have been tested to extend the in vivo half-life of peptides, including fusion to long-lived serum proteins, PEGylation and conjugation to non-covalent albumin-binding ligands. A particularly attractive approach is a âpiggy backâ strategy in which peptides bind via a ligand to albumin without increasing significantly their small size. Acylation with fatty acids is the most successful strategy for delaying peptide clearance. The attachment of either myristic or palmitic acid to insulin and GLP-1 led to the approval of three acylated peptide drugs as daily injectable. Nevertheless, acylated conjugates generally present low solubility and weak affinity for albumin. Peptides have been explored as an alternative, as their format offers multiple approaches for affinity maturation as well as an easier synthesis of the conjugates. However, the albumin-binding peptides developed so far do not overcome the disadvantages faced with fatty acids. The first aim of my project was the development of an albumin-binding tag that combines the favorable properties of both fatty acids and peptides, and overcomes at the same time their limitations. This tag was engineered in order to have i) a high affinity for human albumin, ii) a high solubility in physiological solutions, and iii) an efficient synthesis in conjunction with therapeutic peptides. Towards this goal, I created a chimeric format consisting of a fatty acid combined with a short linear peptide. After iterative rounds of affinity screening, an evolved peptide-fatty acid tag able to bind human, rat and rabbit albumin with Kd values of 39, 220 and 320 nM, respectively, was obtained. This makes it the best albumin-binding tag among all of the peptide- or fatty acid-based ligands that have been developed. This tag can be easily synthesized in conjugation with peptides on standard automated synthesizers. Finally, it exhibits a high solubility due to four negatively charged amino acids in the peptide sequence, despite the presence of a 16-carbon palmitic hydrophobic tail. The second aim of my project was the application of this tag to three bioactive bicyclic peptides, previously developed in the laboratory, in order to improve their in vivo half-lives. I demonstrated that the conjugation has, overall, a mild impact on peptidesâ binding affinity towards their targets and human albumin, and increases their proteolytic stability. Pharmacokinetic experiments showed that the tag extends the elimination half-life of bicyclic peptides in rats and rabbits approximately 25-fold to over seven and five hours, respectively. Tag conjugation to a bicyclic peptide targeting coagulation factor XII led to an efficient inhibition of coagulation in rabbits up to eight hours, suggesting a potential application in humans as anti-thrombotic drug. These results indicated that the tag will efficiently prolong the exposure of target proteins to bioactive peptides in humans due to its higher affinity for human albumin than that of rat and rabbit.

EPFL2017

New methods for developing (bi)cyclic peptides by phage display

Camille Villequey

Cyclic peptide ligands are a promising molecular format for the development of therapeutics. They combine some of the advantages of large protein therapeutics (high affinity and specificity) and of small molecule drugs (accessibility to chemical synthesis and low immunogenicity). With phage display, large combinatorial libraries of more than one billion mono- and polycyclic peptides can be developed and screened against virtually any target. During phage affinity selections, the physical link between phenotype (encoded peptide) and genotype (phage DNA) allows the identification of enriched peptide binders by sequencing of the phage vector DNA. Based on these concepts, our laboratory has established a robust chemistry in which phage displayed peptides containing two or three cysteines are cyclized or bi-cyclized with thiol-reactive linkers. This procedure has been generally applied for the isolation of potent and selective (bi)cyclic peptide ligands with therapeutic potential. The isolation of binders with optimal properties is highly dependent on the possibility to generate (bi)cyclic peptide libraries with high structural diversity and on the ability to thoroughly decode the phage selection output. In my PhD work, I developed new methods to generate large and structurally diverse libraries and to decode more efficiently phage selected peptides. In my first project, I explore the cyclization of peptides with two chemical bridges as a method to provide rapid access to phage-encoded libraries with high scaffold diversity. To this end, a range of twelve structurally diverse chemicals were tested for thiol-reactivity and phage compatibility. Subsequently, peptide libraries of the format XCXnCXnCX (X = random amino acid, C = cysteine, n = 3 or 4) were cyclized with six of these chemical linkers and panned against the protease plasma kallikrein. The selection yielded inhibitors with remarkable affinity (sub-nM KI), target selectivity (>1000-fold) and proteolytic stability (t1/2 in plasma > 3 days) despite a relatively small molecular mass (~ 1200 Da). In the second project, I developed phage-encoded libraries of small cyclic peptides in order to identify ligands that have the potential to be orally absorbed. A major challenge for the generation of such library is the usually low diversity of compounds due to the limited number of amino acids. In this work, we addressed this limitation by taking advantage of a recently discovered peptide cyclization reaction in which the thiol of a cysteine is ligated with the N-terminal amino group using a chemical reagent. We applied this strategies to two phage-encoded peptide libraries of four and five amino acids (XXXC and XXXXC) that were cyclized with eight chemical linkers yielding a collection of more than 1.3 million compounds. Panning the libraries against the two disease targets plasma kallikrein and coagulation factor XIa yielded ligands with single-digit micromolar affinity. In a third project, I developed a strategy to bypass the bacterial infection step in phage display. Bacterial infection is usually crucial for the propagation and identification of enriched clones by DNA sequencing. However, mutations or chemical modifications of the phage coat proteins, sometimes lead to decreased infection rates and therefore compromise the entire procedure. Consequently, there is an interest for developing methods in which this step could be completely omitted.

EPFL2017

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

Christian Heinis, Simon Jan Middendorp, Jonas Alfred Karl Wilbs

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 (K-i = 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, t(1/2)(plasma) =16 +/- 4 h). The inhibitor efficiently blocked activation of the intrinsic coagulation pathway in human blood ex vivo.

2017

Development of an albumin-binding ligand for prolonging the plasma half-life of peptide therapeutics

Alessandro Zorzi

EPFL2017

New methods for developing (bi)cyclic peptides by phage display

Camille Villequey

EPFL2017