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.

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

Screening of structurally diverse bicyclic peptide libraries by phage display

Davide Bertoldo

Bicyclic peptides are an attractive molecule format for the development of therapeutics and research tools. Our laboratory is specialized on the development of bicyclic peptide ligands by phage display. In brief, libraries of randompeptides each containing three cysteines are displayed on the surface of a phage and cyclized with a trivalent thiol-reactive chemical reagent. Ligands of a target of interest are isolated by affinity selection and their sequence identified by DNA sequencing. The first goal of my thesis was to test if more diverse bicyclic peptide libraries can be generated if linear peptides on phage are cyclized in parallel with multiple different chemical cyclization linkers. Towards this end, we cyclized peptide libraries of the format XCXnCXnCX (X = random amino acid, C = cysteine, n = 3 or 4) with three different chemicals and panned the library against the protease urokinase-type plasminogen activator (uPA). Interestingly, different peptide sequences were enriched when the phage peptide library was cyclized with the different chemical linkers, indicating that a larger diversity of bicyclic peptides is generated. Screening the larger library generated binders with a higher binding affinity. In the second project I carried out, I isolated bicyclic peptide ligands to a protein of therapeutic interest, b-catenin. b-catenin is the main translational co-activator of theWnt cell signaling pathway. Pathological upregulation of b-catenin is associated with the majority of colorectal cancer. Blocking b-catenin activity is thus an attractive therapeutic approach. However, so far the development of b-catenin inhibitors has been challenging due to the flat and featureless surface of the protein, lacking binding pockets for synthetic ligands. Screening a phage display library comprising >12 billion bicyclic peptides yielded binders for different protein epitopes. The binding site of four peptides was identified to correspond to that of ICAT (inhibitor of b-catenin and Tcf), which is a prime target site on b-catenin for therapeutic intervention and to which no synthetic ligands could be isolated so far. In the third project, we isolated and characterized bicyclic peptides binding to globular actin (G-actin). G-actin constitutes the fundamental building block of microfilaments (also named F-actin) in eukaryotic cells. Beyond this structural function, G-actin has a role in gene transcription and chromatin remodeling. While specific F-actin probes are available for in vitro applications, G-actin probes are lacking. To fulfil this demand, we screened a library of bicyclic peptides for G-actin binders and isolated ligands with dissociation constants in the low nanomolar range. The isolated peptides were found to bind to the same surface region as thymosin b4 and a range or marine toxins. The bicyclic peptide G-actin ligands allowed measurement of the binding affinity of natural G-actin ligands in fluorescence anisotropy competition assays.

EPFL2016

Site-Specific Polymer Attachment to HR2 Peptide Fusion Inhibitors against HIV-1 Decreases Binding Association Rates and Dissociation Rates Rather Than Binding Affinity

Maarten Danial, Harm-Anton Klok, Frederik Roman Wurm

A popular strategy for overcoming the limited plasma half-life of peptide heptad repeat 2 (HR2) fusion inhibitors against HIV-1 is conjugation with biocompatible polymers such as poly(ethylene glycol) (PEG). However, despite improved resistance to proteolysis and reduced renal elimination, covalent attachment of polymers often causes a loss in therapeutic potency. In this study, we investigated the molecular origins of the loss in potency upon conjugation of linear, midfunctional, and hyperbranched PEG-like polymers to peptides that inhibit HIV-1 host cell membrane fusion. Fluorescence binding assays revealed that polymer conjugation imparted mass transport limitations that manifested as coexistent slower association and dissociation rates from the gp41 target on HIV-1. Furthermore, reduced association kinetics rather than affinity disruption was responsible for the loss in antiviral potency. Finally, the binding assays indicated that the unmodified HR2-derived peptide demonstrated diffusion-limited binding. The observed high potency of the unmodified peptide in HIV-1 inhibition assays was therefore attributed to rapid peptide conformational changes upon binding to the gp41 prehairpin structure. This study emphasizes that the view in which polymer ligation to therapeutic peptides inadvertently leads to loss in potency due to a loss in binding affinity requires scientific verification on a case-by-case basis and that high peptide potency may be due to rapid target binding events.