1,720,986 research outputs found
Post-translational modification of genetically encoded polypeptide libraries
No full textThe genetic encoding of polypeptides with biological display systems enables the facile generation and screening of very large combinatorial libraries of molecules. By post-translationally modifying the encoded polypeptides, chemically and structurally more diverse molecules beyond linear amino acid polymers can be generated. The first post-translational modification applied to encoded polypeptides, the oxidation of cysteine residues to form disulfide bridges, is a natural one and was used to cyclise short peptides soon after the invention of phage display. Recently a range of non-natural chemical strategies for the post-translational modification of encoded polypeptide repertoires were applied to generate optical biosensors, semisynthetic polypeptides, peptide-drug conjugates, redox-insensitive monocyclic peptides or multicyclic peptides, and these strategies are reviewed in this article
Phage display libraries of differently sized bicyclic peptides
No full textPhage selections with combinatorial libraries of uniformly sized bicyclic peptides have recently yielded potent and selective binders of several protein targets. In this work we varied in a combinatorial fashion the ring sizes of bicyclic peptides in phage libraries, expecting that they would yield binders with higher affinities and/or more diverse binding motifs that could be affinity matured. 14 new phage peptide libraries of the format Cys-(Xaa)m-Cys-(Xaa)n-Cys (Xaa are random amino acids, m and n = 3, 4, 5 or 6) were generated and cyclized with tris-(bromomethyl)benzene. Affinity selections against the tumor-associated serine protease urokinase-type plasminogen activator yielded bicyclic peptide inhibitors with a large variety of consensus sequences. Several of the identified consensus sequences were exclusively found in bicyclic peptides having defined ring size combinations. Some of these peptides may bind in orientations that allow affinity maturation of non-conserved regions, while others do not. Having available multiple leads isolated from such bicyclic peptide libraries with variable ring sizes could therefore be a great asset for the generation of high affinity binders
Enzymatic Cyclisation of Peptides with a Transglutaminase
No full textThe ability of microbial transglutaminase to ligate two polypeptides through glutamine and lysine residues was exploited to generate cyclic peptides. Peptides with an N-terminal short glutamine-donor sequence (Ala-Leu-Gln), a variable polypeptide linker and a C-terminal lysine residue could efficiently be cyclised by the enzyme
Peptide Ligands Stabilized by Small Molecules
No full textBicyclic peptides generated through directed evolution by using phage display offer an attractive ligand format for the development of therapeutics. Being nearly 100-fold smaller than antibodies, they promise advantages such as access to chemical synthesis, efficient diffusion into tissues, and needle-free application. However, unlike antibodies, they do not have a folded structure in solution and thus bind less well. We developed bicyclic peptides with hydrophilic chemical structures at their center to promote noncovalent intramolecular interactions, thereby stabilizing the peptide conformation. The sequences of the peptides isolated by phage display from large combinatorial libraries were strongly influenced by the type of small molecule used in the screen, thus suggesting that the peptides fold around the small molecules. X-ray structure analysis revealed that the small molecules indeed formed hydrogen bonds with the peptides. These noncovalent interactions stabilize the peptide-protein complexes and contribute to the high binding affinity
Genetically Encoded Synthetic Polypeptides as Innovative Cancer Therapeutics
No full textLigands based on bicyclic peptides can combine favourable properties of antibodies (good
binding affinity and target specificity) and small molecule ligands (stability, access to
chemical synthesis, diffusion properties) and might be suitable molecular structures for the
development of therapeutics1. By using a combinatorial methodology based on phage display
and a chemical cyclisation reaction2, we isolated a potent (Ki = 53 nM) and selective inhibitor
of human urokinase-type plasminogen activator (uPA), a trypsin-like serine protease that
participates in the turnover of extracellular matrix (ECM) proteins and is implicated in tumor
growth and invasion3. X-ray structure determination of the bicyclic peptide bound to uPA
revealed that both peptide loops engage the target to form a large interaction surface of 701
Å2 with multiple hydrogen bonds and complementary charge interactions, explaining the high
affinity and specificity of the inhibitor. The interface resembles that between two proteins and
suggests that these constrained peptides have the potential to act as small protein mimics.
Moreover, further study revealed that the in vitro-evolved bicyclic peptide are stable in vivo
and remain active for several days overcoming a limitation faced by many in vitro-evolved
peptide leads and promises to be suitable for the generation of long-acting peptide
therapeutics4,5. Its therapeutic effect is currently being tested in vivo
Measuring net protease activities in biological samples using selective peptidic inhibitors
No full textThe measurement of activities from individual proteases in biological samples is difficult because of the numerous proteases, their overlapping activities, and the lack of specific substrates. We applied selective protease inhibitors based on bicyclic peptides (>2000-fold selective over related proteases) to block individual proteases, allowing the quantification of their net activities. In protease mixtures, activity contributions of the serine proteases plasma kallikrein and urokinase-type plasminogen activator (uPA) were accurately quantified. In a tumor extract, we could quantify uPA activity. Because bicyclic peptide inhibitors toward virtually any protease can be generated by phage display, the approach should be applicable to any protease
Bicyclization and Tethering to Albumin Yields Long-Acting Peptide Antagonists
No full textProteolytically stable peptide architectures are required for the development of long-acting peptide therapeutics. In this work, we found that a phage-selected bicyclic peptide antagonist exhibits an unusually high stability in vivo and subsequently deciphered the underlying mechanisms of peptide stabilization. We found that the bicyclic peptide was significantly more stable than its constituent rings synthesized as two individual macrocycles. The two rings protect each other from proteolysis when linked together, conceivably by constraining the conformation and/or by mutually shielding regions prone to proteolysis. A second stabilization mechanism was found when the bicyclic peptide was linked to an albumin-binding peptide to prevent its rapid renal clearance. The bicyclic peptide conjugate not only circulated 50-fold longer (t(1/2) = 24 h) but also became entirely resistant to proteolysis when tethered to the long-lived serum protein. The bicyclic peptide format overcomes a limitation faced by many peptide leads and appears to be suitable for the generation of long-acting peptide therapeutics
Bicyclic Peptide Inhibitor Reveals Large Contact Interface with a Protease Target
No full textFrom a large combinatorial library of chemically constrained bicyclic peptides we isolated a selective and potent (K(i) = 53 nM) inhibitor of human urokinase-type plasminogen activator (uPA) and crystallized the complex. This revealed an extended structure of the peptide with both peptide loops engaging the target to form a large interaction surface of 701 Å(2) with multiple hydrogen bonds and complementary charge interactions, explaining the high affinity and specificity of the inhibitor. The interface resembles that between two proteins and suggests that these constrained peptides have the potential to act as small protein mimics
Chemical Macrocyclization of Peptides Fused to Antibody Fc Fragments
No full textTo extend the plasma half-life of a bicyclic peptide antagonist, we chose to link it to the Fc fragment of the long-lived serum protein IgG1. Instead of chemically conjugating the entire bicyclic peptide, we recombinantly expressed its peptide moiety as a fusion protein to an Fc fragment and subsequently cyclized the peptide by chemically reacting its three cysteine residues with tris-(bromomethyl)benzene. This reaction was efficient and selective, yielding completely modified peptide fusion protein and no side products. After optimization of the linker and the Fc fragment format, the bicyclic peptide was fully functional as an inhibitor (K(i) = 76 nM) and showed an extended terminal half-life of 1.5 days in mice. The unexpectedly clean reaction makes chemical macrocyclization of peptide-Fc fusion proteins an attractive synthetic approach. Its good compatibility with the Fc fragment may lend the bromomethylbenzene-based chemistry also for the generation of antibody-drug conjugates
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