Pattern-Based Sensing of Peptides and Aminoglycosides with a Single Molecular Probe

A coumarin-based molecular probe can be used for the sensing of peptides and aminoglycoside antibiotics. The probe reacts with the primary amine group(s) of the analytes to give a mixture of covalent adducts with distinct colors. Each analyte gives rise to a characteristic UV–vis spectrum. A pattern-based analysis of the spectra allows identifying structurally related analytes. Furthermore, it is possible to obtain information about the quantity and the purity of the analytes

Bicyclization and Tethering to Albumin Yields Long-Acting Peptide Antagonists

Proteolytically 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 (<i>t</i>_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

Phage Selection of Photoswitchable Peptide Ligands

Photoswitchable ligands are powerful tools to control biological processes at high spatial and temporal resolution. Unfortunately, such ligands exist only for a limited number of proteins and their development by rational design is not trivial. We have developed an <i>in vitro</i> evolution strategy to generate light-activatable peptide ligands to targets of choice. In brief, random peptides were encoded by phage display, chemically cyclized with an azobenzene linker, exposed to UV light to switch the azobenzene into <i>cis</i> conformation, and panned against the model target streptavidin. Isolated peptides shared strong consensus sequences, indicating target-specific binding. Several peptides bound with high affinity when cyclized with the azobenzene linker, and their affinity could be modulated by UV light. The presented method is robust and can be applied for the <i>in vitro</i> evolution of photoswitchable ligands to virtually any target

Development of Potent and Selective <i>S. aureus</i> Sortase A Inhibitors Based on Peptide Macrocycles

Sortases are transpeptidase enzymes that anchor surface proteins, including virulence factors, to the cell wall of Gram-positive bacteria, and they are potential targets for the development of anti-infective agents. While several large compound libraries were searched by high-throughput screening, no high-affinity inhibitors of sortases could be developed to date. Here, we applied phage display to screen billions of peptide macrocycles against sortase A (SrtA) of <i>Staphylococcus aureus</i> (<i>S. aureus</i>). We were able to identify potent and selective inhibitors of SrtA that blocked SrtA-mediated anchoring of synthetic substrates to the surface of live <i>S. aureus</i> cells. A region present in all inhibitory peptides (Leu-Pro-Pro) resembled the natural substrates of SrtA (Leu-Pro-Xaa-Thr-Gly), suggesting that the macrocycles bind to the enzyme’s active site and that they form similar molecular contacts as natural substrates. The evolved peptide macrocycles may be used as lead structures for the development of potent peptidomimetic SrtA inhibitors

Bicyclic Peptide Inhibitor Reveals Large Contact Interface with a Protease Target

From a large combinatorial library of chemically constrained bicyclic peptides we isolated a selective and potent (<i>K</i>_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 Ų 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

To 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 (<i>K</i>_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

Bicyclic Peptide Ligands Pulled out of Cysteine-Rich Peptide Libraries

Bicyclic peptide ligands were found to have good binding affinity and target specificity. However, the method applied to generate bicyclic ligands based on phage-peptide alkylation is technically complex and limits its application to specialized laboratories. Herein, we report a method that involves a simpler and more robust procedure that additionally allows screening of structurally more diverse bicyclic peptide libraries. In brief, phage-encoded combinatorial peptide libraries of the format X_<i>m</i>CX_<i>n</i>CX_<i>o</i>CX_<i>p</i> are oxidized to connect two pairs of cysteines (C). This allows the generation of 3 × (<i>m</i> + <i>n</i> + <i>o</i> + <i>p</i>) different peptide topologies because the fourth cysteine can appear in any of the (<i>m</i> + <i>n</i> + <i>o</i> + <i>p</i>) randomized amino acid positions (X). Panning of such libraries enriched strongly peptides with four cysteines and yielded tight binders to protein targets. X-ray structure analysis revealed an important structural role of the disulfide bridges. In summary, the presented approach offers facile access to bicyclic peptide ligands with good binding affinities

Boosting the Sensitivity of Ligand–Protein Screening by NMR of Long-Lived States

A new NMR method for the study of ligand–protein interactions exploits the unusual lifetimes of long-lived states (LLSs). The new method provides better contrast between bound and free ligands and requires a protein–ligand ratio ca. 25 times lower than for established <i>T</i>_1ρ methods, thus saving on costly proteins. The new LLS method was applied to the screening of inhibitors of urokinase-type plasminogen activator (uPA), which is a prototypical target of cancer research. With only 10 μM protein, a dissociation constant (<i>K</i>_D) of 180 ± 20 nM was determined for the strong ligand (inhibitor) UK-18, which can be compared with <i>K</i>_D = 157 ± 39 nM determined by the established surface plasmon resonance method