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 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