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

Genetic, Structural, and Phenotypic Properties of MS2 Coliphage with Resistance to ClO₂ Disinfection

Common water disinfectants like chlorine have been reported to select for resistant viruses, yet little attention has been devoted to characterizing disinfection resistance. Here, we investigated the resistance of MS2 coliphage to inactivation by chlorine dioxide (ClO₂). ClO₂ inactivates MS2 by degrading its structural proteins, thereby disrupting the ability of MS2 to attach to and infect its host. ClO₂-resistant virus populations emerged not only after repeated cycles of ClO₂ disinfection followed by regrowth but also after dilution-regrowth cycles in the absence of ClO₂. The resistant populations exhibited several fixed mutations which caused the substitution of ClO₂-labile by ClO₂-stable amino acids. On a phenotypic level, these mutations resulted in a more stable host binding during inactivation compared to the wild-type, thus resulting in a greater ability to maintain infectivity. This conclusion was supported by cryo-electron microscopy reconstruction of the virus particle, which demonstrated that most structural modification occurred in the putative A protein, an important binding factor. Resistance was specific to the inactivation mechanism of ClO₂ and did not result in significant cross-resistance to genome-damaging disinfectants. Overall, our data indicate that resistant viruses may emerge even in the absence of ClO₂ pressure but that they can be inactivated by other common disinfectants