Functionalized Proline‐Rich Peptides Bind the Bacterial Second Messenger c‐di‐GMP

c‐di‐GMP is an attractive target in the fight against bacterial infections since it is a near ubiquitous second messenger that regulates important cellular processes of pathogens, including biofilm formation and virulence. Screening of a combinatorial peptide library enabled the identification of the proline‐rich tetrapeptide Gup‐Gup‐Nap‐Arg, which binds c‐di‐GMP selectively over other nucleotides in water. Computational and CD spectroscopic studies provided a possible binding mode of the complex and enabled the design of a pentapeptide with even higher binding strength towards c‐di‐GMP. Biological studies showed that the tetrapeptide inhibits biofilm growth by the opportunistic pathogen P. aeruginosa. Target the messenger: Proline‐rich peptides with aromatic and cationic moieties were developed to bind the bacterial second messenger c‐di‐GMP through π–π stacking and hydrogen bonding. The tetrapeptides, which were identified through combinatorial screening, inhibit biofilm formation by the opportunistic pathogen P. aeruginosa.