Identification and validation of a novel anti-virulent that binds to pyoverdine and inhibits its function

Pseudomonas aeruginosa: causes serious infections in patients with compromised immune systems and exhibits resistance to multiple antibiotics. The rising threat of antimicrobial resistance means that new methods are necessary for treating microbial infections. We conducted a high-throughput screen for compounds that can quench the innate fluorescence of the chromophore region of theP. aeruginosasiderophore pyoverdine, a key virulence factor. Several hits were identified that effectively quench pyoverdine fluorescence, and two compounds considerably improved the survival ofCaenorhabditis eleganswhen worms were challenged withP. aeruginosa. Commercially available analogs of the best hit, PQ3, were tested for their ability to rescueC. elegansfromP. aeruginosaand to interact with pyoverdine via fluorescence and solution NMR spectroscopy.H-1-N-15 and(1)H-C-13 HSQC NMR were used to identify the binding site of PQ3c. The structure model of pyoverdine in complex with PQ3c was obtained using molecular docking and molecular dynamics simulations. PQ3c occupied a shallow groove on pyoverdine formed by the chromophore and N-terminal residues of the peptide chain. Electrostatic interactions and pi-orbital stacking drove stabilization of this binding. PQ3c may serve as a scaffold for the development of pyoverdine inhibitors with higher potency and specificity. The discovery of a small-molecule binding site on apo-pyoverdine with functional significance provides a new direction in the search of therapeutically effective reagent to treatP. aeruginosainfections.