Targeting virulence: Structure-guided development of pyocyanin inhibitors against Pseudomonas aeruginosa

Antimicrobial resistance is an escalating healthcare threat, necessitating new anti-infectives targeting novel structures.[1] An emerging strategy focuses on reducing the production of virulence factors to disarm pathogens rather than kill them, potentially mitigating resistance development. Pseudomonas aeruginosa (P. aeruginosa) is a gram-negative, opportunistic human pathogen that is particularly prevalent in nosocomial infections and chronic infections in cystic fibrosis patients.[2] It produces a wide variety of virulence factors, including the phenazine pyocyanin. Pyocyanin plays an important role in the formation of reactive oxygen species and biofilms, as well as in pathogen toxicity.[3]

In this study, we developed virulence factor inhibitors that target the biosynthesis of pyocyanin by inhibiting PhzB, a key enzyme in its production. Using raloxifene, known to reduce pyocyanin production in cellulo[4], we employed a structure-based approach to design and synthesize analogs exploring various aromatic and aliphatic substituents at the 3-position of the benzo[b]thiophene scaffold. To minimize off-target effects on the human estrogen receptor, we also investigated the effect of the phenolic hydroxyl groups attached to the scaffold. Isothermal titration calorimetry indicated that the synthesized compounds bind to BcPhzB (an ortholog of PhzB from Burkholderia cepacia) with low micromolar affinity. Assays on pyocyanin production in P. aeruginosa PA14 demonstrated inhibitory effects within the low micromolar concentration range, indicating a substantial enhancement compared to the parent compound. Additionally, high-resolution X-ray cocrystal structures elucidated the binding modes of several compounds, underscoring the potential for the development of additional analogs.