Design and Evaluation of Multi-Target Anti-Virulence Strategies Against Pseudomonas aeruginosa: Quorum-Sensing Network Inhibition Through Pharmacophore Screening, Molecular Docking, and Laboratory Assessment

Pseudomonas aeruginosa is an important opportunistic pathogen associated with hospital-acquired and chronic infections. Its ability to persist in host environments, produce multiple virulence factors, adapt to environmental stress, and withstand antimicrobial treatment makes infections difficult to manage. A substantial part of its pathogenic behavior is regulated by quorum sensing (QS), a network of cell-to-cell chemical communication systems that links population density and environmental conditions to gene expression. QS controls, directly or indirectly, the production of secreted enzymes, pigments, secondary metabolites, rhamnolipids, and phenotypes associated with biofilm development. Although QS inhibitors have attracted considerable attention, inhibition of a single receptor or signaling branch does not necessarily eliminate all virulence-associated phenotypes. Interactions among the Las, Rhl, and Pqs systems, the regulatory contribution of PqsE, and connections with other networks such as Gac/Rsm and c-di-GMP may preserve residual responses. This manuscript therefore considers a multi-target anti-virulence framework and two complementary research hypotheses: intervention in toxin–antitoxin (TA) regulation, with emphasis on HigBA, and disruption of functional amyloid FapC assembly in the biofilm matrix. The computational component describes pharmacophore-based screening and molecular docking directed at LasR, QscR, and PqsR. Three candidate identifiers—STK649448, STK935680, and STL546559—are discussed on the basis of the available interaction analyses. The laboratory component summarizes recorded procedures for antimicrobial peptide and tobramycin assessment, including MIC testing, combination testing, pyocyanin and protease assays, pyoverdine assessment, RNA extraction, and qRT-PCR. Missing raw measurements and methodological uncertainties are explicitly identified. The available evidence supports candidate selection and describes experiments performed, but does not establish receptor antagonism, statistically significant virulence suppression, TA-mediated bacterial self-destruction, or FapC intracellular trapping. The proposed framework is intended to guide staged validation of complementary anti-virulence targets. Keywords: Pseudomonas aeruginosa; quorum sensing; anti-virulence; LasR; RhlR; PqsR; QscR; pharmacophore; molecular docking; toxin–antitoxin; HigBA; FapC; functional amyloid; biofilm.