Drug repositioning as an antivirulence strategy: Ketoprofen targets quorum sensing and biofilm in Serratia sp.

Serratia sp. are opportunistic Gram-negative bacteria capable of forming robust biofilms and expressing a wide range of quorum sensing (QS)-regulated virulence factors, including motility, protease secretion, and prodigiosin production. The rise of multidrug-resistant strains has emphasized the urgent need for alternative therapeutic strategies targeting bacterial virulence rather than viability. In this context, nonsteroidal anti-inflammatory drugs (NSAIDs) such as ketoprofen have emerged as potential quorum-quenching agents. This study explores the antivirulence activity of ketoprofen against Serratia sp. through a combination of in-vitro phenotypic assays and an advanced in-silico framework. In-vitro assays demonstrated that sub-inhibitory concentrations of ketoprofen significantly impair key virulence traits. Ketoprofen exhibited up to 90.68% inhibition of initial bacterial adhesion and disrupted mature biofilms, reducing biomass by up to 79.1%. Furthermore, motility assays revealed profound inhibition of both swimming and swarming behaviors, alongside a 100% suppression of protease activity and a 60.4% reduction in prodigiosin production, without exerting direct bactericidal effects. To elucidate the molecular basis of this competitive antagonism, an integrative computational approach was deployed. Deep learning-driven molecular docking (GNINA) revealed that ketoprofen targets the LuxR-type QS receptor SmaR with exceptional and reproducible affinity (mean Vina affinity of -9.51 kcal/mol), vastly outperforming natural acyl-homoserine lactone (AHL) autoinducers. Kinetic stability was validated through 5 ns vacuum molecular dynamics (MD), followed by 100 ns explicit solvent MD simulations in independent replicates. The trajectories confirmed remarkable positional retention of the ligand (Ligand RMSD ~ 0.08-0.16 nm) within the binding pocket. Rigorous MM/PBSA free energy calculations on the equilibrated trajectories yielded highly favorable binding free energies (ΔG ranging from - 19.90 to -30.11 kcal/mol), driven by massive enthalpic contributions (ΔH up to -34.20 kcal/mol) and a persistent network engaging 10 to 14 key interacting residues. Overall, these findings demonstrate that ketoprofen acts as a highly stable "molecular plug", effectively outcompeting endogenous AHL signals to lock the SmaR receptor in an inactive state. This highlights ketoprofen's immense potential as a repurposed antivirulence agent for combating biofilm-associated and multidrug-resistant Serratia infections.