Computational and Experimental Validation of 4-Amino-3- Hydroxynaphthalene-1-Sulfonic Acid as a Novel Antibiofilm Agent.

Abstract

Pseudomonas aeruginosa (PA) is a gram-negative opportunistic pathogen, and is well-known for its biofilm-forming ability and antimicrobial resistance, challenging the treatment of infections, particularly in immunocompromised individuals. Biofilms are the complex communities of bacterial cells encased in a protective extracellular matrix, serving as a key survival strategy for PA and enabling them to resist antimicrobial therapies and evade host immune defense mechanisms effectively. This study aims to explore the antibiofilm potential of 4-amino-3-hydroxynaphthalene-1-sulfonic acid (ANS) and its derivatives by targeting the major biofilm-forming pathway, the quorum sensing (QS) system in PA, through in vitro and in silico approaches. Biochemical assays and confocal imaging demonstrated the dose-dependent biofilm-inhibitory activity of ANS in both clinical and standard PA strains. Growth curve analysis further confirmed that ANS exhibited antibiofilm effects without significant antimicrobial activity. Additionally, violacein quantification assays using Chromobacterium violaceum revealed the ANS interferes with the QS pathway. In silico analyses, encompassing molecular docking and molecular dynamic (MD) simulations, confirmed the stable interactions between ANS and LasR regulator, demonstrating higher binding energy compared to other QS regulators (LasI, RhlR, PqsA, PqsC, PqsD). In silico structure-based modifications predicted ANS derivative (L19) with potential antibiofilm activity, further experimental validation is required to confirm the activity of L19. Notably, ANS is non-toxic to HepG2 cells at concentrations up to 800 μg/mL, indicating its potential safety for therapeutic applications. These findings underscore the significance of ANS and its derivatives as promising candidates for the development of novel antibiofilm agents targeting the QS system in PA.