Discovery of β‐nitrostyrene derivatives as potential quorum sensing inhibitors for biofilm inhibition and antivirulence factor therapeutics against <i>Serratia marcescens</i>

Quorum sensing (QS) inhibition has emerged as a promising target for directed drug design, providing an appealing strategy for developing antimicrobials, particularly against infections caused by drug-resistant pathogens. In this study, we designed and synthesized a total of 33 β-nitrostyrene derivatives using 1-nitro-2-phenylethane (NPe) as the lead compound, to target the facultative anaerobic bacterial pathogen <i>Serratia marcescens</i>. The QS-inhibitory effects of these compounds were evaluated using <i>S. marcescens</i> NJ01 and the reporter strain <i>Chromobacterium violaceum</i> CV026. Among the 33 new β-nitrostyrene derivatives, (<i>E</i>)-1-methyl-4-(2-nitrovinyl)benzene (m-NPe, compound 28) was proven to be a potent inhibitor that reduced biofilm formation of <i>S. marcescens</i> NJ01 by 79%. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) results revealed that treatment with m-NPe (50 μg/ml) not only enhanced the susceptibility of the formed biofilms but also disrupted the architecture of biofilms by 84%. m-NPe (50 μg/ml) decreased virulence factors in <i>S. marcescens</i> NJ01, reducing the activity of protease, prodigiosin, and extracellular polysaccharide (EPS) by 36%, 72%, and 52%, respectively. In <i>S. marcescens</i> 4547, the activities of hemolysin and EPS were reduced by 28% and 40%, respectively, outperforming the positive control, vanillic acid (VAN). The study also found that the expression levels of QS- and biofilm-related genes (<i>flhD, fimA, fimC, sodB, bsmB, pigA, pigC</i>, and <i>shlA</i>) were downregulated by 1.21- to 2.32-fold. Molecular dynamics analysis showed that m-NPe could bind stably to SmaR, RhlI, RhlR, LasR, and CviR proteins in a 0.1 M sodium chloride solution. Importantly, a microscale thermophoresis (MST) test revealed that SmaR could be a target protein for the screening of a quorum sensing inhibitor (QSI) against <i>S. marcescens</i>. Overall, this study highlights the efficacy of m-NPe in suppressing the virulence factors of <i>S. marcescens</i>, identifying it as a new potential QSI and antibiofilm agent capable of restoring or improving antimicrobial drug sensitivity.