Design, Synthesis, and Biological Evaluation of Phenyloxadiazole Sulfoxide Derivatives as Potent Pseudomonas aeruginosa Biofilm Inhibitors

Abstract

With the development of antimicrobial agents, researchers have developed new strategies through key regulatory systems to block the expression of virulence genes without affecting bacterial growth. This strategy can minimize the selective pressure that leads to the emergence of resistance. Quorum sensing (QS) is an intercellular communication system that plays a key role in the regulation of bacterial virulence and biofilm formation. Studies have revealed that the QS system controls 4-6% of the total number of P. aeruginosa genes, and quorum sensing inhibitors (QSIs) could be a promising target for developing new prevention and treatment strategies against P. aeruginosa infection. In this study, four series of phenyloxadiazole and phenyltetrazole sulfoxide derivatives were synthesized and evaluated for their inhibitory effects on P. aeruginosa PAO1 biofilm formation. Our results showed that 5b had biofilm inhibitory activity and reduced the production of QS-regulated virulence factors in P. aeruginosa. In addition, silico molecular docking studies have shown that 5b binds to the P. aeruginosa QS receptor protein LasR through hydrogen bond interaction. Preliminary structure-activity relationship and docking studies show that 5b has broad application prospects as an anti-biofilm compound, and further research will be carried out in the future to solve the problem of microbial resistance.

Keywords: Pseudomonas aeruginosa; biofilm; inhibitor; quorum sensing; sulfoxide.

Figure 1

(A) Sulfur compounds found in natural products and their biological activities. (B) Structures and strategies of the design of the target compounds.

Scheme 1

The synthesis route of the title compounds.

Figure 2

The effects of 5a, 5b, 5f, 5j, and 5k on QS system reporter strains (A) PAO1-lasB-gfp, (B) PAO1-rhlA-gfp, and (C) PAO1-pqsA-gfp. The experiments were performed in triplicate.

Figure 3

Effects of 5b on PAO1-lasB-gfp and P. aeruginosa PAO1 biofilm growth and formation. (A) Growth at different concentrations of 5b (20, 10, 5, 2.5, 1.25μM). (B) Dose-dependent inhibition curves of 5b incubated with the QS monitors PAO1-lasB-gfp. (C) IC₅₀ values calculations were based on three biological replicates and performed by nonlinear fitting, using Graphpad Prism 6 software. (San Diego, CA, USA). The IC₅₀ values of 5b were 3.53 ± 0.16 μM for PAO1-lasB-gfp. (D) Growth at different concentrations of 5b (50, 25, 12.5, 5, 2.5 μM) for 16 h. (E) Inhibition of biofilm formation by different concentrations of 5b (50, 25, 12.5, 5, 2.5 μM) for 24 h in microtiter plate. (F) CLSM images of biofilms formed for 24 h with control and 5b (50 μM), with 2-aminobenzimidazole used as positive control (An equal amount of dimethyl sulfoxide solvent was set as the control group). Data represent the average of three independent determinations of triplicate samples.

Figure 4

Effects of 5b on the production of (A) elastase, (B) pyocyanin, and (C) rhamnolipid at different concentrations (50, 25, 12.5 μM, and control group). Error bars are means ± SDs. ** = p < 0.01 versus the control, **** = p < 0.0001 versus the control.

Figure 5

Predicted binding model of compounds (OdDHL, 5b, 5f) and LasR (PDB code: 2uv0). (A) Overlay of the crystal ligand OdDHL (green) with the docked pose of 5b (blue) and 5f (brown) in the LasR LBD domain (PDB ID:2UV0). (B,C) 2D structure-diagrams of protein–ligand complexes.