Computer-aided identification of recognized drugs as Pseudomonas aeruginosa quorum-sensing inhibitors

Abstract

Attenuation of Pseudomonas aeruginosa virulence by the use of small-molecule quorum-sensing inhibitors (referred to as the antipathogenic drug principle) is likely to play a role in future treatment strategies for chronic infections. In this study, structure-based virtual screening was used in a search for putative quorum-sensing inhibitors from a database comprising approved drugs and natural compounds. The database was built from compounds which showed structural similarities to previously reported quorum-sensing inhibitors, the ligand of the P. aeruginosa quorum-sensing receptor LasR, and a quorum-sensing receptor agonist. Six top-ranking compounds, all recognized drugs, were identified and tested for quorum-sensing-inhibitory activity. Three compounds, salicylic acid, nifuroxazide, and chlorzoxazone, showed significant inhibition of quorum-sensing-regulated gene expression and related phenotypes in a dose-dependent manner. These results suggest that the identified compounds have the potential to be used as antipathogenic drugs. Furthermore, the results indicate that structure-based virtual screening is an efficient tool in the search for novel compounds to combat bacterial infections.

FIG. 1.

2D structures of the compounds tested in this study. (A) Structures of the known LasR binding compounds used in the 2D similarity search against the SuperNatural and SuperDrug databases (OdDHL, TP-1, furanone C30, and patulin) and included in the SB-VS for reference (OdDHL) and comparison (all). (B) Structures of the six compounds tested for the ability to inhibit LasR in P. aeruginosa.

FIG. 2.

Dose-response curves of the three drugs salicylic acid (A), nifuroxazide (B), and chlorzoxazone (C) when incubated together with PAO1 lasB::gfp(ASV). The three compounds reduce the RFU level (GFP fluorescence units divided by OD₄₅₀) at various concentrations (right), indicating inhibition of LasR, while displaying no effect on growth (left). Growth is displayed as the increase in OD₄₅₀ compared to the initial level. Salicylic acid and nifuroxazide were tested at concentrations of 400 (▪), 200 (Δ), 50 (•), 12.5 (□), 3.125 (▴), and 0 (○) μg/ml, while chlorzoxazone was tested at 200 (▪), 100 (▵), 20 (•), 6.25 (□), 1.5625 (▴), and 0 (○) μg/ml. Results are representative of three independent experiments.

FIG. 3.

Expression of rhlA::gfp(ASV) (A) and pqsA::gfp(ASV) (B) in wild-type and lasR mutant P. aeruginosa PAO1 treated with the three identified LasR inhibitors. Results are average RFU values taken from a single time point measurement corresponding to maximal induction of the reporters in the late log phase of growth. Inhibitors were added at concentrations of 200 μg/ml for salicylic acid and nifuroxazide and 100 μg/ml for chlorzoxazone. Averages and SDs of eight replicates are shown.

FIG. 4.

Inhibition of exogenous protease production (A), pyoverdine production (B), and rhamnolipid production (C) in P. aeruginosa treated with the three identified LasR inhibitors. 1, untreated; 2, lasI rhlI mutant (QS deficient); 3, salicylic acid treated; 4, nifuroxazide treated; 5, chlorzoxazone treated. Results were taken after 18 h of growth at 37°C. Averages and SDs of five replicates are shown. Relat., relative; activ., activity; prod., production.

FIG. 5.

CLSM pictures of 4-day-old P. aeruginosa biofilms. A, untreated; B, lasI rhlI mutant (QS deficient); C, salicylic acid treated; D, nifuroxazide treated; E, chlorzoxazone treated. The strains were GFP tagged for visualization. The main pictures are top-down 3D projections, while the flanking pictures are vertical sections. Bars, 20 μm.

FIG. 6.

Results of COMSTAT analysis for total biomass calculation of the individual biofilms. 1, untreated; 2, lasI rhlI mutant (QS deficient); 3, salicylic acid treated; 4, nifuroxazide treated; 5, chlorzoxazone treated. Averages and SDs from analysis of 12 images taken at random positions in three different biofilms are shown.

FIG. 7.

Scenes displaying the conformations (magenta) of the identified LasR inhibitors and known LasR inhibitors as proposed by MVD after docking of the compounds. A, salicylic acid; B, nifuroxazide; C, chlorzoxazone; D, 4-NPO, the known LasR inhibitor used as cutoff in the docking; E, furanone C30, a potent LasR and QSI; F, OdDHL, the natural LasR ligand, as it is present in the LasR crystal structure with its hydrogen bonds (black lines) to the protein (green residues). In scenes A to E, hydrogen bonds proposed by MVD to occur upon binding are shown as black dotted lines together with the interaction residues (green). The conformation of OdDHL (white) has been included in scenes A to E for comparison, and the backbone of the LasR ligand binding domain is shown in blue-white with α-helical residues 65 to 72 omitted for clarity.