Quinazoline-Derivatives of Imino-1,2,3-Dithiazoles Promote Biofilm Dispersion of Pseudomonas aeruginosa

Abstract

Background/Objectives: Biofilm-associated infections pose a major clinical challenge since bacteria within biofilms exhibit highly antibiotic tolerance. Pseudomonas aeruginosa forms persistent biofilms that cause chronic infections in vulnerable patients, including those with cystic fibrosis, burns, or medical implants. Such biofilm-associated chronic infections require prolonged treatments that promote antimicrobial resistance. To address this, recent strategies focus on enhancing biofilm dispersion. Methods: Thirty-six N-arylimino-1,2,3-dithiazoles were screened for their biofilm dispersal activity using a crystal violet assay. Their cytotoxicity was assessed on A549 and HaCat eukaryotic cells. Moreover, their influence on bacterial growth and virulence was investigated. Lastly, fluorescence anisotropy was used to measure membrane fluidity to obtain the first insights on the mechanism of action of these chemicals. Results: Our results showed that quinazoline-derivatives of imino-1,2,3-dithiazoles display biofilm dispersion activity. These compounds do not increase virulence through pyocyanin production, do not modify the growth kinetics of P. aeruginosa, and do not show cytotoxicity towards eucaryotic cells. Conclusions: These findings highlight the potential use of N-arylimino-1,2,3-dithiazole-derived compounds as safe and effective dispersal agents of P. aeruginosa biofilms.

Keywords: N-arylimino-1,2,3-dithiazoles; Pseudomonas aeruginosa; biofilm dispersion.

Scheme 3

General procedure for the synthesis of compounds 5a–i.

Scheme 1

Hypothesis on the possibility of anchoring imino-1,2,3-dithiazoles to the surface of a biofilm.

Figure 1

Structures of the first chemical library of N-arylimino-1,2,3-dithiazole derivatives tested for their dispersal activity of P. aeruginosa biofilm. The series 1a–l consists of monosubstituted derivatives, the series 2a–e comprises polyfunctional compounds derived from anthranilic acid (2-aminobenzoic acid), and the compound series 3a–c is formed from anthranilonitrile (2-aminobenzonitrile).

Figure 2

Structures of the second chemical library of N-arylimino-1,2,3-dithiazoles of interest. In series 4, the iminodithiazole ring is associated with a quinazolin-4-one moiety (in blue), while in series 5, it is a quinazoline system (violet).

Figure 3

Effect of N-arylimino-1,2,3-dithiazole-derived compounds on P. aeruginosa biofilm dispersion. The boxplots represent residual biofilm grown in static conditions for 24 h at 37 °C, non-exposed (1% DMSO) or exposed for 2 h to compounds 5a, 5b, and 5c (10 µM, 1 µM, 0.1 µM, 0.01 µM). Bacterial cells within biofilms were stained with crystal violet, and absorbance was read at 595 nm. Data are the results of the analysis of means of twelve technical replicates (control condition), and two technical replicates (for each treated condition) from at least four independent biological experiments (n = 4). Statistics were performed by one-way ANOVA followed by Dunnett’s multiple-comparison test. Values that are significantly different are indicated by asterisks as follows: *, p < 0.05; **, p < 0.01; ns, not significant (p > 0.05).

Figure 4

P. aeruginosa biofilm formation upon exposure to derivatives of N-arylimino-1,2,3-dithiazoles. The boxplots represent biofilms grown in static conditions at 37 °C for 24 h, non-exposed (1% DMSO), or exposed to compounds 5a, 5b, and 5c (10 µM, 1 µM, 0.1 µM, 0.01 µM). Bacterial cells within biofilms were stained with crystal violet, and absorbance was read at 595 nm. Data are the results of the analysis of means of twelve technical replicates (control condition), and two technical replicates (for each treated condition) from at least four independent biological experiments (n = 4). Statistics were performed by one-way ANOVA followed by Dunnett’s multiple-comparison test. Values that are significantly different are indicated by asterisks as follows: *, p < 0.05; ns, not significant (p > 0.05).

Figure 5

Impact of N-arylimino-1,2,3-dithiazoles-derived compounds 5a–c on P. aeruginosa growth kinetics and pyocyanin production. (A) Aerobic growth (A_{580 nm}) in LB medium was monitored at 37 °C during 24 h under shaking conditions, without (non-treated) or with the selected compounds (5a, 5b, and 5c) at 10 µM. Results are the means of three independent biological experiments (n = 3). (B) Pyocyanin production (%) by the PA14 strain was measured (A_{520 nm}) in the non-treated condition (1% DMSO) or upon 2 h exposure to compounds 5a, 5b, and 5c at 10 µM. Data are the results of the analysis of means of twelve technical replicates (non-treated condition), and two technical replicates (for each treated condition) from at least four independent biological experiments (n = 4). Statistics were performed by one-way ANOVA followed by Dunnett’s multiple-comparison test. ns, not significant (p > 0.05).

Figure 6

Cytotoxicity measurements on A549 and HaCat eukaryotic cells upon exposure to the N-arylimino-1,2,3-dithiazole-derived compounds 5a–c. The cytotoxicity of the compounds 5a, 5b, and 5c at 10 µM was evaluated by assessing LDH release (%) by A549 and HaCaT eukaryotic cell lines. Positive control (Triton X-100 solution was added) was considered as 100% of LDH release. Negative control was considered as the minimal LDH release in DMEM medium by A549 and HaCaT. In the non-treated condition, DMSO at 1%, used as solvent vehicle of the compounds, was added. Data are the results of the analysis of four technical replicates for each condition from at least three independent biological experiments (n = 3). Statistics were performed by one-way ANOVA followed by Dunnett’s multiple-comparison test. ns, not significant (p > 0.05).

Figure 7

Membrane fluidity of P. aeruginosa exposed to imino-1,2,3-dithiazole-derived compounds. The bars represent fluorescence anisotropy values (r) for the PA14 strain non-treated (1% DMSO) or treated with compounds 5a, 5b, and 5c for 2 h at 10 µM. DPH fluorescence was measured with an excitation wavelength of 365 nm and an emission wavelength of 425 nm. Data are the results of the analysis of means of twelve technical replicates (control condition), and four technical replicates (for each treated condition) from at least three independent biological experiments (n ≥ 3). Statistics were performed by one-way ANOVA followed by Dunnett’s multiple-comparison test. Values that are significantly different are indicated by asterisks as follows: ****, p < 0.0001; ns, not-significant (p > 0.05).

Scheme 2

Synthesis of 6-aminoquinazolines IIIa–i (isolated yields). Conditions: (a) DMF-DMA (0.4 M), EtOAc (1 M), 70 °C, 30 min. (b) 4-substituted aniline (1.5 equiv), AcOH (1 M), 118 °C, 30 min. (c) Pd/C (10% w/w), HCO₂NH₄ (5.0 equiv), EtOH (0.2 M), reflux, 1 h.