Exploring novel aryl/heteroaryl-isosteres of phenylthiazole against multidrug-resistant bacteria

Abstract

Antimicrobial resistance has become a concern as a worldwide threat. A novel scaffold of phenylthiazoles was recently evaluated against multidrug-resistant Staphylococci to control the emergence and spread of antimicrobial resistance, showing good results. Several structural modifications are needed based on the structure-activity relationships (SARs) of this new antibiotic class. Previous studies revealed the existence of two key structural features essential for the antibacterial activity, the guanidine head and lipophilic tail. In this study, a new series of twenty-three phenylthiazole derivatives were synthesized utilizing the Suzuki coupling reaction to explore the lipophilic part. The in vitro antibacterial activity was evaluated against a range of clinical isolates. The three most promising compounds, 7d, 15d and 17d, with potent MIC values against MRSA USA300 were selected for further antimicrobial evaluation. The tested compounds exhibited potent results against the tested MSSA, MRSA, and VRSA strains (concentration: 0.5 to 4 μg mL^-1). Compound 15d inhibited MRSA USA400 at a concentration of 0.5 μg mL^-1 (one-fold more potent than vancomycin) and showed low MIC values against ten clinical isolates, including linezolid-resistant strain MRSA NRS119 and three vancomycin-resistant isolates VRSA 9/10/12. Moreover, compound 15d retained its potent antibacterial activity using the in vivo model by the burden reduction of MRSA USA300 in skin-infected mice. The tested compounds also showed good toxicity profiles and were found to be highly tolerable to Caco-2 cells at concentrations of up to 16 μg mL^-1, with 100% of the cells remaining viable.

Fig. 1. The design of the current study based on the previous study.

Scheme 1

Fig. 2. Analyzing the toxicity of compounds 7d, 15d and 17d (tested in triplicates at 8, 16 and 32 μg mL⁻¹) against human colorectal cells (Caco-2) using the MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Results are presented as the percentage of viable cells relative to DMSO (negative control) to determine a baseline measure for the cytotoxic impact of each compound. The absorbance values represent an average of three samples analyzed for each compound. Error bars represent the standard deviation values. Data were analyzed via a two-way ANOVA with post hoc Dunnett's test for multiple comparisons. (*) denotes a statistical difference (p < 0.05) between values obtained for the compounds and the DMSO.

Fig. 3. Killing kinetics of compounds (tested in triplicates at 5 × MIC) against methicillin-resistant Staphylococcus aureus NRS123 over a 24 hours incubation period at 37 °C. DMSO (solvent for the compounds) served as a negative control, and linezolid and vancomycin served as control antibiotics. The error bars represent standard deviation values obtained from triplicate samples used for each compound/antibiotic studied. Data were analyzed via a two-way ANOVA with post hoc Dunnett's test for multiple comparisons. An asterisk (*) denotes a statistical difference (p < 0.05) between the values obtained for each test agent as compared to DMSO.

Fig. 4. Multi-step resistance selection of compounds against methicillin-resistant S. aureus USA 400. Bacteria were serially passaged over a 14 days period, and the broth microdilution assay was used to determine the minimum inhibitory concentration of each compound against MRSA after each successive passage. A four-fold shift in MIC would be indicative of bacterial resistance to the test agent.

Fig. 5. In vivo anti-MRSA activity in a murine model of skin infection. Seventy-two hours post-infection with S. aureus USA300, a visible lesion developed at the site of infection. Twice a day topical application of petroleum jelly (PJ), commercial 2% fusidic acid ointment (FA) or 2% 15d in PJ (15d) was carried out for 4 consecutive days. (A) Photographs of representative mice from the three groups on the sacrifice day. Box plots of the bacterial burden recovered from the skin lesions (B) and the spleens (C) of the infected mice of the three groups. In both plots, the whiskers span the difference between the minimum and maximum readings, the horizontal bar represents the median, and the (+) sign represents the mean of the log₁₀CFU mL⁻¹. Statistical analysis was done using the ordinary one-way ANOVA, followed by Tukey's multiple comparisons test. The * indicates a statistical difference (p ≤ 0.05). The charts were generated using GraphPad Prism (version 9.0).