Carmofur Exhibits Antimicrobial Activity Against Streptococcus pneumoniae

Abstract

Background/Objectives:Streptococcus pneumoniae (S. pneumoniae) is a major pathogen causing severe infectious diseases, with an escalating issue of antimicrobial resistance that threatens the efficacy of existing antibiotics. Given the challenges in developing traditional antibiotics, drug repurposing strategies offer a novel approach to address the resistance crisis. This study aims to evaluate the antibacterial and anti-biofilm activities of the approved non-antibiotic anticancer drug carmofur against multidrug-resistant S. pneumoniae, and investigate the mechanism of action, and assess therapeutic potential in vivo. Methods/Results: Antimicrobial tests revealed that carmofur exhibited strong antibacterial activity against multidrug-resistant S. pneumoniae strains, with minimum inhibitory concentrations (MICs) ranging from 0.25 to 1 µg/mL. In the biofilm detection experiments, carmofur not only inhibited the formation of biofilms, but also effectively removed biofilms under high concentration conditions. Mechanistic studies showed that carmofur disrupted bacterial membrane permeability and decreased intracellular ATP levels. Molecular docking and dynamics simulation assays indicated that carmofur could stably bind to thymidylate synthase through hydrogen bonding and hydrophobic interactions, thereby exerting antibacterial effects. Meanwhile, carmofur was able to repress the expression of the thyA gene at the mRNA level. In a mouse infection model, the carmofur treatment group showed a reduction of approximately two log levels in bacterial load in lung tissue and blood, a significant decrease in the levels of inflammatory cytokines TNF-α and IL-6, and an improvement in survival rate to 60%. Conclusions: In summary, carmofur demonstrated significant antibacterial and anti-biofilm activities against multidrug-resistant S. pneumoniae and showed good anti-infective effects in vivo, suggesting its potential clinical application as a therapeutic agent against drug-resistant bacteria.

Keywords: S. pneumoniae; antibacterial; carmofur; mouse pneumonia; multidrug resistant.

Figure 1

Effects of different concentrations of carmofur on the growth of S. pneumoniae strain 17426. (A) The 2D structural formula of carmofur; (B) bacterial growth curves based on optical density (OD 600 nm) over time. Carmofur concentrations were set at 0.25 µg/mL, 0.5 µg/mL, and 1 µg/mL. Untreated control was used as a reference; (C) changes in colony-forming units (CFUs/mL) over time in carmofur-treated groups. Concentrations of 0.5 µg/mL, 1 µg/mL, and 2 µg/mL were tested. Each group consisted of three biological replicates. Data are presented as mean ± standard deviation (Mean ± SD). *** p < 0.001, and n.s. indicates not significant. The statistical analysis at the end of the experiment was performed via Student’s t test.

Figure 2

Anti-biofilm effects of carmofur (0.25, 0.5, 1, 2 and 5 µg/mL) against S. pneumoniae. Auranofin (MIC = 0.5 µg/mL), a known reported drug, served as a positive control. (A) Inhibition of biofilm formation at varying concentrations of carmofur. (B) Eradication effect on pre-formed biofilms. (C) Total bacterial count in mature biofilms, evaluated with acridine orange fluorescence probe at different carmofur concentrations. (D) Reduction in metabolically active cells in mature biofilms, assessed by ATP levels. Each group consisted of three biological replicates. Data are presented as Mean ± SD. Significance levels are indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ‘n.s.’ indicates no significant difference. Statistical analyses were performed via one-way ANOVA.

Figure 3

Antibacterial mechanism of carmofur. (A) PI uptake in S. pneumoniae. (B) Analysis of intracellular ATP levels in S. pneumoniae treated with various concentrations of carmofur. Daptomycin (8 µg/mL) served as a positive control. Each group consisted of three biological replicates. Data are presented as mean ± SD. Significance levels are indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001. The statistical analysis at the end of the experiment was performed via one-way ANOVA.

Figure 4

Molecular docking and interaction analysis of carmofur with target protein. (A) Three-dimensional structure of the target protein, comprising typical α-helices and β-sheets; (B) superimposed results of carmofur’s four primary binding conformations, showing its spatial compatibility; (C) distribution of binding energies (Cdocker energy) across different conformations, with the lowest binding energy (around −25 kJ/mol) marked in the red box as the optimal conformation; (D) detailed interaction analysis of carmofur and 5-fluorouracil within the binding pocket; (E) two-dimensional spatial binding mode of carmofur in the binding pocket, showing multiple interactions.

Figure 5

The effects of carmofur on thymidylate synthase. (A) The root mean square deviation (RMSD) as a function of time, indicating that the system gradually reached stability during the molecular dynamics simulation; (B) the solvent-accessible surface area (SASA) over time, reflecting fluctuations in protein solvent accessibility; (C) the radius of gyration (Rg) as a function of time, demonstrating the overall compactness of the protein conformation; (D) the root mean square fluctuation (RMSF) analysis, revealing the conformational flexibility of different protein residues; (E) the number of hydrogen bonds over time, illustrating the dynamic characteristics of hydrogen bonding interactions within the system; (F) the binding free energy decomposition results, showing the energetic contributions of different residues to ligand binding; (G) the effect of carmofur on relative mRNA expression levels. Data are presented as mean ± standard deviation (mean ± SD); mean ± SD was repeated three times. * p < 0.05, ** p < 0.01; n.s. indicates no significant difference. The statistical analysis at the end of the experiment was performed Via Student’s t test.

Figure 6

Therapeutic efficacy of carmofur in mice infected with S. pneumoniae. (A) Results of blood biochemical indexes to evaluate the effects of carmofur on liver and kidney functions in mice. (B) Survival rate of mice treated with carmofur 1 h post-infection with an inoculum dose of 5 × 10⁸ CFU. (C) Bacterial load in blood and lung tissues of mice. (D) Levels of TNF-α and IL-6 in mouse serum. (E) Histopathological changes in lung tissue post-treatment. Data are presented as mean ± standard deviation (Mean ± SD). ** p < 0.01, *** p < 0.001; n.s. indicates no significant difference. The statistical analysis at the end of the experiment was performed Via Student’s t test.