Repurposing Toremifene for Treatment of Oral Bacterial Infections

Abstract

The spread of antibiotic resistance and the challenges associated with antiseptics such as chlorhexidine have necessitated a search for new antibacterial agents against oral bacterial pathogens. As a result of failing traditional approaches, drug repurposing has emerged as a novel paradigm to find new antibacterial agents. In this study, we examined the effects of the FDA-approved anticancer agent toremifene against the oral bacteria Porphyromonas gingivalis and Streptococcus mutans We found that the drug was able to inhibit the growth of both pathogens, as well as prevent biofilm formation, at concentrations ranging from 12.5 to 25 μM. Moreover, toremifene was shown to eradicate preformed biofilms at concentrations ranging from 25 to 50 μM. In addition, we found that toremifene prevents P. gingivalis and S. mutans biofilm formation on titanium surfaces. A time-kill study indicated that toremifene is bactericidal against S. mutans Macromolecular synthesis assays revealed that treatment with toremifene does not cause preferential inhibition of DNA, RNA, or protein synthesis pathways, indicating membrane-damaging activity. Biophysical studies using fluorescent probes and fluorescence microscopy further confirmed the membrane-damaging mode of action. Taken together, our results suggest that the anticancer agent toremifene is a suitable candidate for further investigation for the development of new treatment strategies for oral bacterial infections.

Keywords: Porphyromonas gingivalis; Streptococcus mutans; biofilms; oral infections; toremifene.

FIG 1

Structure of toremifene (pK_a = 8.0).

FIG 2

(A and B) Reduction of P. gingivalis (A) and S. mutans (B) biofilm formation on titanium disks by toremifene. Shown is the percentage of biofilm formation in the presence of toremifene relative to the untreated control. The values are means and standard deviations (SD) of the results of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with the untreated control. (C) Fluorescence microscopy images of biofilms formed on titanium disks. Live cells stained green, and cells with compromised membranes stained red. The images were processed with an unsharp mask of Zen 2.0. Scale bars, 100 μm.

FIG 3

Time-kill kinetics of toremifene against S. mutans. (A) Exponential-phase cells of S. mutans were treated with 1× the MIC and 4× the MIC of toremifene (TOR), with 1× the MIC and 4× the MIC of chlorhexidine (CHX), or with the solvents of the drugs (DMSO and water, respectively). Samples were taken at 0, 1, 2, 3, 4, 5, and 24 h, and the numbers of CFU per milliliter were determined. The data represent means ± SD from the results of 3 independent experiments. The dashed line indicates the lower limit of detection.

FIG 4

Percentage of incorporation of radiolabeled precursors into macromolecules after treatment of S. mutans with 4× the MIC of toremifene (TOR) or control antibacterials (ciprofloxacin [CIP], rifampin [RIF], tetracycline [TET], and triclosan [TRI]). The data represent the means from at least three independent replicates ± SD.

FIG 5

Effect of toremifene on membrane permeability. (A) Outer-membrane permeabilization of P. gingivalis after treatment with different concentrations of toremifene, assessed by quantifying NPN uptake. Cells treated with 1× the MIC of triclosan (TRI) were used as a positive control (see Table S1 in the supplemental material). (B) Inner-membrane permeabilization of P. gingivalis after treatment with different concentrations of toremifene, determined by measuring Sytox green uptake. Melittin (MEL) (10 μg/ml) was used as a positive control. (C) Effects of increasing concentrations of toremifene on the membrane permeability of S. mutans, monitored by the uptake of Sytox green. Cells treated with melittin (2.5 μg/ml) served as a positive control. For all the panels, cells treated with ciprofloxacin (CIP) (1× the MIC) served as a negative control. The data represent the means from three independent replicates ± SD (**, P < 0.01; ***, P < 0.001). a.u., arbitrary units.

FIG 6

Determination of the binding affinity of toremifene for LPS of P. gingivalis using BC. The concentration-dependent displacement of BC from LPS induced by toremifene is shown. Cells treated with 1× and 4× the MIC of chlorhexidine (CHX) were used as a positive control (see Table S1 in the supplemental material). Cells treated with 1× the MIC of ciprofloxacin (CIP) were used as a negative control (see Table S1). The data represent the means from three independent replicates and SD.

FIG 7

Microscopic visualization of toremifene-induced membrane damage using the lipophilic dye FM4-64. Cells were treated with DMSO (solvent control) or with 4× the MIC of toremifene (TOR) or triclosan (TRI). Scale bars, 2 μm. The images were processed with the unsharp mask of Zen 2.0.

FIG 8

Effect of toremifene on mammalian cells. (A) Dose response of the hemolytic activity of toremifene against red blood cells. Red blood cells were treated with different concentrations of toremifene, and its hemolytic activity was determined in comparison with Triton X-100 (100% hemolysis) and PBS (0% hemolysis). Tests were performed in quadruplicate, and the results are presented as means and SD. (B) Dose response of the cytotoxic activity of toremifene against HOC18 cells. Cytotoxicity was determined in comparison with Triton X-100 (positive control) and supplemented αMEM (0% cytotoxicity). Tests were performed in duplicate, and the results are presented as means and SD.