Molecule Targeting Glucosyltransferase Inhibits Streptococcus mutans Biofilm Formation and Virulence

Objectives: As a major cariogenic bacterium, Streptococcus mutans (S. mutans) has adapted to the biofilm lifestyle, which is essential for pathogenesis of dental caries. There is a critical need to develop new strategies to control the biofilm-based infection. Biofilm formation in S. mutans is promoted by major virulence factors known as glucosyltransferases (Gtfs), which synthesize adhesive extracellular polysaccharides (EPS). The current study was designed to identify novel small molecules that target Gtfs, thereby inhibiting S. mutans biofilm formation and with the potential to prevent dental caries.
Methods: A library of approximately 150,000 commercially available compounds was screened in silico against the substrate-binding site of GtfC to select molecules with high binding potential, followed by high-throughput in vitro screening. The inhibitory effects of the top hit on S. mutans biofilm formation and EPS synthesis were analysed by microbiological and biochemical methods, and confocal laser scanning microscopy. To investigate the mechanism underlying, quantitative RT-PCR and zymogram assay were performed. The in vivo effect of the molecule on the development of caries was also examined in a rat model.
Results: Structure-based virtual screening resulted in the identification of a quinoxaline derivative, named D21 as a potential Gtf inhibitor. In vitro assays showed that D21 (10μg/mL) was capable of inhibiting EPS synthesis (60% reduction, compared with vehicle control) and biofilm formation (79% reduction) in S. mutans by selectively antagonizing Gtfs, instead of killing the bacteria directly. We also found that D21 significantly reduced the incidence and severity of both smooth and sulcal-surface caries in vivo with a concomitant reduction of the percentage of S. mutans in the animals’ dental plaque (P<0.05).
Conclusions: Our study represents the first description of a small molecule identified by structure-based virtual screening that targets Gtfs and has the capacity to inhibit both biofilm formation and cariogenicity of S. mutans.