Promising Therapeutic Agents Selectively Inhibit Streptococcus Mutans Biofilms

Objectives: The tenacious biofilms formed by Streptococcus mutans, the primary etiological agent for dental caries, are resistant to conventional antibiotics and current treatments. There is a growing need for novel therapeutics to selectively inhibit S. mutans biofilms while conserving the normal oral microbiota.
Methods: We have taken an in-silico structure-based approach for the design of small molecules using FlexX LeadIT and SeeSAR. Lead molecules were 1) selected based on energy-minimization calculations and druglike properties; 2) synthesized with overall high yield; 3) tested in vitro against growth and biofilms of S. mutans, three commensal streptococci (Streptococcus sanguinis, Streptococcus parasanguinis, and Streptococcus gordonii), and two non-streptococcal oral bacteria (Aggregatibacter actinomycetemcomitans and Actinomyces naeslundii); and 4) examined for binding kinetics using bio-layer interferometry, HPLC analysis, and fluorescence assays.
Results: Our lead molecules selectivity inhibited S. mutans biofilms within a low micromolar IC₅₀ range and preserved the growth of S. mutans, commensal streptococci species, and non-streptococci oral species with high micromolar selectivity (MIC/IC₅₀). Dichloromethotrexate, a potent dihydrofolate reductase (DHFR) inhibitor which affects cell proliferation, served as our positive control. After bio-layer interferometry analysis, we obtained very low micromolar binding dissociation (K_D) values, highlighting the strong binding affinity of our small molecules to our target enzyme. HPLC assays coupled with optimized fluorescence assays confirmed that our small molecules inhibited our target enzyme’s role in biofilm formation within a low micromolar IC₅₀ range.
Conclusions: Lead molecules examined in vitro agreed with our structure-based drug discovery approach and demonstrate potential as therapeutic agents to prevent dental caries.