Development of Azole Derivatives as Novel Fatty Acid Biosynthesis Inhibitors Against Streptococcus Mutans

Objectives: With the goal of discovering novel antibacterial agents against S. mutans, a library of pyrazole and thiazole compounds were synthesized and screened for their antibacterial activity. These compounds showed effective antimicrobial activity against drug resistant Staphylococcus aureus, Enterococcus faecalis, and other gram-positive bacterial strains. We hypothesized these azole derivatives could effectively inhibit S. mutans and lead to the discovery of novel antibacterial and therapeutic agents against dental caries.
Methods: The antibacterial activity of synthesized compounds was studied using minimum inhibitory concentration (MIC) assay and biofilm inhibition tests. MIC was determined by a microdilution method as recommended by the Clinical and Laboratory Standards Institute and biofilm inhibition was studied using a crystal violet assay. The lead efficacious compounds from antibacterial assays were then profiled for mechanism of inhibition using a CRISPR Interference (CRISPRi) library and qRT-PCR.
Results: Azole derivatives demonstrated antibacterial activity against S. mutans with high potency. The MIC values for several derivatives were found to be as low as 1 µg/ml and active against a panel (6 representative strains) of genomically diverse clinical isolates. These derivatives also effectively inhibited biofilm formation by S. mutans. Mode of action CRISPRi studies showed that several genes related to fatty acid biosynthesis and transcriptional proteins were observed as critical for S. mutans growth in presence of azole compounds. Confirmation of gene hits based on growth curve analysis revealed fatty acid biosynthesis genes fabG, fabZ, fabK and bccP were strongly inhibited followed by transcriptional factors genes rs5 and alaS.
Conclusions: It indicates that azole derivatives may target the synthesis of fatty acids, a major component in cell membrane lipids to inhibit bacterial growth. CRISPRi screening of gene targets provides a predictive tool for mode of action studies and azole derivatives could be a potential drug candidate against dental caries.