Streptococcus mutans diadenylate cyclase: A promising target for preventing dental caries

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Project Summary The tenacious biofilms formed by Streptococcus mutans are resistant to conventional antibiotics and current treatments such as ‘oral rinses’. Current treatments are not ‘biofilm-specific’ and kill pathogenic species as well as commensal species alike. Therefore, there is a growing need for novel therapeutics to selectively inhibit S. mutans biofilms while conserving the oral microenvironment. Recent studies from our lab and others’ have shown that increased levels of cyclic di-AMP (c-di-AMP), an important secondary messenger in S. mutans, favored biofilm formation by upregulating the expression of gtfB, the gene coding for glucosyl transferase B (GtfB). GtfB is responsible for the production of water-insoluble glucans and is critical for biofilm formation and virulence of S. mutans. C-di-AMP is a novel cyclic dinucleotide synthesized from two ATP molecules by the enzyme, diadenylate cyclase (DAC). A suggested mechanism by which c-di-AMP controls the biofilm formation involves a c-di-AMP-binding protein (CabPA)’s interaction with VicR, a transcriptional factor known for regulating gtfB. S. mutans DAC (smDAC) is not an essential enzyme. Therefore, the inhibition of smDAC is a novel strategy to inhibit the S. mutans biofilms without affecting its growth. DAC inhibition should downregulate gtfB expression and reduce the glucan production. S. mutans coexists with other oral microbes and its ability to form biofilms may be influenced by other bacteria. Multi-species biofilms enable the testing of the selectivity of PB8 towards S. mutans along with other commensal streptococci. We have taken a structure-based approach for the design of inhibitors using our recently solved crystal structure of smDAC enzyme. With the help of in-silico screening and preliminary SAR studies, we have identified low micromolar inhibitors of smDAC. The most active compound identified from these studies is a novel small molecule PB8, which inhibits smDAC (IC50 = 17.2 M) and S. mutans biofilm (IC50 = 10.2 M). PB8 inhibited 80 % multi-species biofilm at 50 M. PB8 did not affect the growth of S. mutans and commensal bacteria (S. gordonii, S. sanguinis, and S. parasanguinis) up to 100 µM showing it is a selective biofilm inhibitor. In surface plasmon resonance (SPR) studies, PB8 showed high binding affinity to smDAC (KD = 7.1 M). To facilitate the structure activity relationship (SAR) and lead optimization studies, we have developed a three-step high yielding synthesis of PB8 and conducted preliminary SAR studies. The overall goal of this proposal is to optimize the biofilm inhibitory activity of PB8 and establish its binding affinity to smDAC and its potential as novel selective biofilm inhibitor that can be used for the prevention and treatment of dental caries. The specific aims are: 1) To optimize the biofilm inhibitory activity of PB8 through structure activity relationship studies. Successful completion of the proposed studies will validate smDAC as a novel target for biofilm inhibition and identify novel, non-toxic compounds that can selectively inhibit cariogenic biofilms, while leaving the commensal and beneficial microbes intact. 2) To evaluate smDAC inhibition and biofilm inhibition profiles of PB8 and its synthesized analogs.