Tetracyclic homoisoflavanoid (+)-brazilin: a natural product inhibits c-di-AMP-producing enzyme and Streptococcus mutans biofilms

Abstract

The tenacious biofilms formed by Streptococcus mutans are resistant to conventional antibiotics and current treatments. There is a growing need for novel therapeutics that selectively inhibit S. mutans biofilms while preserving the normal oral microenvironment. Previous studies have shown that increased levels of cyclic di-AMP, an important secondary messenger synthesized by diadenylate cyclase (DAC), favored biofilm formation in S. mutans . Thus, targeting S. mutans DAC is a novel strategy to inhibit S. mutans biofilms. We screened a small NCI library of natural products using a fluorescence detection assay. (+)-Brazilin, a tetracyclic homoisoflavanoid found in the heartwood of Caesalpinia sappan , was identified as one of the 11 “hits,” with the greatest reduction (>99%) in fluorescence at 100 µM. The sm DAC inhibitory profiles of the 11 “hits” established by a quantitative high-performance liquid chromatography assay revealed that (+)-brazilin had the most enzymatic inhibitory activity (87% at 100 µM) and was further studied to determine its half maximal inhibitory concentration (IC 50 = 25.1 ± 0.98 µM). (+)-Brazilin non-competitively inhibits sm DAC’s enzymatic activity ( K i = 140.0 ± 27.13 µM), as determined by a steady-state Michaelis-Menten kinetics assay. In addition, (+)-brazilin’s binding profile with sm DAC ( K d = 11.87 µM) was illustrated by a tyrosine intrinsic fluorescence quenching assay. Furthermore, at low micromolar concentrations, (+)-brazilin selectively inhibited the biofilm of S. mutans (IC 50 = 21.0 ± 0.60 µM) and other oral bacteria. S. mutans biofilms were inhibited by a factor of 10 5 in colony-forming units when treated with 50 µM (+)-brazilin. In addition, a significant dose-dependent reduction in extracellular DNA and glucan levels was evident by fluorescence microscopy imaging of S. mutans biofilms exposed to different concentrations of (+)-brazilin. Furthermore, colonization of S. mutans on a representative model of enamel using suspended hydroxyapatite discs showed a >90% reduction with 50 µM (+)-brazilin. In summary, we have identified a drug-like natural product inhibitor of S. mutans biofilm that not only binds to sm DAC but can also inhibit the function of sm DAC. (+)-Brazilin could be a good candidate for further development as a potent therapeutic for the prevention and treatment of dental caries. IMPORTANCE This study represents a significant advancement in our understanding of potential therapeutic options for combating cariogenic biofilms produced by Streptococcus mutans . The research delves into the use of (+)-brazilin, a natural product, as a potent inhibitor of Streptococcus mutans ’ diadenylate cyclase ( sm DAC), an enzyme crucial in the formation of biofilms. The study establishes (+)-brazilin as a non-competitive inhibitor of sm DAC while providing initial insights into its binding mechanism. What makes this finding even more promising is that (+)-brazilin does not limit its inhibitory effects to S. mutans alone. Instead, it demonstrates efficacy in hindering biofilms in other oral bacteria as well. The broader spectrum of anti-biofilm activity suggests that (+)-brazilin could potentially serve as a versatile tool in a natural product-based treatment for combating a range of conditions caused by resilient biofilms.