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There is a growing need for novel therapeutics that selectively inhibit\n              <jats:italic>S. mutans<\/jats:italic>\n              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\n              <jats:italic>S. mutans<\/jats:italic>\n              . Thus, targeting\n              <jats:italic>S. mutans<\/jats:italic>\n              DAC is a novel strategy to inhibit\n              <jats:italic>S. mutans<\/jats:italic>\n              biofilms. We screened a small NCI library of natural products using a fluorescence detection assay. (+)-Brazilin, a tetracyclic homoisoflavanoid found in the heartwood of\n              <jats:italic>Caesalpinia sappan<\/jats:italic>\n              , was identified as one of the 11 \u201chits,\u201d with the greatest reduction (&gt;99%) in fluorescence at 100 \u00b5M. The\n              <jats:italic>sm<\/jats:italic>\n              DAC inhibitory profiles of the 11 \u201chits\u201d established by a quantitative high-performance liquid chromatography assay revealed that (+)-brazilin had the most enzymatic inhibitory activity (87% at 100 \u00b5M) and was further studied to determine its half maximal inhibitory concentration (IC\n              <jats:sub>50<\/jats:sub>\n              = 25.1 \u00b1 0.98 \u00b5M). (+)-Brazilin non-competitively inhibits\n              <jats:italic>sm<\/jats:italic>\n              DAC\u2019s enzymatic activity (\n              <jats:italic>K<\/jats:italic>\n              <jats:sub>\n                <jats:italic>i<\/jats:italic>\n              <\/jats:sub>\n              = 140.0 \u00b1 27.13 \u00b5M), as determined by a steady-state Michaelis-Menten kinetics assay. In addition, (+)-brazilin\u2019s binding profile with\n              <jats:italic>sm<\/jats:italic>\n              DAC (\n              <jats:italic>K<\/jats:italic>\n              <jats:sub>\n                <jats:italic>d<\/jats:italic>\n              <\/jats:sub>\n              = 11.87 \u00b5M) was illustrated by a tyrosine intrinsic fluorescence quenching assay. Furthermore, at low micromolar concentrations, (+)-brazilin selectively inhibited the biofilm of\n              <jats:italic>S. mutans<\/jats:italic>\n              (IC\n              <jats:sub>50<\/jats:sub>\n              = 21.0 \u00b1 0.60 \u00b5M) and other oral bacteria.\n              <jats:italic>S. mutans<\/jats:italic>\n              biofilms were inhibited by a factor of 10\n              <jats:sup>5<\/jats:sup>\n              in colony-forming units when treated with 50 \u00b5M (+)-brazilin. In addition, a significant dose-dependent reduction in extracellular DNA and glucan levels was evident by fluorescence microscopy imaging of\n              <jats:italic>S. mutans<\/jats:italic>\n              biofilms exposed to different concentrations of (+)-brazilin. Furthermore, colonization of\n              <jats:italic>S. mutans<\/jats:italic>\n              on a representative model of enamel using suspended hydroxyapatite discs showed a &gt;90% reduction with 50 \u00b5M (+)-brazilin. In summary, we have identified a drug-like natural product inhibitor of\n              <jats:italic>S. mutans<\/jats:italic>\n              biofilm that not only binds to\n              <jats:italic>sm<\/jats:italic>\n              DAC but can also inhibit the function of\n              <jats:italic>sm<\/jats:italic>\n              DAC. (+)-Brazilin could be a good candidate for further development as a potent therapeutic for the prevention and treatment of dental caries.\n            <\/jats:p>\n            <jats:sec>\n              <jats:title>IMPORTANCE<\/jats:title>\n              <jats:p>\n                This study represents a significant advancement in our understanding of potential therapeutic options for combating cariogenic biofilms produced by\n                <jats:italic>Streptococcus mutans<\/jats:italic>\n                . The research delves into the use of (+)-brazilin, a natural product, as a potent inhibitor of\n                <jats:italic>Streptococcus mutans<\/jats:italic>\n                \u2019 diadenylate cyclase (\n                <jats:italic>sm<\/jats:italic>\n                DAC), an enzyme crucial in the formation of biofilms. The study establishes (+)-brazilin as a non-competitive inhibitor of\n                <jats:italic>sm<\/jats:italic>\n                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\n                <jats:italic>S. mutans<\/jats:italic>\n                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.\n              <\/jats:p>\n            <\/jats:sec>\n          <\/jats:sec>","DOI":"10.1128\/spectrum.02418-23","type":"journal-article","created":{"date-parts":[[2024,4,9]],"date-time":"2024-04-09T13:04:34Z","timestamp":1712667874000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":4,"title":["Tetracyclic homoisoflavanoid (+)-brazilin: a natural product inhibits c-di-AMP-producing enzyme and\n            <i>Streptococcus mutans<\/i>\n            biofilms"],"prefix":"10.1128","volume":"12","author":[{"given":"Edwin M.","family":"Rojas","sequence":"first","affiliation":[{"name":"School of Dentistry, University of Alabama at Birmingham, Birmingham, Alabama, USA"},{"name":"Department of Chemistry, University of Alabama at Birmingham, Birmingham, Alabama, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hua","family":"Zhang","sequence":"additional","affiliation":[{"name":"Division of Biomaterial &amp; Biomedical Sciences, School of Dentistry, Oregon Health &amp; Science University, Portland, Oregon, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0342-2378","authenticated-orcid":false,"given":"Sadanandan E.","family":"Velu","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Alabama at Birmingham, Birmingham, Alabama, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1694-3954","authenticated-orcid":false,"given":"Hui","family":"Wu","sequence":"additional","affiliation":[{"name":"Division of Biomaterial &amp; Biomedical Sciences, School of Dentistry, Oregon Health &amp; Science University, Portland, Oregon, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","first-page":"661","article-title":"The global burden of oral diseases and risks to oral health","volume":"83","author":"Petersen PE","year":"2005","unstructured":"Petersen PE, Bourgeois D, Ogawa H, Estupinan-Day S, Ndiaye C. 2005. 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