Advisor(s)
Sadanandan Velu
Hui Wu
Committee Member(s)
Eugenia Kharlampieva
Nathaniel Lawson
Outi Keinaenen
School
College of Arts and Sciences
Document Type
Dissertation
Department (new version)
Chemistry
Date of Award
1-7-2025
Abstract
Streptococcus mutans (S. mutans) is the primary etiological agent involved in the initiation and progression of dental caries. S. mutans forms robust biofilms on the tooth surface, and current preventative treatments, such as oral rinses, eliminate both pathogenic and commensal oral species. Therefore, there is an urgent need for therapeutics which selectively inhibit S. mutans biofilms while keeping the oral microbiome intact. The diadenylate cyclase (DAC) enzyme in S. mutans plays a key role in biofilm formation. DAC converts adenosine triphosphate (ATP) into cyclic di-AMP (c-di-AMP), a secondary messenger involved in several cellular pathways. Increased levels of c-di-AMP have shown to induce biofilm formation. Therefore, DAC is a potential druggable target to selectively inhibit biofilm formation by S. mutans. Small-molecule leads were identified using various approaches in this dissertation. Chapter 1 identifies PB8 as an initial lead from an in-silico screening against Thermotoga maritima DNA integrity scanning protein A (tmDisA), a DAC enzyme homologous to Streptococcus mutans diadenylate cyclase (smDAC), until the crystal structure of smDAC was solved. PB8 and a library of analogs were synthesized to evaluate their biochemical and anti-biofilm properties. PB8 selectively inhibits biofilms of S. mutans while preserving the cell viability of S. mutans and commensal streptococci. In a fluorescence turn-on assay, PB8 showed preliminary dose-dependent inhibition of smDAC when decreasing the fluorescence over time. However, when high performance liquid chromatography (HPLC) was used as a secondary assay to quantify conversion of ATP to c-di-AMP, PB8 and its analogs were identified as false-positives since they did not demonstrate any significant inhibition of smDAC’s catalytic activity. A biotinylated analog of PB8 was synthesized to glean PB8’s mechanism of action against biofilm formation of S. mutans using a Streptavidin pull-down assay. Chapter 2 identifies (+)-brazilin, a natural product tetracyclic homoisoflavanoid from the heartwood of Caesalpinia sappan, as a first-generation non-competitive smDAC inhibitor and selective biofilm inhibitor of S. mutans. In this study, a small-molecule library from the National Cancer Institute (NCI) was screened using the HPLC enzymatic inhibitory assay, with (+)-brazilin showing significant inhibition of c-di-AMP production by smDAC. Michaelis-Menten kinetics using HPLC revealed that (+)-brazilin non-competitively binds to the ATP catalytic site in smDAC. A tyrosine intrinsic fluorescence assay provided evidence that (+)-brazilin interacts with tyrosine residues as part of its binding mechanism to smDAC. Crystal violet assays and colony-forming units (CFU’s) assays elucidated (+)-brazilin’s selectivity and single species biofilm inhibitory activity in the sub-micromolar range. Fluorescence imaging showed a decrease in biofilms, glucans, and extracellular DNA (eDNA) when S. mutans was treated with dose-dependent concentrations of (+)-brazilin. (+)-Brazilin inhibited biofilms of S. mutans formed on hydroxyapatite discs, proving that our lead can inhibit biofilms formed on surfaces comparable to that of natural tooth structure. Chapter 3 identifies STL372167 from a high throughput in silico screening study of 1.1 M compounds as a potent inhibitor of smDAC. Filters for “druglikeness” and “leadlikeness” from SeeSAR 13.1, a software program used for lead identification from structure-activity-relationship and docking analyses, were employed to narrow down the docking results and select compounds with commercial availability. After obtaining compounds from Molport.com and evaluating their potential for inhibiting smDAC using our HPLC assay, we identified STL372167 as a potent inhibitor of smDAC’s catalytic activity. Excited by our discovery, we used the coumarin system found in STL372167 as inspiration to construct a library of purchased and synthetic analogs of STL372167 for preliminary structure-activity-relationship studies along with biochemical and microbiological evaluation. Several analogs from this library exhibited smDAC inhibition and S. mutans biofilm inhibition, with the most potent and anticariogenic being 7218310521, 7218310540, AG-I-116, and AG-I-117. Docking models of these compounds disclose key structural motifs required for smDAC inhibition, paving way for the optimization of first-generation coumarins as smDAC inhibitors for preventing dental caries. In summary, this dissertation showcases the computational studies and biochemical assays employed to characterize smDAC, allowing for the identification of small molecules that target smDAC. Microbiological assays were employed to demonstrate selective inhibition of S. mutans biofilms, in vitro. Techniques in organic synthesis enabled the construction of compound libraries with the aim to improve the potency, efficacy, and druglike properties of leads through structure-activity-relationship studies. From this work, smDAC demonstrates to be an enticing druggable target for therapeutics to prevent dental caries.
ProQuest ID
Recommended Citation
Rojas, Edwin Miguel, "Targeting Streptococcus Mutans Diadenylate Cyclase For The Prevention Of Dental Caries" (2025). All ETDs from UAB. 7294.
https://digitalcommons.library.uab.edu/etd-collection/7294
