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

变形链球菌二腺苷酸环化酶：预防龋齿的一个有希望的目标

基本信息

批准号：
10662189
负责人：
Edwin M Rojas
金额：
$ 5.35万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10662189
关键词：
3-Dimensional Actinobacillus actinomycetemcomitans Actinomyces naeslundii Active Sites Affect Affinity Anaerobic Bacteria Antibiotics Architecture Bacteria Binding Binding Proteins Biological Biological Assay Clinic Clinical Code Consumption Dental Enamel Dental caries Development Dietary Sugars Dinucleoside Phosphates Disease Docking Down-Regulation Enzymes Etiology Evaluation Genes Genotype Glucans Glucosyltransferases Glycolysis Goals Growth HA coating Healthcare In Vitro Infection Lactic acid Marketing Microbe Microbial Biofilms Modeling Morus Natural Products Oral Oral cavity Pathogenicity Periodicity Plant Lectins Prevention Production Radiolabeled Reporting Resistance Saliva Signal Pathway Streptococcus Streptococcus gordonii Streptococcus mutans Streptococcus sanguis Structure Structure-Activity Relationship Surface Plasmon Resonance Testing Time Tooth Demineralization Transferase Virulence Water Widespread Disease analog beneficial microorganism commensal bacteria commensal microbes confocal imaging cost dental biofilm design experimental study glucosyltransferase B improved in silico in vitro Assay inhibitor lead optimization meter nanomolar novel novel strategies novel therapeutics oral bacteria oral commensal oral microbiome oral plaque pathogen pathogenic bacteria polymicrobial biofilm prevent resilience screening small molecule targeted agent tooth surface transcription factor

项目摘要

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.

项目概要变形链球菌形成的顽强生物膜对传统抗生素和目前的抗生素具有抗药性目前的治疗方法，如“口腔冲洗”，不是“生物膜特异性的”，也能杀死致病菌。因此，越来越需要新的治疗方法来选择性抑制金黄色葡萄球菌。我们实验室和其他人的最新研究表明，它可以抑制变形菌生物膜，同时保护口腔微环境。环二-AMP (c-di-AMP) 水平的增加（变形链球菌中重要的第二信使）有利于通过上调 gtfB（编码葡萄糖基转移酶 B (GtfB) 的基因）的表达来形成生物膜。负责产生水不溶性葡聚糖，对于生物膜的形成和毒力至关重要 C-di-AMP 是一种新型环状二核苷酸，由两个 ATP 分子通过酶合成，二腺苷酸环化酶（DAC）是 c-di-AMP 控制生物膜形成的机制。 c-di-AMP 结合蛋白 (CabPA) 与 VicR 的相互作用，VicR 是一种已知调节 gtfB 的转录因子。 mutans DAC (smDAC) 不是必需酶，因此，抑制 smDAC 是一种新策略。抑制变形链球菌生物膜而不影响其生长应该下调 gtfB 表达。并减少变形链球菌与其他口腔微生物共存及其形成生物膜的能力。可能受到其他细菌的影响多物种生物膜可以测试 PB8 对细菌的选择性。变形链球菌以及其他共生链球菌我们采用了基于结构的方法进行设计。借助计算机筛选，我们最近解决了 smDAC 酶的晶体结构。和初步的 SAR 研究，我们已经确定了 smDAC 的低微摩尔抑制剂最活跃的化合物。从这些研究中鉴定出一种新型小分子 PB8，它可以抑制 smDAC (IC50 = 17.2 μM) 和 S. 变形菌生物膜（IC50 = 10.2 µM），50 µM PB8 抑制 80% 的多物种生物膜，但不影响生长。变异链球菌和共生细菌（戈氏链球菌、血链球菌和副血链球菌）浓度高达 100 µM PB8 是一种选择性生物膜抑制剂，在表面等离子共振 (SPR) 研究中，PB8 显示出高结合亲和力。 smDAC（KD = 7.1 μM）为了促进结构活性关系（SAR）和先导化合物优化研究，我们开发了PB8的三步高产率合成方法并进行了初步的SAR研究。该提案的目标是优化 PB8 的生物膜抑制活性并建立其与 smDAC 的结合亲和力及其作为新型选择性生物膜抑制剂用于预防和治疗牙科疾病的潜力具体目标是：1）通过结构活性优化PB8的生物膜抑制活性。关系研究的成功完成将验证 smDAC 作为一个新的目标。生物膜抑制并鉴定可以选择性抑制致龋生物膜的新型无毒化合物，同时 2) 评估 smDAC 抑制和生物膜抑制，使共生微生物和有益微生物保持完整。 PB8 及其合成类似物的概况。