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：
10330368
负责人：
Edwin M Rojas
金额：
$ 5.26万
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/10330368
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 Crystallization 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 Hydroxyapatites In Vitro Infection Lactic acid Lead 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 base 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 nanomolar novel novel strategies novel therapeutics oral bacteria oral commensal oral microbiome oral plaque pathogen pathogenic bacteria polymicrobial biofilm prevent screening small molecule targeted agent tooth surface transcription factor

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 Crystallization 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 Hydroxyapatites In Vitro Infection Lactic acid Lead 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 base 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 nanomolar novel novel strategies novel therapeutics oral bacteria oral commensal oral microbiome oral plaque pathogen pathogenic bacteria polymicrobial biofilm prevent screening small molecule targeted agent tooth surface transcription factor

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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.

项目摘要由链球菌突变形成的顽强的生物膜对常规抗生素有抵抗力和电流诸如“口腔冲洗”之类的治疗方法。当前的治疗不是“生物膜特异性”，也不是杀死病原体规格作为共生物种。因此，越来越需要新颖的治疗来选择性抑制S。 Mutans生物膜在保存口服微环境的同时。我们实验室和其他人的最新研究表明循环DI-AMP（C-DI-AMP）的水平增加了，这是S. mutans中的重要次级信使，首选生物膜形成是通过上调GTFB的表达，即编码葡萄糖基转移酶B（GTFB）的基因。 GTFB 负责产生不溶的葡萄糖，对于生物膜形成和病毒至关重要 S. Mutans。 C-DI-AMP是一种新型的环状二核苷酸，由酶从两个ATP分子合成，颗粒化酶环化酶（DAC）。 C-Di-Amp控制生物膜形成的建议机制涉及 C-DI-AMP结合蛋白（CABPA）与VICR的相互作用，VICR是一种调节GTFB的转录因子。 s Mutans DAC（SMDAC）不是必需的酶。因此，抑制SMDAC是一种新颖的策略抑制巨链链球菌生物膜而不影响其生长。 DAC抑制作用应下调GTFB表达并减少葡萄糖产生。 S. Mutans与其他口腔微生物共存及其形成生物膜的能力可能受其他细菌的影响。多种物种生物膜可以测试PB8的选择性 S. mutans以及其他共生链球菌。我们采用了基于结构的设计方法使用我们最近解决的SMDAC酶晶体结构的抑制剂。借助于内部筛查和初步的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确定的是一种新型的小分子PB8，它抑制了SMDAC（IC50 =17.2M）和S. Mutans生物膜（IC50 = 10.2M）。 PB8抑制50℃的80％多种物种生物膜。 PB8不影响增长 S. utans and Comensal细菌（S. gordonii，S。sanguinis和S. parasanguinis）最多100 µm 是一种选择性生物膜抑制剂。在表面等离子体共振（SPR）研究中，PB8表现出高的结合亲和力 SMDAC（KD = 7.1M）。为了促进结构活动关系（SAR）和铅优化研究，我们已经开发了三步的高屈服合成PB8并进行了初步SAR研究。总体该建议的目标是优化PB8的生物膜抑制活性，并建立与SMDAC的结合亲和力它作为新型选择性生物膜抑制剂的潜力，可用于预防和治疗牙齿龋齿。具体目的是：1）通过结构活性优化PB8的生物膜抑制活性关系研究。成功完成拟议的研究将验证SMDAC作为新的目标生物膜抑制并鉴定可以选择性抑制商源性生物膜的新型无毒化合物，而使共生和有益的微生物完好无损。 2）评估SMDAC抑制和生物膜抑制 PB8及其合成的类似物的轮廓。