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

项目摘要 变形链球菌形成的坚韧的生物膜对常规抗生素具有抗性， 治疗方法如“漱口水”。目前的治疗方法不是“生物膜特异性的”，也可以杀死致病物种 就像一个共生物种一样。因此，对选择性抑制S. 变形菌生物膜，同时保护口腔微环境。我们实验室和其他实验室最近的研究表明， 增加了环二腺苷酸（c-di-AMP）的水平，这是S.变种人，青睐 通过上调编码葡糖基转移酶B（Gtf B）的基因gtf B的表达来形成生物膜。GtfB 负责水不溶性葡聚糖的产生，并且对于生物膜的形成和细菌的毒力至关重要。 S.变异人C-di-AMP是由两个ATP分子通过酶合成的新型环状二核苷酸， 二腺苷酸环化酶（DAC）。c-di-AMP控制生物膜形成的建议机制包括： c-di-AMP结合蛋白（CabPA）与VicR（已知用于调节gtfB的转录因子）的相互作用。S. 变形杆菌DAC（smDAC）不是必需酶。因此，抑制smDAC是一种新的策略， 抑制S.而不影响其生长。DAC抑制应下调gtfB表达 并减少葡聚糖的产生。S.变形杆菌与其他口腔微生物共存， 可能受到其他细菌的影响。多物种生物膜使得能够测试PB 8对 S.变异沿着与其他链球菌。我们采用了基于结构的设计方法 使用我们最近解决的smDAC酶的晶体结构的抑制剂。在计算机模拟筛选的帮助下 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的小分子PB 8，其抑制smDAC（IC 50 = 17.2 μ M）和S. 变形杆菌生物膜（IC 50 = 10.2 μ M）。PB 8在50 μ M下抑制80%的多物种生物膜。PB 8不影响生长 色葡萄变形杆菌和大肠杆菌（S. gordonii，S. sanguinis和S. parasanguinis）高达100 µM， 是选择性生物膜抑制剂。在表面等离子体共振（SPR）研究中，PB 8显示出与人IL-10的高结合亲和力。 smDAC（KD = 7.1 μ M）。为了促进构效关系（SAR）和先导化合物优化研究，我们 已经开发了PB 8的三步高产率合成，并进行了初步的SAR研究。整体 该提议的目标是优化PB 8的生物膜抑制活性并建立其与smDAC的结合亲和力 以及其作为新型选择性生物膜抑制剂的潜力，其可用于预防和治疗牙 龋齿具体目的是：1）通过结构活性优化PB 8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作为一种新的目标， 生物膜抑制和鉴定可选择性抑制致龋生物膜的新型无毒化合物， 使微生物和有益微生物保持完整。2)评价smDAC抑制和生物膜抑制 PB 8及其合成类似物的特征。

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.