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和电流具有耐药性 治疗，如‘口腔漱口’。目前的治疗方法不是生物膜专一性的，也会杀死致病物种。 作为共生物种。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 变形链球菌中重要的第二信使--环二AMP(c-di-AMP)水平的增加是受欢迎的 通过上调葡萄糖转移酶B(GTFB)的编码基因GTFB的表达来形成生物膜。GTFB 负责生产水不溶的葡聚糖，对生物被膜的形成和致病力至关重要 变形链球菌。C-二-AMP是一种新型的环状二核苷酸，由两个ATP分子通过酶合成， 二腺苷环化酶(DAC)。C-di-AMP控制生物膜形成的一个建议的机制包括 一个c-二-AMP结合蛋白(CabPA)的S与已知调节GTFB的转录因子VICR相互作用。S. 变形杆菌DAC(SmDAC)不是一种必需的酶。因此，抑制smDAC是一种新的策略 在不影响其生长的情况下抑制变形链球菌的生物膜。DAC抑制应下调GTFB的表达 并减少葡聚糖的生产。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。多物种生物膜能够测试PB8对 变形链球菌和其他共生链球菌。我们采取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。借助硅胶筛查技术 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PB8，它能抑制SmDAC(IC_(50)=17.2M)和S. 变形杆菌生物膜(IC_(50)=10.2M)。在50时，Pb8对多菌种生物膜的抑制率为80%。Pb8对生长无影响 变形链球菌和共生菌(戈登链球菌、血链球菌和副血链球菌)高达100微米。 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表现出与 SmDAC(Kd=7.1M)。为了促进结构活性关系(SAR)和领导优化研究，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。整体而言 该方案的目的是优化PB8的生物被膜抑制活性，并建立其与smDAC的结合亲和力 以及它作为一种新型的选择性生物膜抑制剂用于预防和治疗牙周炎的潜力 龋齿。具体目标是：1)通过结构活性优化PB8的生物被膜抑制活性 关系研究。拟议研究的成功完成将证明SMDAC是一项新的目标 抑制生物膜，并确定新的无毒化合物，可以选择性地抑制致龋性生物膜，而 使共生和有益的微生物完好无损。2)评价smDAC抑制作用和生物膜抑制作用 PB8及其合成类似物的图谱。

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.