基于葡萄糖基转移酶C结构的变异链球菌生物膜小分子抑制剂的筛选

A majority of infectious diseases, including dental caries, are associated with bacterial biofilms. Caries develops when a range of different bacteria create a cariogenic environment in which the plaque biofilm is repeatedly exposed to carbohydrates. Streptococcus mutans(S. mutans) is the major etiological agent of dental caries. It is believed that decreasing the number of cariogenic bacteria in dental biofilms and preventing S. mutans biofilm formation are the keys to devise therapeutic and preventive strategies for dental caries. Glucosyltransferase (Gtfs) enzymes produced by S. mutans are important virulence factors in the pathogenesis of dental caries. Gtfs can synthesize large amounts of exopolysaccharides (EPS) from sucrose, which promotes formation of cariogenic biofilms. Antibodies against Gtfs protect teeth from caries development in animal models. The structure of GtfC catalytic domain complex has been solved. Structural information on this class of enzyme could facilitate development of novel inhibitors of Gtfs. In this study, we will utilize the structure of GtfC catalytic domain in the complex with acarbose and maltose to identify small molecules that target Gtfs, which may have therapeutic potential of treating S. mutans derived caries. A small molecule library of 500,000 compounds will be screened using the predicted affinity to GtfC based on in silico docking analysis and the receptor ligands that have calculated affinity with GtfC will be identified. The biological activities of those ligands will be examined through S. mutans single-species biofilm formation assay, EPS production detection and the Gtfs activities assay in order to screen potent Gtfs inhibitor. And the underlying mechanism of inhibition will be explored. After that, the microcosm biofilm model and animal experiment will be used to verify the effects and inhibitions of the potent compounds to discover the potential small biological molecules. The aim of this project is to identify potent and selective Gtfs inhibitors which can inhibit the cariogenic biofilm formation, and thus to demonstrate the feasibility to structure-based design of therapeutic drugs and explore the novel therapeutic and preventive strategies for dental caries.

变异链球菌（S. mutans）是公认的重要致龋菌。S. mutans 可利用其产生的重要毒力因子葡萄糖基转移酶（Gtfs）代谢蔗糖产生胞内外多糖，构成牙菌斑生物膜基质，促进致龋性牙菌斑生物膜的形成和成熟，导致龋病的发生发展。寻找以关键酶作为靶蛋白的防龋制剂是生态防龋的重要途径。本课题以GtfC与阿卡波糖及鼠李糖复合晶体三维立体结构为基础，采用分子对接技术对ZINC数据库中约500,000小分子进行虚拟筛选，评估并选择其中与GtfC匹配度高的配体小分子；然后利用S. mutans单菌种生物膜进行生物活性检测筛选出Gtfs抑制剂并研究其抑制机制；再利用体外牙菌斑微生态系和体内实验动物龋模型对小分子进行生物学验证，观察其对龋及对牙菌斑微生态系的影响及调节作用。期待能筛选出具有防龋潜能的生物小分子，或以筛选出的小分子结构为基础，合成更有效、更具活性的生物小分子，旨在进一步探索生态防龋的新策略。

本项目《基于葡萄糖基转移酶C结构的变异链球菌生物膜小分子抑制剂的筛选》已完成原计划目标，并在原有研究基础上进行了扩展，研究内容包括：①以葡萄糖基转移酶C结构为基础的变异链球菌生物膜小分子抑制剂的筛选的研究。采用分子对接技术对ZINC数据库中约500,000小分子进行虚拟筛选，评估并选择其中与GtfC高度匹配的配体小分子，通过生物膜模型实验、荧光显微镜分析、酶谱法分析、qRT-PCR、生物膜干涉技术、DOCK分析、动物模型体内实验等研究筛选出的小分子，研究发现：#G43小分子苯环上酰胺基可结合S. mutans GtfC的氨基酸位点 Glu515, Asp477和Asp588，而特异性的抑制S. mutans生物膜的形成，且不影响共生菌S. sanguinis 和S. gordonii的生长，动物模型体内实验显示#G43可抑制S. mutans生物膜形成，且并未产生细胞毒性，本研究证明#G43是可特异性抑制S. mutans GtfC，是具有防龋潜能的生物活性小分子；②生物小分子三甲曲沙（trimetrexate, TMQ）抑制变异链球菌生物膜的研究，研究发现：TMQ衍生物#66小分子可通过特异性的结合二氢叶酸还原酶（Dihydrofolate reductase, DHFR）而抑制变异链球菌生物膜的形成；③ 三苯甲咪唑（clotrimazole，CTR）和双氯甲氧苯咪唑（econazole, ECO）抑制变异链球菌生物膜及其毒力因子的研究，研究发现：CTR和CEO均可通过降低胞外多糖（Exopolysaccharide，EPS）的产生来抑制S. mutans生物膜形成，并可通过抑制ldh的表达来降低S. mutans的产酸和耐酸性。原项目计划中的小部分内容因为实验方法的调整而有所修改，本项目支持的研究已筛选出可特异性的抑制S. mutans生物膜形成的生物小分子，同时也提示以关键酶作为靶向蛋白可筛选出特异性的生物小分子抑制剂，或合成更有效、更具活性的生物小分子，是探索生态防龋的新策略。研究成果已发表SCI论文4篇，中文核心期刊论文2篇，另有1篇SCI文章已接收。