绿原酸对金黄色葡萄球菌粘附蛋白酶SrtA的抑制作用及其靶位确证

The rise in antibiotic-resistant bacteria is a major concern, in particular because it includes many different species of pathogenic microbes and has evolved against virtually every antibiotic deployed. There seems to be few new antibiotics in the drug discovery pipeline.The development of new classes of antibiotics has lagged far behind our growing need for such drugs. .Rather than focusing on therapeutics that target in vitro viability, much like conventional antibiotics, an alternative approach is to target functions essential for infection, such as virulence factors required to cause host damage and disease. This approach has several potential advantages including expanding the repertoire of bacterial targets, preserving the host endogenous microbiome, and exerting less selective pressure, which may result in decreased resistance..The conventional concept of virulence is defined by the ability of a pathogen to cause disease. Thus, efforts to develop anti-virulence therapies are geared at 'disarming' the pathogen by inhibiting virulence factors that can cause direct harm to the host. A potential advantage of this approach is that new antimicrobials aimed at inhibiting virulence rather than growth may impose weaker selective pressure for the develop-ment of antibiotic resistance relative to current antibiotics.The assembly of surface proteins and pili in the cell wall envelope of Gram-positive bacteria is catalyzed by sortase. Sortase substrates function as adhesins, internalins, blood clotting and immune evasion factors, and transporters for nutrients across the microbial cell wall envelope; without them, most pathogens cannot sustain an infection. .Chlorogenic acid from Lonicera japonica Thunb.was found to be a inhibitor of sortase and exert antibiotic activity. The active component isolated was characterized by spectroscopic analysis, and their inhibitory effect against SrtA and minimum inhibitory concentrations (MICs) against S. aureus also has been determined. By the method of HPLC, SPR,site direct mutates and the elucidation of protein-inhibitor complex structure will give further insight into the mechanism of action of this kind of chemical components. We will also elucidate the potential of chemical components for inhibition of S. aureus and other gram-positive bacteria cell adhesion to fibronectin via fibronectin-binding protein and the anti-infection action in vivo.This will provide valuable information for the development of new anti-infection drug targeting virulence facotrs of bacteria.

毒力因子是病原菌感染畜禽的关键因素，且多不是细菌生命活动所必需。因此以毒力因子等为靶干预病原菌感染过程的新机制抗菌药物的开发成为研究热点。SrtA是介导以金黄色葡萄球菌为代表的革兰氏阳性菌毒力相关表面蛋白细胞壁锚定的重要蛋白酶，对其功能的抑制可有效干预病原菌对宿主细胞的侵袭。本课题组前期构建了金葡菌SrtA抑制筛选和粘附抑制作用评价模型，开展了天然产物活性成分的追踪分离，获得高活性单体化合物绿原酸。本项目拟进行绿原酸SrtA抑制作用的多角度评价；采用HPLC和SPR等方法研究作用机制和动力学相关特性，结合氨基酸点突变确定作用/结合靶蛋白的关键区域；利用结构生物学方法解析与该化合物作用的靶标蛋白及复合物的晶体结构，明确绿原酸抑制SrtA的作用靶点。最终阐明其抑制SrtA的构效关系，为以病原菌毒力为靶的新一代抗感染创新药物研发打下基础。

SrtA 是介导以金黄色葡萄球菌为代表的革兰氏阳性菌毒力相关表面蛋白细胞壁锚定的重要蛋白酶，对其功能的抑制可有效干预病原菌对宿主细胞的侵袭。以其作为靶标的抗感染新型药物的研发备受关注。. 本课题研究完成了SrtA蛋白活性结构域的克隆表达和SrtA体外活性评价研究，建立SrtA小分子抑制剂筛选平台，系统开展了中药小分子活性化合物的筛选，获得多种活性小分子化合物；完成了绿原酸及其苯丙素类衍生物抑制SrtA构效关系研究，阐明该类化合物抑制SrtA的分子机制。绿原酸及其类似物主要结合于由Asn114,Ile182，Cys184，Gly192和Arg197等残基构成的活性区域，抑制剂的羟基部分（C1）和Asn114残基之间的静电作用力是小分子与蛋白结合的最重要作用力。完成活性小分子化合物芦丁、槲皮素和刺槐素等的SrtA抑制作用分子机制和作用靶点的分析确证；阐明了SrtA在金葡菌所致乳腺炎和菌血症中的作用研究；完成金葡菌感染动物模型的建立和活性化合物体内药效评价。为以病原菌毒力为靶的新一代抗感染创新药物研发打下基础。. 申请SrtA研究相关专利3项；发表SCI收录论文6篇,中文期刊论文1篇（其中基金为第一标注的4篇）；以课题研究结果为部分内容获得教育部自然科学二等奖一项；以相关研究为学位论文内容毕业研究生3名。.