基于LasR受体在线筛选中药黄芩中细菌生物膜抑制活性成分的研究

The formation of biofilm (BF) is one of important reasons for bacterial resistance behavior. Quorum sensing inhibitors (QSIs), which could interfere with the quorum sensing system and therefore inhibit BF formation without causing resistance, represent an important breakthrough in the development of new antibacterial drugs. Traditional Chinese medicine (TCM) as an important potential source of QSIs, exhibits some advantages such as low toxicity, low cost, and low tendency to induce resistance. However, current screening strategies and methods have some shortcomings, such as time consumption, low efficiency, high cost, etc., which seriously restricts the possibility of obtaining novel QSIs from TCM. In a previous study, a well-known TCM, Scutellaria baicalensis Georgi, has proved to inhibit BF formation. In this research, the QS receptor protein LasR, which is closely related to Pseudomonas aeruginosa BF formation, will be immobilized onto a monolithic column for the first time. In order to screen BF inhibitors from Scutellaria baicalensis Georgi extracts, a platform, which combines LasR-affinity monolith chromatography (LasR-AMC), RP-HPLC, and mass spectrometry (MS) technologies, will be established. The BF inhibition activity of the screened active ingredients will be verified by means of an in vitro activity test. In addition, a comparative analysis between AMC and other screening methods, such as QSI selectors and computer-aided drug design (CADD), will be carried out. This research will be useful to elucidate the scientific connotation of TCM in the fight against drug-resistant bacteria. Moreover, it can not only provide an effective screening tool, but also a significant basis for the development of novel, safe, and effective TCM drugs against resistant bacteria.

细菌生物膜（BF）是导致耐药行为的重要原因。群体感应抑制剂（QSI）通过拮抗细菌自身的群感系统而抑制BF的形成进而避免耐药性产生，是研发新型抗菌药物的重要突破口。中药是获得QSI的重要来源，具有低毒价廉、不易引起耐药等优势。然而，现有的筛选策略和方法存在着耗时低效、费用高昂等不足，严重制约了从中药复杂成分中发现QSI。前期，我们发现中药黄芩可以抑制铜绿假单胞菌（PA）的BF形成。本研究拟首次基于PA群感系统中与BF形成密切相关的重要受体蛋白LasR，建立LasR亲和整体色谱­纳流液相色谱­质谱二维在线筛选方法，寻找中药黄芩中具有抑制BF活性的新型QSI，并通过体外BF抑制活性试验验证，结合QSI选择器筛选法和计算机辅助手段进行相互验证。本项目旨在提供一种“筛选、分离、鉴别”一体化的中药QSI研究方法，为揭示中药抗耐药菌的科学内涵，开发安全、有效的抗耐药菌中药新药提供科学依据。

细菌生物膜（biofilm，BF）是导致耐药行为的重要原因。群体感应抑制剂（quorum sensing inhibitor，QSI）通过拮抗细菌自身的群感系统而抑制BF的形成进而避免耐药性产生，是研发新型抗菌药物的重要突破口。本研究基于铜绿假单胞菌（Pseudomonas aeruginosa，PA）群感系统中与BF形成密切相关的重要受体蛋白LasR，成功建立了LasR亲和整体色谱-纳流液相色谱-质谱二维在线筛选方法，获得中药黄芩中具有抑制BF活性的新型QSI。所取得的重要成果如下：一、将LasR蛋白通过环氧基开环反应，固定在poly(GMA-co-PEGDA) 整体材料之上，成功获得固定了LasR蛋白的亲和整体色谱柱 (LasR-AMC)；通过微径液相方法评估其活性，通过红外光谱及扫描电镜实验表征了LasR-AMC的理化性质；二、成功构建了固定化LasR亲和整体色谱-纳流液相色谱-质谱联用的在线筛选、分离、检测平台；首次采用了柱切换技术将固定化 LasR 亲和整体色谱柱（第一维）与反相 C18 柱（第二维）和质谱仪相连建立在线筛选平台；三、活性筛选指导下对黄芩提取物进行筛选，得到一系列QSI，包括baicalin, scutellarin 以及 wogonoside等，并完成结构鉴定；以最低抑菌浓度评估抑菌作用，以原子力显微镜和扫描电镜观察其对生物膜形态结构的影响，以结晶紫染色法测定对生物膜的抑制率；此外，通过分子对接等计算机辅助药物设计手段，分析LasR与化合物之间的作用机制。综上所述，本研究提供了一种“筛选、分离、鉴别”一体化的中药QSI研究方法，也为揭示中药抗耐药菌的科学内涵，开发安全、有效的抗耐药菌中药新药提供科学依据。