基于网络药理学策略的多靶点阵列式亲和色谱技术在线筛选群体感应抑制剂的研究

The formation of biofilm (BF) is one of the most important causes of bacterial resistance behavior. Quorum sensing inhibitors (QSIs), which could interfere with quorum sensing system and therefore inhibit BF formation without causing resistance, is becoming one of the innovated treatment against drug-resistance bacteria. Natural product is an important source for discovering new QSIs. However, current strategies and methods for screening QSIs from natural products are limited to single-target, which overlooked the network features of QS system, and easily led to interference failure and even new resistance. According to the principle of network pharmacology, three target proteins i.e. LasR, RhlR, and MvfR, are considered as the most closely related to Pseudomonas aeruginosa (PA) BF formation. In this research, a monolithic column array will be developed for the first time by combining LasR/RhlR/MvfR-affinity onto polymer-based monolithic support. In order to screen multi-target QSIs from TCM extracts, an on-line platform, which integrates LasR/RhlR/MvfR-affinity monolithic column array, nano-HPLC, and mass spectrometry (MS), will be established. The anti-biofilm and anti-bacterial activity of the screened active ingredients will be verified by means of in vitro activity tests. Moreover, a comparative study between the proposed strategy and the computer virtual screening approach will be also carried out. This research will be useful for elucidating the scientific connotation of TCM against drug-resistant bacteria. It can not only provides a powerful tool for screening novel QSIs, but also shows great meaning in elucidating theoretical basis for the development of novel, safe, and effective drugs against resistant bacteria.

细菌生物被膜（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来抑制BF的形成进而避免耐药性的产生，已成为抗耐药菌治疗的全新手段。多样性的天然产物是获取QSI的重要来源，然而现有QSI筛选策略都是基于单一靶点进行，不符合QS系统网络特征，容易导致QSI失效甚至引起新的抗性。本研究拟基于网络药理学原理，选取铜绿假单胞菌（PA）中与BF形成密切相关的三种QS信号节点蛋白LasR/RhlR/MvfR，建立固定化多靶点亲和整体色谱阵列-纳流液相色谱-质谱在线筛选方法，寻找天然产物中新型多靶点QSI活性成分，并通过体外活性验证，结合虚拟筛选手段进行对比评估。本项目旨在提供一种“筛选、分离、鉴定”一体化的多靶点QSI活性成分研究方法，提高QSI药物筛选自动化程度及效率；为揭示QSI抗耐药菌的科学内涵，开发安全有效的抗菌新药提供理论依据。

细菌生物被膜（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来抑制BF的形成进而避免耐药性的产生，已成为抗耐药菌治疗的全新手段。本研究基于网络药理学原理，选取铜绿假单胞菌（Pseudomonas aeruginosa，PA）中与BF形成密切相关的多种QS信号节点LuxR家族蛋白，建立固定化多靶点亲和整体色谱阵列-纳流液相色谱-质谱在线筛选方法，寻找天然产物中新型多靶点QSI活性成分，并通过体外活性验证，结合虚拟筛选手段进行对比评估。所取得的重要成果如下：一、开发了一系列有机聚合物整体材料的先进合成方法；将靶点蛋白通过环氧基开环反应，固定在有机聚合物整体材料之上，成功获得固定了相关亲和整体色谱柱；通过微径液相方法评估其活性与米氏常数，通过红外光谱及扫描电镜实验表征了其理化性质；二、成功建立了亲和整体色谱阵列-纳流液相色谱-质谱联用的在线筛选、分离、检测方法，进行亲和活性成分筛选；三、活性筛选指导下对中药提取物进行筛选，发现黄芩苷与QS系统多个靶点具有显著亲和作用，在亚最低抑菌浓度下明显抑制铜绿假单胞菌BF的形成并增强各种常规抗生素体外杀菌作用的能力；对QS调节的铜绿假单胞菌毒力表型（蛋白酶、弹性蛋白酶、绿脓菌素、鼠李糖脂和外毒素A等）具有剂量依赖性抑制作用；QS调节基因（包括lasI、lasR、rhlI、rhlR、pqsR和pqsA）的表达水平受到抑制提示其介导的QS抑制改变基因和蛋白质表达的能力。此外，通过分子对接等计算机辅助药物设计手段，分析靶标与黄芩苷之间的多靶点作用机制。综上所述，本研究提供了一种“筛选、分离、鉴定”一体化的多靶点QSI活性成分研究方法，提高QSI药物筛选自动化程度及效率；为揭示QSI抗耐药菌的科学内涵，开发安全有效的抗菌新药提供理论依据。