新型广谱核酸适体类MBLs抑制剂的筛选及其功能研究

Due to the absence of clinically available inhibitor against metallo-β-lactamases (MBLs), this project will innovatively screening a broad-spectrum aptamer-inhibitor that targets a common character MBLs molecule structure and can effectively inhibit MBLs activity. A cavity of highly similar spatial conformation and founction is located on the surface of active centre of all B1 type MBLs. This project will adopt the technique of SELEX, together with the tactics of "toggle selection" and "subtractive selection" to obtain an aptamer that can specifically bind this conformation. Then the affinities of the aptamer for several typical MBLs will be assayed with surface plasmon resonance, the interaction model with MBLs will be analyzed by using bioinformatics techniques, meanwhile，the impact on enzymetic kinetic parameter of several typical MBLs we studied to analyze the blockage effect of aptamer against MBLs activity centre. The technique of quantum dot probe will be used to find the sub-cell structure location of aptamer. Finally, the inhibitory effect against MBLs will be evaluated through in vitro culture and in animal infectious model. This project will not only obtain a novel and broad-spectrum inhibitor against B1 type MBLs, but the interaction model will provide new ideas for further development of MBLs inhibitors.

针对当前临床上尚无金属-β-内酰胺酶（MBLs）抑制剂可用的局面，本项目将结合MBLs结构的共性，研究能高效、广谱抑制B1型MBLs酶活性的核酸适体类抑制剂。B1型MBLs不同亚型的活性中心表面均存在一个结构和功能高度相似的穴状空间构象，我们即以该构象作为靶点，采用SELEX技术，配合"切换筛选"和"消减筛选"策略，获得一种能与B1型MBLs的上述构象特异性结合的核酸适体。然后采用表面等离子共振技术检测适体与多种典型MBLs的亲和力，同时研究其对多种典型MBLs酶反应动力学的影响，以分析适体对MBLs活性中心的封闭作用；进而采用生物信息学手段研究适体与MBLs相互作用的模式，并采用量子点探针技术对适体进行细菌亚结构定位；最终通过体外细菌培养和动物感染模型，评价适体对MBLs的抑制效果。本项目将不仅获得MBLs新型广谱，而且核酸适体-MBLs作用模式将为MBLs抑制剂的研究提供新思路。

我们从挑选NDM-1和VIM-2作为靶标，采用毛细管电泳SELEX作为基本筛选技术，配合“切换筛选”和“消减筛选”策略进行核酸适体筛选，进行了5轮筛选。共对100个克隆进行测序，最终共获得有效序列27个。按照序列相似性，可将所得核酸适体分为5组。从每一组中挑选第一个序列合成适体，并采用竞争ELISA技术检测亲和力；G1-1、G2-1、G3-1、G4-1和G5-1与NDM-1的亲和力Kd值分别为Kd = 11pM， Kd = 11nM，Kd = 8.7nM，Kd = 337pM，Kd = 430pM，与VIM-2的亲和力分别为0.625、20nM、1.25nM、40nM和5nM。上述5个核酸适体中只有G3-1和G4-1表现出对NDM-1和VIM-2的抑制活性，其余核酸适体不能抑制NDM-1或VIM-2的活性。G3-1和G4-1与等摩尔浓度的NDM-1或VIM-2混合，能分别抑制31%、45%和25%、37%的酶活性。采用分别表达NDM-1、VIM-2或IMP-1-1的大肠杆菌作为对象，观察适体G3-1对抗生素最低抑制浓度（minimal inhibitory concentraion，MIC）。全长G3-1 不能降低β-内酰胺类抗生素的MIC值，而截短型的G3-1则可以降低MIC值。采用荧光显微镜检测发现截短后的G3-1能够结合细菌的细胞壁，进一步表明截短型的核酸适体G3-1可能能够进入细菌细胞内。我们采用核酸适体G3-1进行抗感染实验。动物感染模型中的抗感染实验表明，在采用核酸适体治疗后，动物的血液、接种部位、次级病变区域、脾和肝脏中的细菌量均显著减少，动物的存活率明显提高。本项目为β-内酰胺酶抑制剂的研究提供了新的思路。