超广谱β-内酰胺酶CTX-M-14中药抑制剂的筛选及芸香苷抑酶作用研究

Screening ofβ-lactamase CTX-M-14 Chinese Medicine Inhibitor and Enzyme Inhibition of Rutin

β-内酰胺酶的产生是细菌产生耐药的常见机制,其主要代表为超广谱β-内酰胺酶,其中CTX-M-14在全世界呈现流行趋势,为寻找一种CTX-M-14抑制剂,使细菌恢复对β-内酰胺类抗生素的敏感性,本试验首先构建了CTX-M-14的原核表达载体,并以CTX-M-14为靶点借助虚拟筛选工具Autodock Vina进行筛选,得到结合作用较好的中药单体抑制剂芸香苷,通过DS Visualizer分析其相关作用力;通过联合抑菌试验验证芸香苷与抗生素的联合抑菌作用;通过酶动力学试验比较抑酶剂芸香苷与克拉维酸的抑酶作用与方式。结果表明,芸香苷与超广谱β-内酰胺酶CTX-M-14结合部位形成多个氢键和范德华力,其结合作用力为9.9 kcal/mol;芸香苷与头孢噻肟钠对大肠杆菌E320呈协同作用(FICI=0.236);0.312 5 mg/mL的芸香苷与头孢噻肟钠对CTX-M-14蛋白阳性重组菌呈协同作用(FICI≤0.375);1.25 mg/mL的芸香苷与头孢噻肟钠对导入空质粒pET-28a(+)的BL-21呈无关作用(FICI=1.5);抑酶剂芸香苷与克拉维酸均为竞争性抑制剂,但克拉维酸抑酶作用优于芸香苷。本试验证明,芸香苷可以竞争性抑制CTX-M-14活性,具有β-内酰胺酶抑制剂潜质。

The production ofβ-lactamases is a common mechanism for bacteria to develop drug resistance,and its main representatives are extended-spectrumβ-lactamases,of which CTX-M-14 is showing an epidemic trend worldwide.In order to find an inhibitor to CTX-M-14 for restoring the susceptibility of bacteria toβ-lactam antibiotics,in this experiment firstly a prokaryotic expression vector of CTX-M-14 was constructed,and CTX-M-14 was used as the target site for screening with the help of virtual screening tool Autodock Vina,and finally a monomeric inhibitor of Chinese medicine,rutin,with good binding effect was obtained.Its relevant potency by DS Visualizer was analyzed,the combined inhibitory effect of rutin and antibiotics was verified by co-inhibition test,and the enzyme inhibiting effect of rutin and clavulanic acid were compared by enzyme kinetic test.The results showed that rutin formed multiple hydrogen bonds and van der Waals forces at the binding site of CTX-M-14,and its binding force was 9.9 kcal/mol.Rutin and cefotaxime sodium showed synergistic effect on Escherichia coli E320(FICI=0.236);0.3125 mg/mL of rutin and cefotaxime sodium showed synergistic effect CTX-M-14 protein(FICI≤0.375);1.25 mg/mL of rutin and cefotaxime sodium had no effect on BL-21 introduced into empty plasmid pET-28a(+)(FICI=1.5);both rutin and clavulanic acid were competitive inhibitors,but clavulanic acid had better enzyme inhibitory effect than rutin.This experiment demonstrates that rutin may competitively inhibit the CTX-M-14 activity and has a potential asβ-lactamase inhibitor.