基于药效团模型筛选CTX-M-14型超广谱β-内酰胺酶抑制剂

doi:10.13560/j.cnki.biotech.bull.1985.2023-1228

摘要/Abstract

摘要：

【目的】 开发针对头孢噻肟-水解酶-14（CTX-M-14）型超广谱β-内酰胺酶（extended-spectrum β-lactamase，ESBLs）的抑制剂，用以缓解细菌耐药带来的严重危害。【方法】 使用DiscoveryStudio Visualizer（DS Visualizer）构建基于受体结构的药效团模型（structure-based pharmacophore, SBP）和基于配体共同特征的定性药效团模型（common feature pharmacophore generation, HIPHOP），并将验证后的模型作为查询条件对ZINC数据库进行以CTX-M-14蛋白为靶标的虚拟筛选，得到拟合分数良好的中药单体成分甘草酸（glycyrrhizic acid，GL），对其进行相关作用力分析、分子动力学模拟和结合自由能计算，分析甘草酸与CTX-M-14蛋白的结合模式、结合能力及稳定性；最后通过联合抑菌试验及酶动力学试验考察甘草酸的抗菌增敏活性、抑酶作用及抑酶方式。【结果】 中药单体成分甘草酸主要与CTX-M-14蛋白活性中心多个氨基酸残基形成氢键和范德华作用力，两者的对接分数与结合自由能分别为-10 kcal/mol及-22.06 kcal/mol；甘草酸与头孢噻肟钠联用呈协同作用（FICI≤0.5）；甘草酸可竞争性抑制β-内酰胺酶对底物抗生素的水解作用，对头孢噻肟钠的抑酶保护率可达58.53％，与克拉维酸接近（60.98％）。【结论】 中药单体甘草酸可与CTX-M-14蛋白稳定结合，通过竞争性抑制β-内酰胺酶对底物抗生素的水解作用，提高多重耐药大肠杆菌E320和重组蛋白阳性菌BL-21对头孢噻肟钠的敏感性，实现抗生素的减量增效。

关键词: 甘草酸, CTX-M-14, 药效团模型, 分子动力学模拟, 联合抑菌

Abstract:

【Objective】 Development of inhibitors against cefotaxime-hydrolase-14（CTX-M-14）-type ultra-broad-spectrum β-lactamases（ESBLs）to mitigate the serious harm caused by bacterial drug resistance. 【Method】 DiscoveryStudio Visualizer（DS Visualizer）was used to construct a receptor structure-based pharmacophore（SBP）and a ligand common feature-based qualitative pharmacophore model（HIPHOP）, and the validated models were used as query conditions for virtual screening of the ZINC database targeting CTX-M-14 protein, and glycyrrhizic acid（GL）, a monomer component of traditional Chinese medicine（TCM）with good fitting scores, was obtained. The related force analysis, molecular dynamics simulation and binding free energy calculation were carried out to analyse the binding mode, binding capacity and stability of glycyrrhizic acid and CTX-M-14 protein. Finally, the antibacterial sensitizing activity, enzyme inhibition and enzyme inhibition of glycyrrhizic acid were investigated by the combined bacterial inhibition assay and enzyme kinetic assay. 【Result】 Glycyrrhizic acid, a single component of traditional Chinese medicine, mainly formed hydrogen bonds and van der Waals forces with multiple amino acid residues in the active centre of CTX-M-14 protein, and the docking fraction and binding free energy of the two were -10 kcal/mol and -22.06 kcal/mol, respectively. Glycyrrhizic acid synergistically interacted with cefotaxime sodium（FICI ≤ 0.5）; glycyrrhizic acid competitively inhibited the hydrolysis of the substrate antibiotic by β-lactamase, and the enzyme inhibition and protection rate of cefotaxime sodium reached 58.53%, which was close to that of clavulanic acid（60.98%）. 【Conclusion】 The Chinese medicine monomer glycyrrhizic acid can bind stably to CTX-M-14 protein and increase the sensitivity of multi-drug-resistant Escherichia coli E320 and recombinant protein-positive bacterium BL-21 to cefotaxime sodium by competitively inhibiting the hydrolysis of the substrate antibiotic by β-lactamase, so as to achieve a reduction in the quantity and increase in the effectiveness of the antibiotic.

Key words: glycyrrhizic acid, CTX-M-14, pharmacophore modelling, simulation of molecular dynamics, combined bacterial inhibition

王梦帆, 赵子玉, 王春光, 刘廷玉, 陈曦, 张铁. 基于药效团模型筛选CTX-M-14型超广谱β-内酰胺酶抑制剂[J]. 生物技术通报, 2024, 40(6): 319-329.

WANG Meng-fan, ZHAO Zi-yu, WANG Chun-guang, LIU Ting-yu, CHEN Xi, ZHANG Tie. Screening of CTX-M-14-type Ultra-broad-spectrum β-lactamase Inhibitors Based on Pharmacophore Modelling[J]. Biotechnology Bulletin, 2024, 40(6): 319-329.

图/表 14

图1 CTX-M-14蛋白抑制剂训练集化合物（A）和测试集化合物（B）2D结构

Fig. 1 2D structures of CTX-M-14 protein inhibitor training set compound（A）and test set compound（B）

图2 基于CTX-M-14蛋白受体（A）及配体共同特征（B）药效团模型绿色箭头和绿色网状球形，氢键受体；紫色箭头和紫色网状球形，氢键供体；浅蓝色圆球和蓝色网状球形，疏水作用力；深蓝色圆球和深蓝色网状球形，负电荷中心；灰色网状球形，排除体积

Fig. 2 Pharmacophore modelling based on CTX-M-14 protein receptor（A）and ligand co-features（B） Green arrow and green reticulated sphere, hydrogen bond acceptor; purple arrow and purple reticulated sphere, hydrogen bond donor; light blue orb and blue reticulated sphere, hydrophobic forces; dark blue orb and dark blue reticulated sphere, negative charge centre; grey reticulated sphere, excluded volume

图3 SBP模型（A）和HIPBOP 模型（B）ROC曲线图

Fig. 3 ROC curves of SBP model（A）and HIPBOP model（B）

图4 SBP模型（A）和HIPHOP模型（B）与测试集化合物拟合示意图，甘草酸与SBP模型（C）和HIPHOP模型（D）拟合示意图

Fig. 4 Schematic of SBP model (A) and HIPHOP model (B) fitted to the test set of compounds and glycyrrhizic acid fitted to the SBP model (C) and HIPHOP model (D)

表1 SBP和HIPHOP模型同时命中的小分子化合物及其FitValue

Table 1 Small molecule compounds hit simultaneously by SBP and HIPHOP models and their FitValues

ZINC ID	名称Name	拟合值FitValue
ZINC ID	名称Name	SBP	HIPHOP
ZINC901518	茶渍酸Lecanoric acid	2.6156	2.32539
ZINC96015174	甘草酸Glycyrrhizin	2.03995	2.44671
ZINC4164596	富马前冰岛酸Fumarprotocetraric acid	1.25871	2.61666

表2 SBP模型和HIPHOP模型同时命中的候选化合物与CTX-M-14蛋白对接分数

Table 2 Docking scores of candidate compounds and CTX-M-14 proteins hit simultaneously by the SBP model and HIPHOP model

ZINC ID	名称 Name	结合自由能Affinity*/ （kcal·mol^-1）	均方根偏差/RMSD
ZINC ID	名称 Name	结合自由能Affinity*/ （kcal·mol^-1）	最小值l.b.	最大值u.b.
ZINC901518	茶渍酸Lecanoric acid	-8.3	0	0
ZINC96015174	甘草酸Glycyrrhizin	-10.0	0	0
ZINC4164596	富马前冰岛酸Fumarprotocetraric acid	-9.4	0	0

图5 甘草酸与CTX-M-14蛋白分子对接示意图绿色、粉色和黄色圆形，氨基酸残基；绿色、粉色和黄色虚线，相互作用力；灰色线形，甘草酸配体；灰色粗棒，甘草酸配体；灰色细棒，氨基酸残基；黑色字体，氨基酸残基名称

Fig. 5 Schematic diagram of molecular docking of glycyrrhizic acid with CTX-M-14 protein+ Green, pink and yellow circles, amino acid residues; green, pink and yellow dashed lines, interacting forces; grey lines, glycyrrhizic acid ligands; grey thick rods, glycyrrhizic acid ligands; grey thin rods, amino acid residues; black lettering, amino acid residue names

图6 甘草酸与CTX-M-14蛋白复合物分子动力学模拟的RMSD（A）和RMSF（B）

Fig. 6 RMSD（A）and RMSF（B）of molecular dynamics simulations of glycyrrhizic acid complexed with CTX-M-14 protein

图7 甘草酸与CTX-M-14蛋白复合物动力学模拟的平衡轨迹采样（A）和结合自由能分解（B）

Fig. 7 Equilibrium trajectory sampling（A）and binding free energy decomposition（B）for kinetic simulations of glycyrrhetinic acid complexes with CTX-M-14 protein

图8 甘草酸与CTX-M-14蛋白相互作用中关键氨基酸残基及其结合自由能分解

Fig. 8 Key amino acid residues and their binding free energy decomposition in the interaction between glycyrrhetinic acid and CTX-M-14 protein

表3 甘草酸和抗菌药物对大肠杆菌的 MIC

Table 3 MIC of glycyrrhetinic acid and antibiotics against Escherichia coli

药物Drug/（mg·mL^-1）	E320	蛋白重组阳性菌BL-21 Protein recombinant positive bacterial strain BL-21
甘草酸Glycyrrhizic acid	>25	>25
苯唑西林钠Benzoxacillin sodium	>256（R）	64（R）
氨苄西林钠Ampicillin sodium	>256（R）	64（R）
头孢唑林钠Cefazolin sodium	>256（R）	16（R）
头孢他啶Ceftazidime	>256（R）	8（I）
头孢呋辛Cefuroxime	4（S）	16（I）
头孢曲松钠Ceftriaxone sodium	>256（R）	32（R）
头孢噻肟钠Cefotaxime sodium	1024（R）	>1024（R）

表4 甘草酸与头孢噻肟钠联合抑菌试验

Table 4 Combination antibacterial test of glycyrrhetinic acid and cefotaxime sodium

菌株Strain	药物Drug	单药MIC MIC of single drug	联合用药MIC MIC of combined drugs	FICI	结果判定Result determination
E320	甘草酸Glycyrrhizic acid（mg·mL^-1）	>25	3.125	<0.25	协同作用 Synergy
E320	头孢噻肟钠Cefotaxime（μg·mL^-1）	1 024	128	<0.25	协同作用 Synergy
CTX-M-14蛋白重组阳性菌CTX-M-14 Protein recombinant positive bacteria	甘草酸Glycyrrhizic acid（mg·mL^-1）	>25	3.125	<0.375	协同作用 Synergy
	头孢噻肟钠Cefotaxime（μg·mL^-1）	1 024	256	<0.375	协同作用 Synergy
蛋白重组阴性对照菌Protein recombinant negative control bacteria	甘草酸Glycyrrhizic acid（mg·mL^-1）	>25	1.5625	<1.0625	无关作用 Unrelated effects
蛋白重组阴性对照菌Protein recombinant negative control bacteria	头孢噻肟钠Cefotaxime（μg·mL^-1）	8	8	<1.0625	无关作用 Unrelated effects

表5 CTX-M-14蛋白酶动力学参数

Table 5 CTX-M-14 protease kinetic parameters

组别Group	K_m	V_max	K_i	K_m/V_max
空白对照组Blank control group	7.13	21.33	-	0.33
溶液对照组Solution control group	7.11	20.97	-	0.34
克拉维酸组Clavulanic acid group	8.76	17.79	3.78	0.50
甘草酸Glycyrrhetinic acid group	8.39	18.03	9.69	0.47

表6 甘草酸对头孢噻肟钠的抑酶保护率

Table 6 Enzyme-inhibition protection rate of glycyrrhetinic acid against cefotaxime sodium

组别Group	初始OD值 Initial OD value	OD值 OD value	OD值变化量 OD value variation	抑酶保护率 Enzyme-inhibition protection rate/%
空白对照组Blank control group	1.15±0.035	0.33±0.015	0.82±0.026	-
溶液对照组Solution control group	1.14±0.020	0.32±0.025	0.81±0.015	-
克拉维酸组Clavulanic acid group	1.19±0.025	0.87±0.020	0.32±0.020*	60.98
甘草酸组Glycyrrhetinic acid group	1.31±0.017	0.98±0.010	0.34±0.022*	58.53

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