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

doi:10.13560/j.cnki.biotech.bull.1985.2021-1105

Abstract

Abstract:

The production of β-lactamases is a common mechanism for bacteria to develop drug resistance,and its main representatives are extended-spectrum β-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 to β-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）;0.312 5 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 as β-lactamase inhibitor.

Key words: virtual screening, CTX-M-14, rutin, combined antibacterial, enzyme kinetics

ZHAO Zi-yu, WANG Chun-guang, LV Jian-cun, LI Ji-kai, ZHANG Tie. Screening of β-lactamase CTX-M-14 Chinese Medicine Inhibitor and Enzyme Inhibition of Rutin[J]. Biotechnology Bulletin, 2022, 38(6): 235-244.

Figures/Tables 12

Fig. 1 Gel electrophoresis of PCR amplified product M：Marker;1：Escherichia coli E320;2：E. coli 25922

Fig. 2 Double enzyme digestion of transformed plasmid M：Marker;1：after double enzyme digestion;2：E320

Fig. 3 Alignment of CTX-M-14 sequencing results Plasmid：sequencing result;ctx：predicted gene sequence

Fig. 4 Expression detection of recombinant protein A：CTX-M-14 recombinant protein（M：Protein marker;1：before induction;2：after induction）. B：Soluble detection of recombinant protein（M：Protein marker;1：supernatant;2：precipitation）

Fig. 5 Optimization of protein expression conditions A：Optimization of induction time（M：Protein marker;1：before induction;2：3 h;3：4 h;4：5 h;5：6 h）. B：Optimization of induction temperature（M：Protein marker;1：before induction;2：26℃;3：31℃;4：34℃;5：37℃）. C：Optimization of IPTG concentration（M：Protein marker;1：before induction;2：0.2 mmol/L;3：0.4 mmol/L;4：0.6 mmol/L;5：0.8 mmol/L;6：1.0 mmol/L;7：1.2 mmol/L）

Fig. 6 Purification and activity identification of recomb-inant protein A：Protein purification and identification（M：Protein marker;1：before purification）;2：after purification. B：Identification of protein activity. Top row：CTX-M-14 recombinant protein group is added. Lower row：Blank control group

Table 1 Scoring results of some Chinese herbal medicinal ingredients screened virtually by AutoDock Vina

配体Ligand	结合能Binding affinity/（kcal·mol^-1）	均方根偏差/最大值RMSD/UB	均方根偏差/最小值RMSD/LB
Rhein diglucoside	-11.5	0	0
Agastachin	-10.5	0	0
Glycyrrhizin	-10	0	0
Timosaponin A3	-10	0	0
Biepiasterolide	-10	0	0
Sennidin A	-9.9	0	0
Rutin	-9.9	0	0
Ciwujianoside	-9.9	0	0
Galloylpaeoniflorin	-9.8	0	0
Acaciin	-9.8	0	0

Fig. 7 Interaction analysis of rutin with extended-spectrum β-lactamases CTX-M-14 A：2D structure diagram of the binding of rutin with CTX-M-14;B：3D schematic diagram of the binding structure of rutin with CTX-M-14

Table 2 MIC of E. coli E320 and recombinant protein positive BL-21

药物Drug	E320	重组蛋白阳性菌BL-21 Recombinant protein-positive bacterium BL-21
芸香苷/（mg·mL^-1）	2.5	2.5
苯唑西林钠/（μg·mL^-1）	>256（R）	64（R）
氨苄西林钠/（μg·mL^-1）	>256（R）	64（R）
头孢唑啉钠/（μg·mL^-1）	>256（R）	16（R）
头孢他啶/（μg·mL^-1）	>256（R）	8（I）
头孢呋辛/（μg·mL^-1）	4（S）	16（I）
头孢曲松钠/（μg·mL^-1）	>256（R）	32（R）
头孢噻肟钠/（μg·mL^-1）	1024（R）	>1024（R）

Table 3 Combined antibacterial test of rutin and cefotaxime sodium

菌株 Strain	药物 Drug	单药MIC MIC of single drug	联合用药MIC MIC of combination drugs	FICI	结果判定 Result determination
E320	芸香苷/（mg·mL^-1）	2.5	0.625	0.236	协同作用
E320	头孢噻肟钠/（μg·mL^-1）	1 024	16	0.236	协同作用
CTX-M-14蛋白阳性重组菌	芸香苷/（mg·mL^-1）	2.5	0.312 5	<0.375	协同作用
CTX-M-14蛋白阳性重组菌	头孢噻肟钠/（μg·mL^-1）	>1 024	256	<0.375	协同作用
导入空白质粒pET-28a（+）的BL-21	芸香苷/（mg·mL^-1）	2.5	1.25	1.5	无关作用
导入空白质粒pET-28a（+）的BL-21	头孢噻肟钠/（μg·mL^-1）	8	8	1.5	无关作用

Table 4 Enzyme kinetic parameters

组别Group	K_m	V_max	K_i	K_m/V_max
空白对照组	7.13	21.33	-	0.33
溶剂对照组	7.11	20.97	-	0.34
芸香苷组	8.76	17.63	11.38	0.50
克拉维酸组	8.45	17.79	3.78	0.47

Table 5 Inhibition rate of enzyme

组别 Group	初始OD值 Initial OD value	处理后OD值 OD value after treatment	OD值变化量 OD value change amount	抑酶保护率 Enzyme inhibition protection rate/%
空白对照组	1.15±0.035	0.33±0.015	0.82±0.026	-
溶剂对照组	1.14±0.020	0.32±0.025	0.81±0.015	-
克拉维酸组	1.19±0.025	0.87±0.020	0.32±0.020*	60.98
芸香苷组	1.50±0.030	1.14±0.010	0.35±0.020*	57.31

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