Team:TJUSLS China/Demonstrate
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Demonstrate
Demonstrate
In our previous work, we have made the circuit with beta-lactamase perform in our engineering expression system and purified our target protein, we have successfully got activated metallo-beta-lactamases. And then effective inhibitors have been screened out with metallo-beta-lactamase and the specific fluorescent probe CDC-1. Enzyme kinetic parameter and inhibition constant have been measured as well.
After the screening in vitro on biochemical level, we will repeat the process of screening the inhibitors in E. coli on cellular level in order to further simulate the drug environment and achieve the therapeutic effect of our dual-drug combination. In order to verify the inhibitors we screened out can inhibit beta-lactamase in vivo of E. coli, activated E. coli with the metallo-beta-lactamase gene was used as a reality and measured the inhibitory effect of the inhibitors on metallo-beta-lactamase.
We determined the inhibitory effect of inhibitors on enzymatic decomposition of antibiotics by analyzing the changes of specific absorption peaks of antibiotics. First, we cultured E.coli with metallo-beta -lactamase gene and transferred them into phosphate buffer to ensure its activity. Then with quantitative measurement system, we made the E. coli bacterial fluid, antibiotics cefazolin and inhibitor react together. After that, through the continuous determination of change of antibiotics cefazolin’s specific absorption peak at 273 nm, we evaluated the inhibition effect of our inhibitors in activated circumstances and the therapeutic effect of the inhibitor in the real situation [1].
Take NDM-23 as an example. Through high-throughput screening, we screened out inhibitors with better inhibitory effects: corilagin and tannic acid. The measurement system is 100 μL including 94 μL E. coli bacterium fluid, 1 μL inhibitor or DMSO and 5 μL antibiotics cefazolin. We firstly determined the antibiotic decomposition ability of the bacteria solution, and the bacteria solution with the decomposition ability of 500 μL in an hour was used as the basis for the evaluation of the inhibitor effect. The results are as follow.
Fig.1
The stability of four antibiotics in the presence of the NDM-23 E.coli cells. The hydrolysis of the drugs at 273, 300, 307 and 360 nm for cefazolin, meropenem and faropenem, andteacycline, respectively in the kineticmode (120 min, triplicatescansper 5 min). The starting concentration of antibiotics was 250 μM.
Then, the inhibitory effect of corilagin on NDM-23 in the presence of live bacteria was determined. The results are as follow.
Fig.2
Inhibition of cefazolin hydrolysis in E.coli cells expressing NDM-23 by Corilagin in the concentration range of 0-160 μM, and IC50 measurements for NDM-23. For each experiment, the E.coli cells expressing NDM-23 (OD600 = 0.02) were first incubated with the inhibitor for 60 min and 250 μM cefazolin was subsequently added.
From our statistics, we were surprised to find that there is an inhibitor Tannic acid which is broad spectrum to all our four beta-lactamases. In addition, Tannic acid is from Chinese Traditional Drug Library.
We also determined the inhibitory effect of tannic acid on different lactamases in living bacteria.
Fig.3
Inhibition of cefazolin hydrolysis in E.coli cells expressing NDM-23 by Tannic acid in the concentration range of 0-160 μM, and IC50 measurements for NDM-23. For each experiment, the E.coli cells expressing NDM-23 (OD600 = 0.02) were first incubated with the inhibitor for 60 min and 250 μM cefazolin was subsequently added
Fig.4
Inhibition of cefazolin hydrolysis in E.coli cells expressing SPG-1 by Tannic acid in the concentration range of 0-160 μM, and IC50 measurements for SPG-1. For each experiment, the E.coli cells expressing SPG-1 (OD600=0.4) were first incubated with the inhibitor for 60 min and 250 μM cefazolin was subsequently added
Fig.5
Inhibition of cefazolin hydrolysis in E.coli cells expressing ElBla Ⅱ by Tannic acid in the concentration range of 0-160 μM, and IC50 measurements for ElBla Ⅱ. For each experiment, the E.coli cells expressing ElBla Ⅱ (OD600 = 0.1) were first incubated with the inhibitor for 60 min and 250 μM cefazolin was subsequently added
Fig.6
Inhibition of cefazolin hydrolysis in E.coli cells expressing AFM-1 by Tannic acid in the concentration range of 0-160 μM, and IC50 measurements for AFM-1. For each experiment, the E.coli cells expressing ADM-1 (OD600=0.1) were first incubated with the inhibitor for 60 min and 250 μM cefazolin was subsequently added
After the experiment, we found that the IC50 value of Corilagin and Tannic acid become higher than those in vitro. The reasons varies. For one thing, pumps remove the inhibitors inside E.coli and decrease their activity, manifesting as higher IC50 value [2]. For another thing, porin in bacteria prevents the contact of inhibitors with beta-lactamase and lead to the same phenomenon [3].
In order to enhance the effect of inhibitors in vivo, we aim to change the structure of small molecule compounds to improve their ability of transmembrane. We are also going to research detailed reaction role of small molecule compounds with beta-lactamase, then screen the compounds with same mechanism for detecting. At the same time we will take high-throughput screening method to select more effective inhibitors.
Reference
[1]Ke-Wu Yang, Yajun Zhou, Ying Ge and Yuejuan Zhang, Real-time activity monitoring of New Delhi metallo-b-lactamase-1 in living bacterial cells by UV-Vis spectroscopy,Chem. Commun., 2017, 53, 8014--8017
[2]Sarah M. Drawz and Robert A. Bonomo, Three Decades of β-Lactamase Inhibitors, Clin Microbiol Rev. 2010 Jan; 23(1): 160–201.
[3]Y. Ye, X. Liu, Z. Zhang, Q. Wu, B. Jiang, L. Jiang, X. Zhang, M. Liu, G. Pielak and C. Liu, Chem. – Eur. J., 2013, 19, 12705.
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tju2019igem@163.com
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No.92 Weijin Road, Nankai District, Tianjin, China