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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2015, Vol. 35 Issue (2): 52-58    DOI: 10.13523/j.cb.20150208
技术与方法     
以Neprilysin蛋白酶为靶标的药物筛选模型的建立及应用
田聪会1,2, 唐延婷2,3, 王权2,3, 周红刚4
1. 天津科技大学生物工程学院 天津 300457;
2. 天津市国际生物医药联合研究院 天津 300457;
3. 旭和(天津)医药科技有限公司 天津 300457;
4. 南开大学药学院 天津 300457
Estabilishment and Application of a Model for Drug Screening Targeting Neprilysin Proteinase
TIAN Cong-hui1,2, TANG Yan-ting2,3, WANG Quan2,3, ZHOU Hong-gang4
1. College of Biological Engineering, Tianjin University of Science & Technology, Tianjing 300457, China;
2. Tianjin International Joint Academy of Biotechnology & Medicne, Tianjin 300457, China;
3. MDCbiotechnology Co.Ltd, Tianjin 300457, China;
4. College of Pharmacy, Nankai University, Tianjin 300457, China
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摘要：

目的:建立以蛋白酶Neprilysin(NEP)为靶点的高通量药物筛选模型,应用该模型筛选抑制剂。方法:利用毕赤酵母表达系统。构建重组质粒pPICZα-A-NEP,表达载体通过与酵母菌X-33基因组染色体发生同源重组,将外源基因整合于染色体后实现目的蛋白的表达。应用荧光共振能量转移法(FRET)检测蛋白酶活性,优化反应条件,建立药物筛选体系,筛选抑制剂。结果:成功构建表达载体pPICZα-A-NEP;建立了以NEP为靶标的药物筛选模型,获得模型反应动力学参数Vmax=3.6μM/s, Kcat/Km=4.5×105M-1s-1,测定模型Z-因子为0.89,说明体系稳定可用于以NEP为靶标的药物的高通量筛选;并用该模型对天然产物组分库进行筛选,在0.5mg/ml的药物浓度下,得到抑制率较高的药物为4种,并测得半数抑制浓度IC50值,其中MDCNCL01000242的IC50值最低,为(8.31±0.03)μg/ml。结论:建立的药物筛选模型较为理想,适用于NEP抑制剂的筛选,可促进药物的研发。
关键词： 毕赤酵母表达;  抑制剂;  高通量筛选;  NEP    
Abstract:

Objective: To establish a viable high-throughput drug screening model targeting on NEP proteinase, and applied the model to screen inhibitors. Methods: Target protein was obtained using Pichia expression system. The gene of NEP was amplified with PCR and cloned into the expression vector pPICZα-A, and using the X-33 Pichia strain as the host for expression of recombinant proteins. Then, proteinase activity of target protein was examined by fluorescence resonance energy transfer(FRET)assays. Finally, a model for drug screening was established and optimized before abundant inhibitors screening. Results: It was not only successfully constructed the expression vector pPICZα-A-NEP, but also established a model for drug screening targeting NEP. Several related kinetic parameters were also obtained.The determination of Z-factor in model was 0.89, indicating that a reliable and stable model for drug screening was established. Furthermore, natural component library was screened, and four inhibitors with high inhibition ratio was finally obtained when the drug concentration was less than 0.5mg/ml. Moreover,the IC50 of MDCNCL01000242 is minimal, namely 8.31 μg/ml. Conclusion: The established model targeting NEP proteinase is suitable for inhibitors screening, which can be used to promote research and development of drug.
Key words: NEP    Pichia expression system    Inhibitors    High Throughput Screening
收稿日期: 2014-10-09 出版日期: 2015-02-25
ZTFLH:  Q819  
基金资助:

国家自然科学基金(81102374)、天津市科技支撑计划(2C2DSY03300)、天津市科技计划(132C2DSY02600)资助项目
通讯作者: 周红刚     E-mail: honggang.zhou@htmdc.org
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引用本文:

田聪会, 唐延婷, 王权, 周红刚. 以Neprilysin蛋白酶为靶标的药物筛选模型的建立及应用[J]. 中国生物工程杂志, 2015, 35(2): 52-58.

TIAN Cong-hui, TANG Yan-ting, WANG Quan, ZHOU Hong-gang. Estabilishment and Application of a Model for Drug Screening Targeting Neprilysin Proteinase. China Biotechnology, 2015, 35(2): 52-58.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150208    或    https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I2/52


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