以Neprilysin蛋白酶为靶标的药物筛选模型的建立及应用

发布时间：2018-02-26 21:38

本文关键词： NEP 毕赤酵母表达抑制剂高通量筛选　出处：《中国生物工程杂志》2015年02期 　论文类型：期刊论文

【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p PICZα-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p PICZα-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。
[Abstract]:Objective: to establish a high-throughput drug screening model targeting the protease Neprilysin nep. Methods: the recombinant plasmid p PICZ 伪 -A-NEP was constructed by using Pichia pastoris expression system. The expression vector was homologous recombined with the genome chromosome of yeast X-33. The expression of the target protein was realized by integrating the exogenous gene into the chromosome. The protease activity was detected by fluorescence resonance energy transfer method, the reaction conditions were optimized, and the drug screening system was established. Results: the expression vector p PICZ 伪 -A-NEP was successfully constructed, and a drug screening model targeting NEP was established. The kinetic parameters of the model reaction were obtained: Vmax=3.6 渭 M / s / Kcat / KMT 4.5 脳 105M-1s-1, and the model Z- factor was 0.89, which indicated that the system was stable and could be used for the high-throughput screening of drugs targeting NEP, and the natural product component library was screened by the model at the concentration of 0.5mg / ml. Four kinds of drugs with high inhibition rate were obtained, and half inhibitory concentration (IC50) was measured. The IC50 value of MDCNCL01000242 was the lowest (8.31 卤0.03) 渭 g / ml. Conclusion: the established drug screening model is ideal and suitable for the screening of NEP inhibitors and can promote the development of drugs.
【作者单位】：天津科技大学生物工程学院;天津市国际生物医药联合研究院;旭和(天津)医药科技有限公司;南开大学药学院;
【基金】：国家自然科学基金(81102374) 天津市科技支撑计划(13ZCZDSY03300) 天津市科技计划(13ZCZDSY02600)资助项目
【分类号】：R96

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