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.
NETL
NSTL
维普
万方数据
