The Prokaryotic Expression Of Human Caspase-3 Gene And The Screening Of Its Inhibitor

Caspase family is a key factor of apoptosis,which abnormal activation would lead to excessive apoptosis and cause a variety of diseases such as neurodcgenerative diseases and cardiovascular diseases.Isatin derivatives have a variety of biological activities,and a class of these compounds are more popular in pharmaceutical research.This study intends to establish a prokaryotic expression system of caspase-3 and use purified caspase-3 protein to establishkinase inhibitory activity screening system in vitro.We tested 38 isatin derivatives to inhibit the activity of caspase-3 and analyzed its structure-activity relationship.Firstly,the prokaryotic expression system of caspase-3 was established.The soluble expression protein was induced by plasmid pET-21b/Caspase-3 in BL21(DE3)Plyss Escherichia coli with 1 mM IPTG at 16? for 5 h.The protein was purified by Ni-beads and eluted by imidazole to obtain recombinant caspase-3.Next,the in vitro caspase-3 kinase activity test platform was constructed with purified caspase-3.We determine the best test conditions:the concentration of substrate Ac-DEVD-AMC and caspase-3 is 0.05 mM and 5 ng/mL respectively,at 25 ? reaction 3h.Finally,we tested 38 isatin derivatives of caspase-3 inhibitory activity and further analysed the theire structure-activity relationship.It was found that the satin has very weak inhibitory activity of caspase-3(IC50>100?M).There were 7 compounds with a stronger caspase-3 inhibitory activity(IC50<0.1?M),in which the inhibitory activity of the compound HKL-2-1-7(IC50=0.29?M)which C-5 position is a benzene and the compound LFX-A021-133-01(IC50=0.38?M)which C-5 position is a nitro exhibited the most potent value.Compared with isatin,The inhibitory activity of these two compounds improved 345 multiples and 263 multiples,respectively.The results suggest that the spatial structure relatively large substituents or the strong electron withdrawing group in C-5-positions can significantly improve caspase-3 inhibitory activity.In summary,we studied the expression of caspasc-3 in prokaryotic system,and the detected isatin derivatives of caspase-3 kinase inhibitory activity in vitro by purified protein,then analysed structure-activity relationship of compounds.The results provide a good experimental basis to study the caspase-3 inhibitor.