七种蛋白酶抑制剂对多房棘球蚴DNA损伤诱导样1蛋白活性的影响

The Effect of Seven Protease Inhibitors on Activity of DNA Damage Inducible 1 Proteinin Echinococcus multilocularis

目前,多房棘球蚴病(alveolar echinococcosis, AE)尚无有效药物治疗手段,迫切需要开发新型治疗药物。前期研究表明,HIV蛋白酶抑制剂(HIV protease inhibitors, HIV PIs)具有潜在抗寄生虫功能。本文旨在研究HIV PIs对Echinococcus multilocularis(Emu)DNA损伤诱导样1蛋白(DNA damage inducible 1 protein, Ddi1)活性的影响。本研究通过构建真核表达重组载体pFastBac1-Emu Ddi1,在昆虫细胞系Sf9细胞中表达筛选出P1代和P2代,纯化出可溶性Ddi1重组蛋白,然后与目的蛋白的荧光底物检测纯化蛋白的活性,进一步检测沙奎那韦(saquinavir, SQV)、利托那韦(ritonavir, RTV)、安普那韦(amprenavir, APV)、阿扎那韦(atazanavir, ATV)、洛匹那韦(lopinavir, LPV)、福沙那韦(fosamprenavir, Fos)、达芦那韦(darunavir, DRV)等7种HIV PIs对Emu Ddi1重组蛋白活性的抑制能力。结果显示:细胞系内真核表达产物的酶活Km为1.422μmol·L^(-1),具有良好的亲和力和活性,最终筛到沙奎那韦对Ddi1蛋白二聚体活性位点的抑制率达67%,其IC_(50)为34,说明沙奎那韦对Emu Ddi1重组蛋白酶活性具有良好的抑制效果。以上结果提示:沙奎那韦抑制重组蛋白Ddi1的活性,可能成为Ddi1的靶向药物,以期为替代药物或开发联合用药提供基础。

As no effective treatment for alveolar echinococcosis(AE)is currently available,the therapeutic drugs are needed urgently.Early stage studies have shown that the protease inhibitors of HIV could also be anticancer and anti-parasitic.The purpose of this paper is to study the effect of the inhibitors(HIV PIs)on the activity of DNA damage inducible 1 protein of Echinococcus multilocularis(Emu Ddi1).The recombinant vector pFastBac1-Emu Ddi1 was constructed and expressed in Sf9 cell,and the soluble protein was successfully purified in P2 generations.Then the enzyme activity of Ddi1 protein was detected with the specific fluorescent substrates,and the inhibition rate of seven HIV PIs including saquinavir(SQV),ritonavir(RTV),amprenavir(APV),atazanavir(ATV),lopinavir(LPV),fosamprenavir(Fos),tipranavir(TPV)and darunavir(DRV)on Ddi1 protein activity was further examined.The results showed that Emu Ddi1had high affinity and activity,and saquinavir showed the highest inhibition rate at 67%to inhibit protease activity of Emu Ddi1 with a IC 50 at 34.These results suggested that saquinavir is effcetive to inhibit the activity of Ddi1,and could be used to develop a potential targeting drug for AE.