基于Hsp90 ATPase活性的抑制剂筛选模型研究

Study on a Screening Model for Inhibitor of Hsp90 ATPase Activity

热休克蛋白90(Heat shock protein,Hsp90)作为分子伴侣,参与调控肿瘤细胞多条信号通路,对肿瘤细胞生长有重要作用.以pGEX-4T-1为载体,构建了Hsp90表达载体于宿主大肠杆菌中表达.通过孔雀绿磷钼酸铵-无机磷检测法,测定异源表达的Hsp90 ATPase活性.实验得出Hsp90对ATP的米氏常数(Km)为369.1μmol/L,在Hsp90和ATP浓度分别为0.18,1 mmol/L时转换效率常数(kcat)为1.6 min-1,最大反应速率(Vmax)为1.0μmol/(L.min).基于该方法,建立Hsp90 ATPase活性抑制剂筛选模型,以Geldanamycin和Radicicol为阳性对照,发现Rifamycin、Rugulosin及My-coE也有较强的Hsp90 ATPase抑制活性.该方法可用于筛选抗肿瘤候选化合物的研究,同时也可为具有ATPase活性蛋白的抑制剂筛选提供参考.

As a molecular chaperone,Hsp90 regulates multiple signaling pathways of tumor cells and plays an important role in the growth of tumor cells.In this paper,recombinant vector of Hsp90-pGEX-4T-1 was constructed for Expression of Hsp90,and fusion protein was expressed in E.coli（DE3）.A colorimetric assay for inorganic phosphate which based on the formation of a phosphomolybdate complex and subsequent reaction with malachite green were used for determination of Hsp90 ATPase activity.The Km for ATP determined in this assay was 369.1 μmol/L,the Vm and kcat were 1.0 μmol/（L·min） and 1.6 min-1,when the concentration of Hsp90 and ATP were 0.18 μmol/L and 1 mmol/L respectively.Based on this method,a screening model for inhibitor of Hsp90 ATPase activity was established.Later study found that Rifamycin,Rugulosin and MycoE showed inhibition of Hsp90 ATPase activity,with the IC50 values were 18.04,22.77 and 38.45 μmol/L,but the IC50 valus were higher than the IC50 of GA and RDC.This method can not only be used to discover anti-cancer drug,but also to screen inhibitor of protein with ATPase activity.