Study on a Screening Model for Inhibitor of Hsp90 ATPase Activity

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.