Rugulosin对N-Hsp90的抑制与结合作用  

Inhibitation and Binding of Rugulosin with N-Hsp90

作　　者：陈俊杰[1] 易玉婷[1] 于淼[1] 薛少龙[1] 熊茜茜[1] 贺晓萌[1] 张连茹[1] 

机构地区：[1]厦门大学生命科学学院,厦门361005

出　　处：《高等学校化学学报》2011年第1期88-94,共7页Chemical Journal of Chinese Universities

基　　金：国家自然科学基金(批准号:30873148;30973566;30921005;90913024);国家"重大新药创制"科技重大专项(批准号:2009ZX09103-083)资助

摘　　要：研究表明,来源于真菌的Rugulosin对热休克蛋白90(Hsp90)的三磷酸腺苷酶(ATPase)活性有较强的抑制作用.本文通过Western Blot实验发现,Rugulosin对Hsp90的客户蛋白Akt和Raf-1具有一定的降解作用.进一步应用荧光、ForteBio Octet、圆二色和紫外-可见吸收等波谱法研究Rugulosin与Hsp90 N端的ATP结合结构域(N-Hsp90)的相互作用机制发现,Rugulosin与N-Hsp90之间存在较强的相互作用,二者通过氢键等作用力形成静态复合物,二者间的距离约为3.4 nm.其解离常数KD由荧光实验和ForteBio Octet实验计算,分别为22.4和85.9μmol/L;Rugulosin使N-Hsp90的α-螺旋含量由15.0%降至11.6%;紫外-可见吸收光谱实验结果同样表明,Rugulosin使N-Hsp90的二级结构发生了改变.该研究对于Rugulosin的进一步开发以及将其作为分子探针进行与Hsp90相关的信号通路的研究具有重要的意义.Rugulosin firstly isolated from the metabolites of Penicillium rugulosum Thom has a strong inhibitory activity on Streptococcus and Corynebacterium.Heat shock protein 90（Hsp90） is a molecular chaperone required for the stability and function of a number of signalling proteins and protein kinases that promote cancer cell growth or survival.By molecular docking,we found the combination between Rugulosin and N-Hsp90（N-terminal of Hsp90,the ATPase activity domain）.Rugulosin showed strong inhibition to ATPase activity of Hsp90 and the IC50 was 22.3 μmol/L in vitro experiments.In this study,the interaction mechanism of Rugulosin with N-Hsp90 was investigated by fluorescence,ForteBio Octet,circular dichroism,UV-Visible absorption spectroscopy,SPR technology and Western Blot.Fluorescence spectra experiment results showed that Rugulosin caused a strong fluorescence quenching on intrinsic fluorescence of Hsp90 by static quenching method.Thermodynamic analysis signified the binding mechanism was hydrogen forces and dissociation constant was 22.4 μmol/L,which was 85.9 μmol/L calculated from ForteBio Octet method.Circular dichroism spectra detected the α-helix of N-Hsp90 reduced as the concentration of Rugulosin increased.Rugulosin could also down-regulate the expression of the client protein Akt and Raf-1 in HeLa cells.This study illustrates that Ruglulosin,which inhibited the ATPase activity of Hsp90 through interacting with N-Hsp90,may be a potential inhibitor of Hsp90.

关 键 词：Rugulosin HSP90 荧光光谱 圆二色光谱 表面等离子体共振 

分 类 号：O621[理学—有机化学]