热休克蛋白内质网亚型Grp94特异性荧光探针的设计、合成与应用

Design,synthesis and biological application of affinity-based small molecular probe for Hsp90 endoplasmic reticulum paralogue of Grp94

葡萄糖调节蛋白94(glucose-regulated protein 94,Grp94)是Hsp90在内质网中的亚型,其调控的客户蛋白较专一。选择性抑制Grp94已经成为靶向Hsp90伴侣系统开发药物的新方向。本研究以前期得到的高活性、高选择性Grp94抑制剂DDO-5813为基础,设计并合成得到了Grp94特异性荧光探针Grp94-Probe。采用荧光偏振实验和细胞内与ER-Red共染色实验证明Grp94-Probe与Grp94特异性结合作用。荧光偏振实验结果表明,Grp94-Probe能够较好地与Grp94~N结合(EC_(50)=117.9 nmol/L),且其作为探针分子可以较好的区分化合物对Grp94~N抑制活性的强弱。细胞内荧光成像实验结果显示,Grp94-Probe能够在细胞内质网与ER-Red共染色,表明Grp94-Probe能在细胞内与Grp94结合。该荧光探针为开发Grp94抑制剂提供了活性测试工具分子,为探索细胞内Grp94的化学生物学功能提供了有利工具。

Glucose-regulated protein 94 (Grp94),an endoplasmic reticulum resident Hsp90 paralog,has a limited set of client proteins.Selective inhibition of Grp94 has emerged as a new direction for the development of drugs targeting the Hsp90 chaperone system.Now Grp94-Probe,an affinity-based probe of Grp94,was designed and synthesized based on DDO-5813,a most potent Grp94-selective inhibitor we found previously.Using fluorescence polarization (FP) assay and double staining assay with ER-Red in cells,we confirmed the binding of Grp94-Probe with ER Grp94.The FR results showed that the probe exhibited high affinity for Grp94 N (EC 50 =117.9 nmol/L) without exhibiting obvious Hsp90α inhibition,Moreover,as a fluorescence probe molecule,Grp94-Probe could better distinguish the inhibitory activity of compounds for Grp94 N.The results of fluorescence analysis in cells showed that Grp94-Probe could co-stain with ER-Red in the endoplasmic reticulum,and the fluorescence did not decay rapidly with time after 4 h of staining,which further indicated the binding of Grp94-Probe with Grp94 in cells.This Grp94 selective probe can be further used for biology evaluation of Grp94 inhibitor and exploration of Grp94 biological functions.