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
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摘要: 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Grp94N结合(EC50=117.9 nmol/L)，且其作为探针分子可以较好的区分化合物对Grp94N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。Abstract: 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 Grp94N(EC50=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 Grp94N. 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.
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