Formation of 17-allylamino-demethoxygeldanamycin (17-AAG) hydroquinone by NAD(P)H:quinone oxidoreductase 1:: Role of 17-AAG hydroquinone in heat shock protein 90 inhibition.
Formation of 17-allylamino-demethoxygeldanamycin (17-AAG) hydroquinone by NAD(P)H:quinone oxidoreductase 1:: Role of 17-AAG hydroquinone in heat shock protein 90 inhibition.
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DOI：
10.1158/0008-5472.can-05-2029
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发表时间：
2005-11-01
期刊：
影响因子：
11.2
通讯作者：
Ross, D
中科院分区：
文献类型：
--
作者：
Guo, WC;Reigan, P;Ross, D
我们研究了NAD（P）H：醌氧化还原酶1（NQO 1）在17-烯丙氨基-脱甲氧基格尔德霉素（17-AAG）生物还原代谢中的作用。重组人NQO1代谢17-AAG的高效液相色谱（HPLC）分析显示形成了极性更强的代谢物17-AAGH（2）。17-AAGH2的形成是NQ01依赖性的，并且其形成可以通过添加NQO1的基于机制的（自杀）抑制剂5-甲氧基-1，2-二甲基-3-[（4-硝基苯氧基）甲基]吲哚-4，7-二酮（ES 936）来抑制。通过串联液相色谱-质谱法证实17-AAG还原为相应的氢醌17-AAGH2。17-AAGH2相对稳定，仅在数小时内缓慢地自动氧化回17-AAG。为了研究NQ01在细胞中17-AAG代谢中的作用，我们使用了一对仅在NQO1水平上不同的同基因人乳腺癌细胞系。由于遗传多态性，MDA468细胞缺乏NQ01，并且MDA468/NQ16细胞是表达高水平NQ01蛋白的稳定转染的克隆。使用细胞超声处理物和完整细胞进行的17-AAG代谢的HPLC分析表明，17-AAGH2由MDA 468/NQ16细胞形成，并且17-AAGH2的形成可被ES936抑制。在超声处理物或完整的MDA 468细胞中未检测到17-AAGH2。在用17-AAG处理4小时后，与MDA468细胞相比，MDA468/NQ16细胞对生长抑制的敏感性高12倍。更重要的是，用ES936预处理MDA468/NQ16细胞可以消除其对17-AAG的敏感性增加。通过免疫印迹分析测定热休克蛋白（Hsp）90抑制、Hsp 70诱导和Raf-1降解的细胞标志物。在MDA468/NQ16细胞中观察到显著的Hsp70诱导和Raf-1降解，但在MDA468细胞中未观察到。类似地，下游Raf-1信号分子丝裂原活化蛋白激酶/细胞外信号调节激酶（ERK）激酶和ERK在MDA468/NQ16细胞中也显示出磷酸化水平降低，但在MDA468细胞中没有。检测了17-AAG和17-AAGH2抑制纯化的酵母和人Hsp90 ATP酶活性的能力。在NQO1存在下观察到最大的17-AAG诱导的ATP酶抑制，并且可以被ES 936消除，表明17-AAGH（2）是比17-AAG更有效的Hsp90抑制剂。分子模拟研究还表明，由于氢醌和Hsp90蛋白之间的氢键增加，17-AAGH2在酵母和人Hsp90模型中与ATP结合位点结合更紧密。总之，这些研究表明，NQO 1还原17-AAG可生成17-AAGH（2），这是一种相对稳定的对苯二酚，具有上级Hsp90抑制作用。
We have examined the role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in the bioreductive metabolism of 17-allylamino-demethoxygeldanamycin (17-AAG). High-performance liquid chromatography (HPLC) analysis of the metabolism of 17-AAG by recombinant human NQO1 revealed the formation of a more polar metabolite 17-AAGH(2). The formation of 17-AAGH2 was NQ01 dependent, and its formation could be inhibited by the addition of 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936), a mechanism-based (suicide) inhibitor of NQO1. The reduction of 17-AAG to the corresponding hydroquinone 17-AAGH2 was confirmed by tandem liquid chromatography-mass spectrometry. 17-AAGH2 was relatively stable and only slowly underwent autooxidation back to 17-AAG over a period of hours. To examine the role of NQ01 in 17-AAG metabolism in cells, we used an isogenic pair of human breast cancer cell lines differing only in NQO1 levels. MDA468 cells lack NQ01 due to a genetic polymorphism, and MDA468/NQ16 cells are a stably transfected clone that express high levels of NQ01 protein. HPLC analysis of 17-AAG metabolism using cell sonicates and intact cells showed that 17-AAGH2 was formed by MDA468/NQ16 cells, and formation of 17-AAGH2 could be inhibited by ES936. No 17-AAGH2 was detected in sonicates or intact MDA468 cells. Following a 4-hour treatment with 17-AAG, the MDA468/NQ16 cells were 12-fold more sensitive to growth inhibition compared with MDA468 cells. More importantly, the increased sensitivity of MDA468/NQ16 cells to 17-AAG could be abolished if the cells were pretreated with ES936. Cellular markers of heat shock protein (Hsp) 90 inhibition, Hsp70 induction, and Raf-1 degradation were measured by immunoblot analysis. Marked Hsp70 induction and Raf-1 degradation was observed in MDA468/NQ16 cells but not in MDA468 cells. Similarly, downstream Raf-1 signaling molecules mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase and ERK also showed decreased levels of phosphorylation in MDA468/NQ16 cells but not in MDA468 cells. The ability of 17-AAG and 17-AAGH2 to inhibit purified yeast and human Hsp90 ATPase activity was examined. Maximal 17-AAG-induced ATPase inhibition was observed in the presence of NQO1 and could be abrogated by ES936, showing that 17-AAGH(2) was a more potent Hsp90 inhibitor compared with 17-AAG. Molecular modeling studies also showed that due to increased hydrogen bonding between the hydroquinone and the Hsp90 protein, 17-AAGH2 was bound more tightly to the ATP-binding site in both yeast and human Hsp90 models. In conclusion, these studies have shown that reduction of 17-AAG by NQO1 generates 17-AAGH(2), a relatively stable hydroquinone that exhibits superior Hsp90 inhibition.