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

Abstract

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-AAGH 2 was NQO1 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 mechanismbased (suicide) inhibitor of NQO1. The reduction of 17-AAG to the corresponding hydroquinone 17-AAGH 2 was confirmed by tandem liquid chromatography-mass spectrometry. 17-AAGH 2 was relatively stable and only slowly underwent autooxidation back to 17-AAG over a period of hours. To examine the role of NQO1 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 NQO1 due to a genetic polymorphism, and MDA468/NQ16 cells are a stably transfected clone that express high levels of NQO1 protein. HPLC analysis of 17-AAG metabolism using cell sonicates and intact cells showed that 17-AAGH 2 was formed by MDA468/NQ16 cells, and formation of 17-AAGH 2 could be inhibited by ES936. No 17-AAGH 2 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-AAGH 2 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-AAGH 2 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. (Cancer Res 2005; 65(21): 10006-15)

Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues

“…This is in contrast to 17-AAG, where cancer cell lines with high NQO1 levels were much more sensitive to 17-AAG due to the reduction of the quinone moiety to the more active hydroquinone metabolite (15,16). Similarly, growthinhibitory effects of another resorcinol-containing HSP90 inhibitor (radicicol) were similar in a different pair of isogenic cell lines, differing only in their NQO1 levels (16).…”

“…Effects of NQO1/DT-Diaphorase on Cellular Sensitivity Cellular activity of 17-AAG is dependent on the levels of NQO1/DT-diaphorase due to NQO1-mediated reduction to the more potent hydroquinone (15,16). The melanoma cell lines used here express varying NQO1 levels, with SKMEL 5 and WM266.4 exhibiting the highest activity, SKMEL 28 showing intermediate activity, and SKMEL 2 exhibiting the lowest level (15).…”

“…16), on the sensitivity of cancer cells to the HSP90 inhibitors. As shown previously (15,27), increased sensitivity of 17-AAG was observed in the BE2 line with high NQO1 levels (Table 1B).…”

“…1) is in phase II clinical trials but is difficult to formulate, has low oral bioavailability, is subject to polymorphic metabolism by cytochrome P450 CYP3A4 (14) and NAD(P)H:quinone oxidoreductase 1 (NQO1/DT-diaphorase; refs. 15,16), and is effluxed by P-glycoprotein (15). Although the 17-dimethylaminoethylamino analogue 17-DMAG ( Fig.…”

Up-regulation of Heat Shock Protein 27 Induces Resistance to 17-Allylamino-Demethoxygeldanamycin through a Glutathione-Mediated Mechanism

“…Kelland et al (38) showed that 17-AAG is an NQO1 substrate and that NQO1 expression increased cell resistance to 17-AAG. Subsequently, Guo et al (39) identified an NQO1-dependent metabolite of 17-AAG that would potentially retain Hsp90-binding ability. However, Brunton et al (40) suggest that NQO1 expression in cell lines does not correlate with their sensitivity to 17-AAG.…”