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 Wenchang Guo, Philip Reigan, David Siegel, Joseph Zirrolli, Daniel Gustafson, and David Ross Department of Pharmaceutical Sciences, School of Pharmacy and Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado Abstract We have examined the role of NAD(P)H:quinone oxidoreduc- tase 1 (NQO1) in the bioreductive metabolism of 17-allyla- mino-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 mechanism- based (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 phosphory- lation 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 hydroqui- none 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) Introduction Heat shock protein (Hsp) 90 has been developed as a potential anticancer target and is an attractive target for several reasons (1–3). Hsp90 is a protein chaperone that uses the hydrolysis of ATP to assist in the folding of early nascent forms of client proteins to their mature, correctly folded forms. Once the client protein has been correctly folded, Hsp90 is released, and as such, it functions as a true protein ‘‘catalyst.’’ The basis for Hsp90 as an anticancer target is that this chaperone assists in the folding of many oncogenic proteins. Such proteins include ErbB2, Raf-1, mutant p53, estrogen, and steroid receptors. Thus, by inhibiting Hsp90, one can target a large number of downstream proteins and thereby attack the neoplastic process at several points (2–4). The first Hsp90 inhibitor used clinically was geldanamycin, which did not move forward in clinical trials due to liver toxicity. Second- generation derivatives, such as 17-allylamino-demethoxygeldana- mycin (17-AAG) and 17-dimethylaminogeldanamycin (17-DMAG), do not induce liver toxicity, have completed phase I, and are currently entering phase II clinical trials (2, 3, 5, 6). The product of cytochrome P 450–mediated dealkylation of 17-AAG and 17-DMAG at the 17 position, 17-AG, retains its quinone functionality and is also a Hsp90 inhibitor (7). Because 17-AAG and related benzoquinone ansamycins contain a quinone moiety, bioreduction to semiquinone and hydroquinone species is a possible metabolic pathway within tumor cells, and formation of these species will depend on the levels of bioreductive enzymes. Among the bioreductive enzymes expressed in cancer cells poised to have the greatest influence on 17-AAG metabolism is NAD(P)H:quinone oxidoreductase 1 (NQO1; DT-diaphorase, EC 1.6.99.2). This flavoenzyme can use either NADH or NADPH as reducing cofactors and can catalyze the direct two-electron reduction of quinones to hydroquinones (8). NQO1 is expressed at high levels in many human cancers, including lung, colon, stomach, pancreatic, and breast (9–11). NQO1 has also been shown to Requests for reprints: David Ross, Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Denver, CO 80262. Phone: 303-315-6077; Fax: 303-315-0274; E-mail: david. ross@uchsc.edu. I2005 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-2029 Cancer Res 2005; 65: (21). November 1, 2005 10006 www.aacrjournals.org Research Article Research. on February 9, 2016. © 2005 American Association for Cancer cancerres.aacrjournals.org Downloaded from