The Bioreduction of a Series of Benzoquinone Ansamycins by NAD(P)H:Quinone Oxidoreductase 1 to More Potent Heat Shock Protein 90 Inhibitors, the Hydroquinone Ansamycins □ S 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 Received April 12, 2006; accepted July 6, 2006 ABSTRACT We have previously evaluated the role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in the bioreductive metabolism of 17-(allylamino)-demethoxygeldanamycin (17AAG) to the corre- sponding hydroquinone, a more potent 90-kDa heat shock protein (Hsp90) inhibitor. Here, we report an extensive study with a series of benzoquinone ansamycins, which includes gel-danamycin, 17-(amino)-17-demethoxygeldanamycin, and 17-demethoxy-17-[[2-(dimethylamino)ethyl]amino]-geldanamy- cin. The reduction of these benzoquinone ansamycins by re- combinant human NQO1 to the corresponding hydroquinone ansamycins was monitored by high-performance liquid chro- matography (HPLC) and confirmed by liquid chromatography/ mass spectrometry. Inhibition of purified yeast Hsp90 ATPase activity was augmented in the presence of NQO1 and abro- gated by 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl- ]indole-4,7-dione (ES936), a mechanism-based inhibitor of NQO1, showing that the hydroquinone ansamycins were more potent Hsp90 inhibitors than their parent quinones. An isogenic pair of human breast cancer cell lines, MDA468 and MDA468/ NQ16, differing in expression of NQO1, was used, and HPLC analysis showed that hydroquinone ansamycins were formed by the MDA468/NQ16 cells, which could be prevented by ES936 pretreatment. The MDA468/NQ16 cells were more sensitive to growth inhibition after treatment with the benzoquinone ansamy- cins compared with the MDA468 cells; this increased sensitivity could be reduced by ES936 pretreatment. The increased duration of benzoquinone ansamycin exposure showed increased potency and -fold inhibition in MDA468/NQ16 cells relative to the parental MDA468 cells. Computational-based molecular modeling studies displayed additional contacts between yeast Hsp90 and the hy- droquinone ansamycins, which translated to greater interaction energies compared with the corresponding benzoquinone ansa- mycins. In conclusion, these studies show that the reduction of this series of benzoquinone ansamycins by NQO1 generates the corresponding hydroquinone ansamycins, which exhibit en- hanced Hsp90 inhibition. The 90-kDa heat shock protein (Hsp90) is a molecular chaperone responsible for the ATP-dependent folding, stabil- ity, and function of a number of “client” proteins that are involved in the development and progression of cancer (Ma- loney and Workman, 2002; Isaacs et al., 2003); these proteins include ErbB2, Raf-1, Cdk4, Met, mutant p53, telomerase hTERT, Hif-1, and the estrogen and androgen receptors. The function of Hsp90 has been shown to be dependent on its ability to bind and hydrolyze ATP (Obermann et al., 1998; Panaretou et al., 1998; Pearl and Prodromou, 2001), and competitive inhibition of ATP binding by the natural product geldanamycin (GM), a benzoquinone ansamycin antibiotic isolated from Streptomyces hygroscopicus, leads to the deg- radation of the client proteins by the ubiquitin-proteosome pathway (Whitesell et al., 1994; Schulte et al., 1995; An et al., 1997), resulting in cell cycle arrest, differentiation, and apo- ptosis (Hostein et al., 2001; Munster et al., 2001). Therefore, This work was supported by National Institutes of Health grant R01- CA51210. W.G. and P.R. contributed equally to this work. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.025643. □ S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: Hsp90, heat shock protein 90; GM, geldanamycin; 17AAG, 17-(allylamino)-17-demethoxygeldanamycin; 17DMAG, 17- demthoxy-17-[[2-(dimethylamino)ethyl]amino]-geldanamycin; 17AG, 17-(amino)-17-demethoxygeldanamycin; NQO1, NAD(P)H:quinone oxi- doreductase; 17AAGH 2 , 17-(allylamino)-17-demethoxygeldanamycin hydroquinone; ES936, 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl- ]indole-4,7-dione; 17AEP-GA, 17-demethoxy-17-[[2-(pyrrolidin-1-yl)ethyl]amino]-geldanamycin; DCPIP, 2,6-dichlorophenol-indophenol; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; BSA, bovine serum albumin; rhNQO1, recombinant human NQO1; HPLC, high- performance liquid chromatography; LC/MS, liquid chromatography/mass spectrometry; DMSO, dimethyl sulfoxide. 0026-895X/06/7004-1194 –1203$20.00 MOLECULAR PHARMACOLOGY Vol. 70, No. 4 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 25643/3139514 Mol Pharmacol 70:1194–1203, 2006 Printed in U.S.A. 1194 http://molpharm.aspetjournals.org/content/suppl/2006/07/10/mol.106.025643.DC1.html Supplemental material to this article can be found at: at ASPET Journals on February 9, 2016 molpharm.aspetjournals.org Downloaded from