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

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 corresponding 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]-geldanamycin. The reduction of these benzoquinone ansamycins by recombinant human NQO1 to the corresponding hydroquinone ansamycins was monitored by high-performance liquid chromatography (HPLC) and confirmed by liquid chromatography/ mass spectrometry. Inhibition of purified yeast Hsp90 ATPase activity was augmented in the presence of NQO1 and abrogated 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 ansamycins 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 hydroquinone ansamycins, which translated to greater interaction energies compared with the corresponding benzoquinone ansamycins. In conclusion, these studies show that the reduction of this series of benzoquinone ansamycins by NQO1 generates the corresponding hydroquinone ansamycins, which exhibit enhanced Hsp90 inhibition.The 90-kDa heat shock protein (Hsp90) is a molecular chaperone responsible for the ATP-dependent folding, stability, and function of a number of "client" proteins that are involved in the development and progression of cancer (Maloney 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 degradation 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 apoptosis (Hostein et al., 2001;M...

Role for NAD(P)H:quinone Oxidoreductase 1 and Manganese-Dependent Superoxide Dismutase in 17-(Allylamino)-17-demethoxygeldanamycin-Induced Heat Shock Protein 90 Inhibition in Pancreatic Cancer Cells

“…A role for NQO1 in jpet.aspetjournals.org generating substantially greater intracellular concentrations of both 17AAG and 17AAGH 2 was also demonstrated. The increased intracellular concentrations of 17AAG and 17AAGH 2 , in combination with the more efficient inhibition of Hsp90 by 17AAGH 2 (Guo et al, 2005(Guo et al, , 2006Maroney et al, 2006), are most likely the mechanisms whereby NQO1 contributes to the increased cytotoxicity of 17AAG. The NQO1 activity in the MiaPaCa-2 cell line was approximately 2-fold greater than the BxPC-3 and Panc-1/C5 cell lines; however, there was a more than 15-fold difference in the IC 50 values between MiaPaCa-2 cells and Panc-1/C5 cells, suggesting that the sensitivity to 17AAG in pancreatic cancer cell lines is not linearly related to NQO1 and other susceptibility factors in addition to NQO1 are involved.…”

“…Hydroquinone forms of 17AAG and 17DMAG are relatively stable but have been shown to be sensitive to copper-mediated reoxidation (Guo et al, 2008). Hydroquinone forms of 17AAG and 17DMAG have also been shown to be more potent inhibitors of Hsp90 in in vitro studies using purified Hsp90 compared with their parent quinones (Guo et al, 2006;Maroney et al, 2006). An important feature of the hydroquinone of 17AAG (17AAGH 2 ) is increased water solubility, and this feature has been exploited in an attempt to reduce vehicle-related toxicities associated with the administration of the more hydrophobic 17AAG.…”

“…Previous studies have demonstrated that many of the benoquinone ansamycins, including 17AAG, can undergo direct twoelectron reduction by NAD(P)H:quinone oxidoreductase 1 (NQO1) to their corresponding hydroquinone anasamycins (Kelland et al, 1999;Guo et al, 2005Guo et al, , 2006. NQO1 is an FAD-dependent direct two-electron reductase that can use either NADH or NADPH as reducing cofactor and reduces quinones directly to hydroquinones.…”

Preclinical Antitumor Activity of the Orally Available Heat Shock Protein 90 Inhibitor NVP-BEP800

“…Some of the formulations required to administer 17-AAG may contribute to its dose-limiting toxicity (16). In addition, it is also of concern that resistance to 17-AAG can be acquired through P-glycoprotein upregulation or by mutation or loss of the NQO1 gene [NAD(P)H:quinone oxidoreductase 1, DT-diaphorase], which is required for the bioreduction of 17-AAG into its more potent hydroquinone metabolite (17)(18)(19)(20). To overcome the limitations of the ansamycinderived Hsp90 inhibitors, the identification of a non-ansamycin, fully synthetic small-molecule inhibitor binding in the ATP pocket of Hsp90 would be of great therapeutic interest.…”

19-Substituted Benzoquinone Ansamycin Heat Shock Protein-90 Inhibitors: Biological Activity and Decreased Off-Target Toxicity

“…AST activity was measured using an assay kit (Teco Diagnostics, Anaheim, CA). Viability was also assessed using reduction of MTT to formazan (Guo et al, 2005(Guo et al, , 2006.…”