Received for publication April 14, 2006.
Revised July 5, 2006.
Accepted for publication July 6, 2006.

The bioreduction of a series of benzoquinone ansamycins by NAD(P)H:quinone oxidoreductase 1 (NQO1) to more potent heat shock protein 90 (Hsp90) 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 Hsp90 inhibitor. Here, we report an extensive study with a series of benzoquinone ansamycins, that include geldanamycin, 17-(amino)-17-demethoxygeldanamycin, 17-(allylamino)-17-demethoxygeldanamycin and 17-demthoxy-17-[[2-(dimethylamino)ethyl]amino]-geldanamycin. The reduction of these benzoquinone ansamycins by recombinant human NQO1 to the corresponding hydroquinone ansamycins was monitored by HPLC and confirmed by LC-MS. 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, demonstrating 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, were utilized and HPLC analysis demonstrated that hydroquinone ansamycins were formed by the MDA468/NQ16 cells, which could be prevented by ES936 pre-treatment. The MDA468/NQ16 cells were more sensitive to growth inhibition following treatment with the benzoquinone ansamycins, compared to the MDA468 cells; this increased sensitivity could be reduced by ES936 pre-treatment. The increased duration of benzoquinone ansamycin exposure demonstrated 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 when compared to the corresponding benzoquinone ansamycins. In conclusion, these studies demonstrate that the reduction of this series of benzoquinone ansamycins by NQO1 generates the corresponding hydroquinone ansamycins which exhibit enhanced Hsp90 inhibition.

Key words: Structure-activity relationships and modeling, Enzymology, Quinone oxidoreductase, Protein targets

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