The role of NQO1 in the metabolism of benzoquinone ansamycin Hsp90 inhibitors and development of novel Hsp90 inhibitors as anticancer agents

Heat shock protein 90 (Hsp90) is a chaperone protein which is over-expressed in many solid tumors. Hsp90 plays an important role in the folding and stability of a number of oncogenic proteins including ErbB2, Raf-1, Akt, Hif-1alpha, mutant p53 and estrogen/androgen receptors. Inhibition of Hsp90 activity leads to depletion of these oncogenic proteins resulting in growth inhibition and tumor cell death. Hsp90 inhibitors represent a novel class of antitumor agents and 17AAG is now entering phase II clinical trials. Previous work has demonstrated that NQO1 reduces geldanamycin (GM) and related quinones and expression of NQO1 in cancer cell lines generally increased their sensitivity to 17AAG. These studies were extended to examine in greater detail the role of NQO1 in GM, 17AG, 17AAG, 17DMAG and 17AEP-GA metabolism using isogenic cell systems varying in the expression of NQO1 in combination with suicide inhibitors of NQO1, and recombinant Hsp90. Liver microsomal systems and glutathione were also used in studies of benzoquinone ansamycin (BA) metabolism. BA Hsp90 inhibitors could be reduced by NQO1 to their corresponding hydroquinone ansamycins. Hydroquinone ansamycins are more water-soluble and more potent Hsp90 inhibitors than their corresponding quinones. The oxidation of 17AAG hydroquinone could be stimulated by copper while 17AAG hydroquinone oxidation could be prevented by copper chelators such as D-penicillamine and human serum albumin. 17AAG quinol sulfate was generated by chemical reduction of 17AAG with sodium dithionite. 17AAG quinol sulfate may represent a potential prodrug which could generate 17AAG hydroquinone in cells with high sulfatase activity such as breast cancer cells. The relative rates of one-electron redox cycling of BA Hsp90 inhibitors in human and mouse liver microsomes as well as their conjugation with glutathione at the 19 position were also examined. The redox properties of BA and their ability to interact with glutathione may relate to their dose-limiting liver toxicity. These results have implications for future development of prodrugs of hydroquinone ansamycins and novel 19-substituted BA.