A potential role for NAD(P)H:quinone oxidoreductase 1 (NQO1) in the mechanism of action of the HSP90 inhibitors geldanamycin and 17AAG

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Geldanamycin and 17AAG exhibit antitumor activity primarily through the inhibition of Hsp90. Tandom LCMS studies with purified recombinant human NQO1 demonstrated that geldanamycin, and 17AAG could be reduced by NQO1/NADH to their corresponding hydroquinones. To examine the role of NQO1 in 17AAG metabolism in cells we utilized the NQO1-null human breast cancer cell line MDA468 and MDA468/NQ16, a stably transfected clone that expresses high levels of NQO1 protein. Following treatment with 17AAG the MDA468\NQ16 cell line was 20-fold more sensitive to growth inhibition compared to the MDA468 cell line. The increased sensitivity of the MDA468\NQ16 cell line to 17AAG could be abolished if the cells were pretreated with ES936, a mechanism-based inhibitor of NQO1. HPLC analysis of cells in culture treated with 17AAG demonstrated higher concentrations of 17AAG hydroquinone in MDA468\NQ16 cells compared to MDA468 cells and interestingly MDA468\NQ16 cells also contained greater concentrations of 17AAG. Formation of 17AAG hydroquinone and increased 17AAG accumulation in MDA468/NQ16 cells could be inhibited by pretreatment with ES936. Immunoblot analysis following 17AAG treatment revealed increased Hsp70 and decreased Raf-1 proteins levels in MDA468/NQ16 cells but not MDA468 cells. In molecular modeling studies of the ATP binding site of Hsp90 we examined docking of both quinone and hydroquinone forms of geldanamycin and 17AAG. As expected, the ansamycin ring of both the quinone and hydroquinone forms adopts a C-like conformation with the carbamate group at the bottom of the binding site and the quinone/hydroquinone ring system at the solvent-exposed top of the pocket. The interactions between the residues of the binding site and the quinone and hydroquinone forms were similar to those observed in the literature for geldanamycin. However, the extensive van der Waals contacts between the ansamycin ring of hydroquinone ligands and binding pocket residues were supplemented by additional hydrogen-bonding interactions with other substituents of the macrocycle, which resulted in more favourable binding energies. These results suggest a role for 17AAG hydroquinone in Hsp90 inhibition. (Supported by CA51210)