Enzymatic reduction and glutathione conjugation of benzoquinone ansamycin heat shock protein 90 inhibitors: relevance for toxicity and mechanism of action.

Two-electron reduction of benzoquinone ansamycin (BA) heat shock protein (Hsp) 90 inhibitors by NAD(P)H:quinone oxidoreductase 1 (NQO1) to hydroquinone ansamycins (BAH2s) leads to greater Hsp90 inhibitory activity. BAs can also be metabolized by one-electron reductases and can interact with glutathione, reactions that have been associated with toxicity. Using a series of BAs, we investigated the stability of the BAH2s generated by NQO1, the ability of BAs to be metabolized by one-electron reductases, and their conjugation with glutathione. The BAs used were geldanamycin (GM), 17-(allylamino)-17-demethoxygeldanamycin (17AAG), 17-demethoxy-17-[[2-(dimethyl amino)ethyl]amino]-geldanamycin (17DMAG), 17-(amino)-17-demethoxygeldanamycin (17AG), and 17-demethoxy-17-[[2-(pyrrolidin-1-yl)ethyl]amino]-geldanamycin (17AEP-GA). The relative stabilities of BAH2s at pH 7.4 were GM hydroquinone>17AAG hydroquinone>17DMAG hydroquinone>17AG hydroquinone and 17AEP-GA hydroquinone. Using human and mouse liver microsomes and either NADPH or NADH as cofactors, 17AAG had the lowest rate of one-electron reduction, whereas GM had the highest rate. 17DMAG demonstrated the greatest rate of redox cycling catalyzed by purified human cytochrome P450 reductase, whereas 17AAG again had the slowest rate. GM formed a glutathione adduct most readily followed by 17DMAG. The formation of glutathione adducts of 17AAG and 17AG were relatively slow in comparison. These data demonstrate that GM, the most hepatotoxic BAs in the series had a greater propensity to undergo redox cycling reactions catalyzed by hepatic one-electron reductases and markedly greater reactivity with thiols when compared with the least hepatotoxic analog 17AAG. Minimizing the propensity of BA derivatives to undergo one-electron reduction and glutathione conjugation while maximizing their two-electron reduction to stable Hsp90 inhibitory hydroquinones may be a useful strategy for optimizing the therapeutic index of BAs.