Received for publication April 24, 2008.
Revised July 15, 2008.
Accepted for publication July 16, 2008.

Enzymatic reduction and glutathione conjugation of benzoquinone ansamycin Hsp90 inhibitors: Relevance for toxicity and mechanism of action

Abstract

Two electron reduction of benzoquinone ansamycin (BA) Hsp90 inhibitors by NAD(P)H:Quinone Oxidoreductase 1 (NQO1) to hydroquinone ansamycins (BAH₂) leads to greater Hsp90 inhibitory activity. BA can also be metabolized by one-electron reductases and can interact with glutathione, reactions which have been associated with toxicity. Using a series of BA we investigated the stability of the BAH₂ generated by NQO1, the ability of BA to be metabolized by one electron reductases and their conjugation with glutathione. The BA used were GM (geldanamycin), 17AAG (17-(allylamino)-17-demethoxygeldanamycin), 17DMAG (17-demthoxy-17-[[2-(dimethylamino)ethyl]amino]-geldanamycin), 17AG (17-(amino)-17-demethoxygeldanamycin) and 17AEP-GA (17-demethoxy-17-[[2-(pyrrolidin-1-yl)ethyl]amino]-geldanamycin).The relative stabilities of BAH₂ at pH 7.4 were GMH₂>17AAGH₂>17DMAGH₂>17AGH₂, 17AEP-GAH₂. Using human and mouse liver microsomes, and either NADPH or NADH as cofactors, 17AAG had the lowest rate of one-electron reduction while GM had the highest rate. 17DMAG demonstrated the greatest rate of redox cycling catalyzed by purified human cytochrome P450 reductase while 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 BA 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 to 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 BA.

Key words: cytochrome b5, glutathione conjugates, NAD(P)H-quinone oxidoreductase, NADPH cytochrome P450 reductase, reductases