Enzymatic Reduction and Glutathione Conjugation of Benzoquinone Ansamycin Heat Shock Protein 90 Inhibitors: Relevance for Toxicity and Mechanism of Action

Abstract

ABSTRACT:Two-electron reduction of benzoquinone ansamycin (BA) heat shock protein (Hsp) 90 inhibitors by NAD(P)H:quinone oxidoreductase 1 (NQO1) to hydroquinone ansamycins (BAH 2 s) 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 BAH 2 s 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 BAH 2 s 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 oneelectron 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.Hsp90 is a chaperone protein that is critical for the folding and stability of a number of oncogenic proteins, including Raf-1, mutant p53, ErBb2, Hif-1␣, topoisomerase II, and androgen/estrogen receptors (Selkirk et al., 1994;Schulte et al., 1995;Minet et al., 1999;Xu et al., 2002;Xu and Neckers, 2007). Inhibition of Hsp90 leads to depletion of these "client" proteins via the ubiquitin-proteasome pathway (Schulte et al., 1997;Imamura et al., 1998); therefore, many oncogenic signals can be blocked simultaneously by inhibition of Hsp90 (Powers and Workman, 2006). Benzoquinone ansamycins (BAs) (Scheme 1) are a class of Hsp90 inhibitors that bind to the N-terminal ATP binding pocket of Hsp90 to block Hsp90 ATPase activity (Stebbins et al., 1997). Geldanamycin (GM) was the first drug in this class but was withdrawn from clinical trials because of liver toxicity (Supko et al., 1995). 17-(Allylamino)-17-demethoxygeldanamycin (17AAG) and 17-demethoxy-17-[[2-(dimethylamino)ethyl]amino]-geldanamycin (17DMAG) are analogs of GM, which maintained Hsp90 inhib...

Benzoquinone ansamycin 17AAG binds to mitochondrial voltage-dependent anion channel and inhibits cell invasion

“…Such compounds do not degrade NQO1 but still show HSP90 inhibition (39), and they might be more efficacious with reduced toxicity. It is possible that the benzoquinone moiety binding to mitochondria may explain the liver toxicity of 17AAG observed clinically (5,40,41). Others have shown that tumor cells organize an intramitochondrial HSP90 and TRAP-1 chaperone network and regulate mitochondrial permeability transition (8).…”

“…17AAG and all GA derivatives have quinone moieties that are metabolized by NAD(P)H: quinone oxidoreductase1 (NQO1) (4,5). Kelland et al (6) first reported the relationship between levels of NQO1 and the sensitivity to 17AAG.…”

The Novel HSP90 Inhibitor, IPI-493, Is Highly Effective in Human Gastrostrointestinal Stromal Tumor Xenografts Carrying Heterogeneous KIT Mutations

“…Impaired liver function is commonly observed under treatment with BAderived HSP90-i (10). However, the molecular scaffold of IPI-493, which is related to 17-AAG, shows improved pharmacologic properties (35). According to in vitro data, IPI-493 presents higher potency in comparison with other HSP90-i (17).…”

HSP90 Chaperoning in Addition to Phosphoprotein Required for Folding but Not for Supporting Enzymatic Activities of Measles and Nipah Virus L Polymerases

“…Considering the similar effects of the two different approaches (interfering RNA and drug treatment in different cells), we are confident that the apparent common antiviral effect is the result of the specific inhibition of cellular HSP90 activity. Indeed, the two chemically distinct HSP90 inhibitors we used decrease the chance of observing a biological effect related to any "off-target" activity, as reported in the case of 17-DMAG (49,50). Furthermore, the mechanism of action of both drugs is well characterized, as shown by their perfect docking into the ATPase site of HSP90 (44,51).…”