Reduction of a series of benzoquinone ansamycin Hsp90 inhibitors by one and two electron reductases, and their interaction with reduced glutathione

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Hsp90 is a chaperone protein which is expressed at high levels in many tumors. Hsp90 assists in the folding and stability of a number of oncogenic proteins so that multiple oncogenic signals can be blocked simultaneously by Hsp90 inhibition. We have shown that benzoquinone ansamycin (BA) Hsp90 inhibitors such as 17-(allylamino)-17-demethoxygeldanamycin (17AAG) can undergo two electron reduction by NAD(P)H:Quinone Oxidoreductase 1 (NQO1) to 17AAG hydroquinone (17AAGH₂) which is a more effective Hsp90 inhibitor. BAs may also be metabolized by one-electron reductases such as cytochrome P450 reductase (CYP450R) and cytochrome b5 reductase (Cb5R) and may also interact with glutathione, reactions which may contribute to toxicity. In this study, we investigated the ability of geldanamycin (GM), 17AAG, 17-demthoxy-17-[[2-(dimethylamino)ethyl]amino]-geldanamycin (17DMAG), 17-(amino)-17-demethoxygeldanamycin (17AG) and 17-demethoxy-17-[[2-(pyrrolidin-1-yl)ethyl]amino]-geldanamycin (17AEP-GA) to be reduced via one electron reductases, their interaction with glutathione and the stability of the respective hydroquinone ansamycins generated via two electron reduction. The relative stabilities of hydroquinone ansamycins at room temperature and pH 7.4 in 50mM potassium phosphate buffer were GMH₂>17AAGH₂>17DMAGH₂>17AGH₂, 17AEP-GAH₂. Using human and mouse liver microsomes, and either NADPH or NADH as cofactors, we found that 17AAG had the lowest potential to be metabolized via one electron reductases while GM had the highest rate of microsomal reduction. We also investigated the interaction of BA with glutathione. At room temperature and pH 7.4, GM formed a glutathione adduct most readily followed by 17DMAG. The formation of glutathione adducts of 17AAG and 17AG were relatively slow in comparison. Glutathione adduct formation could be blocked by recombinant human NQO1. 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 these compounds. (Supported by National Institute of Health grant R01 CA51210).