Research during the past year focused primarily on the cellular pharmacology of the geldanamycins , a family of benzoquinoid ansamycins that include 17-AAG and 17-DMAG, two agents currently undergoing clinical evaluation and for which hepatotoxicity is a dose-limiting toxicity. Metabolism studies utilizing human hepatocytes and their extracts, we found, unexpectedly, that the geldanamycins react chemically (i.e., non-enzymatically) with glutathione under physiologic conditions. We characterized the reaction and identified the reaction products using LC/MS and NMR to be the glutathione addition products to the 19 position of the benzoquinone ring (Reference 1). These data suggest that cellular glutathione could play a role in modulating the cellular toxicity of the geldanamycins and therefore a factor in their mechanism of differential toxicity.Using HPLC, and a column that separates proteins by molecular weight, we found that 17-DMAG binds covalently to cellular proteins of hepatocytes obtained from human livers, presumably through their thiol groups. There are two large peaks containing protein-17-DMAG complexes along with multiple smaller peaks. 17-DMAG was found, also, to attach covalently to proteins of HT-29 cells. Covalent attachment to critical cellular proteins could be important to the mechanism of toxicity of this new class of anticancer agents.In studies of the hepatic metabolism of 17-DMAG, we found that 17-DMAG is rapidly converted, intacellularly and in cell homogenates, to the hydroquinone, which we identified by LC/MS. Our data indicate that 17-DMAG exists intracellularly as the hydroquinone, not the parent compound. We found that purified cytochrome- P450- reductase and DT-diaphorase can efficiently carry out the conversion of 17-DMAG to its hydroquinone form. The reactivity of 17-DMAG-hydroquinone with glutathione and hepatic proteins is markedly different from the reactivity of 17-DMAG. Also, 17-DMAG-hydroquinone, formed either enzymatically or chemically, quickly converts back to 17-DMAG under aerobic conditions. Current studies will determine if reactive species are generated by the inter-conversion of 17-DMAG and its hydroquinone and, if so, their contribution to hepatotoxicity and tumor cell toxicity.
|Cysyk, Richard L; Parker, Robert J; Barchi Jr, Joseph J et al. (2006) Reaction of geldanamycin and C17-substituted analogues with glutathione: product identifications and pharmacological implications. Chem Res Toxicol 19:376-81|
|Klecker, Raymond W; Cysyk, Richard L; Collins, Jerry M (2006) Zebularine metabolism by aldehyde oxidase in hepatic cytosol from humans, monkeys, dogs, rats, and mice: influence of sex and inhibitors. Bioorg Med Chem 14:62-6|