Biotransformation of Geldanamycin and 17-Allylamino-17-Demethoxygeldanamycin by Human Liver Microsomes: Reductive versus Oxidative Metabolism and Implications

Abstract

ABSTRACT:Comparative metabolite profiling of geldanamycin and 17-allylamino-17-demethoxygeldanamycin (17AAG) using human liver microsomes in normoxia and hypoxia was conducted to understand their differential metabolic fates. Geldanamycin bearing a 17-methoxy group primarily underwent reductive metabolism, generating the corresponding hydroquinone under both conditions. The formed hydroquinone resists further metabolism and serves as a reservoir. On exposure to oxygen, this hydroquinone slowly reverts to geldanamycin. In the presence of glutathione, geldanamycin was rapidly converted to 19-glutathionyl geldanamycin hydroquinone, suggesting its reactive nature. In contrast, the counterpart (17AAG) preferentially remained as its quinone form, which underwent extensive oxidative metabolism on both the 17-allylamino sidechain and the ansa ring. Only a small amount (<1%) of 19-glutathione conjugate of 17AAG was detected in the incubation of 17AAG with glutathione at 37°C for 60 min. To confirm the differential nature of quinone-hydroquinone conversion between the two compounds, hypoxic incubations with human cytochrome P450 reductase at 37°C and direct injection analysis were performed. Approximately 89% of hydroquinone, 5% of quinone, and 6% of 17-O-demethylgeldanamycin were observed after 1-min incubation of geldanamycin, whereas about 1% of hydroquinone and 99% of quinone were found in the 60-min incubation of 17AAG. The results provide direct evidence for understanding the 17-substituent effects of these benzoquinone ansamycins on their phase I metabolism, reactivity with glutathione, and acute hepatotoxicity.The benzoquinone ansamycins geldanamycin and 17-allylamino-17-demethoxygeldanamycin (17AAG) (Fig. 1) are potent antitumor agents that have undergone preclinical (Supko et al., 1995;Eiseman et al., 1997) and clinical (Banerji et al., 2005;Ramanathan et al., 2005) evaluations for their potential value in fighting cancers. The mechanism regarding the antineoplastic activity of these ansamycins has been extensively studied and showed to proceed through specific binding to the cytoplasmic heat shock protein 90 (HSP90) (Stebbins et al., 1997;Schulte and Neckers, 1998) and its endoplasmic reticulum homolog, glucose-regulated protein 94 (Chavany et al., 1996). Interaction of these ansamycins with the chaperone protein folding machinery results in disruption of heteroprotein complexes and blocking of the refolding and conformational maturation of numerous oncogenic client proteins, i.e., p185 erbB2 (Miller et al., 1994; Schnur et al., 1995a,b) , which are involved in signal transduction in regulation of cell proliferation, apoptosis, and angiogenesis. It is known that the unfolded client proteins are susceptible to degradation via the ubiquitination-proteasome pathway. However, recent findings from electron spin resonance studies of the production of reactive oxygen species (i.e., superoxide anion and hydrogen peroxide) mediated by geldanamycin leading to oxidative stress and cell death argued against the HSP90 inhi...

17-O-demethylreblastatin, a subnormal intermediate in geldanamycin biosynthesis

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“…In contrast, the clinical development of geldanamycin, which inhibits the activity of heat shock protein 90 (HSP 90), was discontinued after the appearance of dose-limiting liver toxicity, which was shown to be related to its chemical structure and not the result of HSP 90 inhibition (Supko et al 1995). Subsequent efforts to identify a clinically useful HSP 90 inhibitor focused on structural modifications to remove the liver toxicophore (Campillos et al 2008;Lang et al 2007;Nowakowski et al 2006). Pharmacological effects are related to a specific moiety on the molecular structure known as a pharmacophore.…”

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“…Both GA and 17-AAG are known to undergo extensive metabolism (Egorin et al, 1998;Musser et al, 2003;Guo et al, 2005Guo et al, , 2006Lang et al, 2007). Although GA and 17-AAG are structurally similar (see Fig.…”

“…Although GA and 17-AAG are structurally similar (see Fig. 1), their metabolite profiles in liver microsomes are different (Lang et al, 2007). GA is primarily (40 -73%) reduced into geldanamycin hydroquinone (GAH 2 ) (Lang et al, 2007;Guo et al, 2008).…”