Abstract

The benzoquinone ansamycin (BQA) are a valuable class of antitumor agents that serve as inhibitors of heat shock protein-90 (Hsp90). However, clinical use of BQAs has resulted in off-target toxicities including concerns of hepatotoxicity. Mechanisms underlying the toxicity of quinones include their ability to redox cycle and/or arylate cellular nucleophiles at the unsubstituted 19-position of the molecule. We have therefore designed 19-substituted BQAs to prevent glutathione conjugation and non-specific interactions with protein thiols as an approach to minimize off-target effects and reduce hepatotoxicity. 19-phenyl and 19-methyl substituted versions of GA and its derivatives, 17-AAG and 17-DMAG, did not react with glutathione, while marked reactivity was observed using parent BQAs. Importantly, while 17-DMAG induced cell death in primary and cultured mouse hepatocytes, 19-phenyl and 19-methyl DMAG showed reduced toxicity, validating the overall approach. Furthermore, our data suggest that arylation reactions rather than redox cycling are a major mechanism contributing to BQA hepatotoxicity. 19-phenyl BQAs inhibited purified Hsp90 in an NAD(P)H oxidoreductase 1 (NQO1)-dependent manner, demonstrating increased inhibitory efficacy of the hydroquinone ansamycin relative to its parent quinone. Molecular modeling supported increased stability of the hydroquinone form of 19-phenyl-DMAG in the active site of human Hsp90. In human breast cancer cells, 19-phenyl BQAs induced growth inhibition also dependent upon metabolism via NQO1 with decreased expression of client proteins and compensatory induction of Hsp70. These data demonstrate that 19-substituted BQAs are unreactive with thiols and display reduced hepatotoxicity when compared to their parent quinones, but retain Hsp90 and growth inhibitory activity in human breast cancer cells although with diminished potency relative to parent BQAs