Geldanamycin Leads to Superoxide Formation by Enzymatic and Non-enzymatic Redox Cycling

Abstract

The ansamycin antibiotic geldanamycin has frequently been used as an inhibitor of heat shock protein 90 (Hsp90), and this agent has been widely employed as a probe to examine the interactions of Hsp90 with endothelial nitric-oxide synthase. Geldanamycin contains a quinone group, which may participate in redox cycling. When geldanamycin was exposed to the flavin-containing enzyme cytochrome P-450 reductase, both semiquinone and superoxide (O2minusdot) radicals were detected using electron spin resonance. The treatment of endothelial cells with geldanamycin resulted in a dramatic increase in O2minusdot generation, which was independent of endothelial nitric-oxide synthase, because it was not inhibited by N-nitro-L-arginine methyl ester and also occurred in vascular smooth muscle cells. Diphenylene iodinium inhibited this increase in O2minusdot by 50%, suggesting that flavin-containing enzymes are involved in geldanamycin-induced O2minusdot generation. In the absence of cells, geldanamycin directly oxidized ascorbate, consumed oxygen, and produced O2minusdot. Geldanamycin decreased the bioavailable nitric oxide generated by 3,4-dihydrodiazete 1,2-dioxide in smooth muscle cells by 50%, whereas pretreatment with superoxide dismutase inhibited the effect of geldanamycin. These findings demonstrate that geldanamycin generates O2minusdot, which scavenges nitric oxide, leading to loss of its bioavailability. This effect is independent of the inhibition of Hsp90 and indicates that geldanamycin cannot be used as a specific inhibitor of Hsp90. In light of these findings, the studies using geldanamycin as an inhibitor of Hsp90 should be interpreted with caution.