Geldanamycin leads to superoxide formation by enzymatic and non-enzymatic redox cycling - Implications for studies of hsp90 and endothelial cell nitric-oxide synthase

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 (O-2(.-)) radicals were detected using electron spin resonance. The treatment of endothelial cells with geldanamycin resulted in a dramatic increase in 07 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 O-2(.-) by 50%, suggesting that flavin-containing enzymes are involved in geldanamycin-induced O-2(.-) generation. In the absence of cells, geldanamycin directly oxidized ascorbate, consumed oxygen, and produced O-2(.-). 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 O-2(.-) 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.