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 O(2)(*)(-) 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.
Dikalov
Sergey
S
Division of Cardiology, Emory University School of Medicine and Atlanta Veterans Administration Hospital, Georgia 30322, USA. dikalov@emory.edu
Landmesser
Ulf
U
Harrison
David G
DG
D001205:Ascorbic Acid / Q000378:metabolism; D016227:Benzoquinones; D002478:Cells, Cultured; D004578:Electron Spin Resonance Spectroscopy; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D019811:Hydroxylamine / Q000378:metabolism; D047029:Lactams, Macrocyclic; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D009569:Nitric Oxide / Q000037:antagonists & inhibitors; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010084:Oxidation-Reduction; D011809:Quinones / Q000494:pharmacology*; D013481:Superoxides / Q000378:metabolism*
D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D011809:Quinones; D013481:Superoxides; D019811:Hydroxylamine; D009569:Nitric Oxide; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D009251:NADPH-Ferrihemoprotein Reductase; D001205:Ascorbic Acid; C001277:geldanamycin