In vitro metabolism of the phosphatidylinositol 3-kinase inhibitor, wortmannin, by carbonyl reductase.
The phosphatidylinositol 3-kinase inhibitor, wortmannin, is extensively used in molecular signaling studies and has been proposed as a potential antineoplastic agent. The failure to detect wortmannin in mouse plasma after i.v. administration prompted in vitro studies of wortmannin metabolism. Wortmannin was incubated with mouse tissue homogenates, homogenate fractions, or purified, recombinant human carbonyl reductase in the presence of specified cofactors and inhibitors. Reaction products were characterized and quantified with liquid chromatography (LC)/mass spectrometry. Reaction rates were characterized using Michaelis-Menten kinetics. Wortmannin was metabolized to a material 2 atomic mass units greater than wortmannin. Liver homogenate had the highest metabolic activity. Some metabolism occurred in kidney and lung homogenates. Very little metabolism occurred in brain or red blood cell homogenates. Liver S9 fraction and cytosol metabolized wortmannin in the presence of NADPH and, to a much lesser extent, in the presence of NADH. Microsomal metabolism of wortmannin was minimal. Purified, recombinant human carbonyl reductase metabolized wortmannin. Quercetin, a carbonyl reductase inhibitor, greatly decreased wortmannin metabolism by S9, cytosol, and carbonyl reductase. The K(M) for wortmannin metabolism by purified, recombinant human carbonyl reductase was 119 +/- 9 microM, and the V(max) was 58 +/- 9 nmol/min/mg of protein. LC-tandem mass spectrometry spectra indicated that carbonyl reductase metabolized wortmannin to 17-OH-wortmannin. Wortmannin reduction by carbonyl reductase may partly explain why wortmannin is not detected in plasma after being administered to mice. Metabolism of wortmannin to 17-OH-wortmannin has mechanistic, and possibly toxicologic, implications because 17-OH-wortmannin is 10-fold more potent an inhibitor of phosphatidylinositol 3-kinase than is wortmannin.
Holleran
Julianne L
JL
University of Pittsburgh Cancer Institute, Room G27E, Hillman Research Pavilion, 5117 Centre Avenue, Pittsburgh, PA 15213-1863.
Fourcade
Julien
J
Egorin
Merrill J
MJ
Eiseman
Julie L
JL
Parise
Robert A
RA
Musser
Steven M
SM
White
Kevin D
KD
Covey
Joseph M
JM
Forrest
Gerald L
GL
Pan
Su-Shu
SS
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D000730:Androstadienes / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000378:metabolism; D005260:Female; D006801:Humans; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D000077191:Wortmannin
D000429:Alcohol Oxidoreductases; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D000730:Androstadienes; D004791:Enzyme Inhibitors; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D000077191:Wortmannin; C000614209:AKR7A5 protein, mouse