Catechol estrogen 4-hydroxyequilenin is a substrate and an inhibitor of catechol-O-methyltransferase.
Redox and/or electrophilic metabolites formed during estrogen metabolism may play a role in estrogen carcinogenesis. 4-Hydroxyequilenin (4-OHEN) is the major phase I catechol metabolite of the equine estrogens equilenin and equilin, which are components of the most widely prescribed estrogen replacement formulation, Premarin. Previously, we have found that 4-OHEN rapidly autoxidized to an o-quinone in vitro and caused toxic effects such as the inactivation of human detoxification enzymes. 4-OHEN has also been shown to be a substrate for catechol-O-methyltransferase (COMT) in human breast cancer cells. In the present study, we demonstrated that 4-OHEN was not only a substrate of recombinant human soluble COMT in vitro with a K(m) of 2.4 microM and k(cat) of 6.0 min(-)(1) but it also inhibited its own methylation by COMT at higher concentrations in the presence of the reducing agent dithiothreitol. In addition, 4-OHEN was found to be an irreversible inhibitor of COMT-catalyzed methylation of the endogenous catechol estrogen 4-hydroxyestradiol with a K(i) of 26.0 microM and a k(2) of 1.62 x 10(-)(2) s(-)(1). 4-OHEN in vitro not only caused the formation of intermolecular disulfide bonds as demonstrated by gel electrophoresis, but electrospray ionization mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry also showed that 4-OHEN alkylated multiple residues of COMT. Peptide mapping experiments further indicated that Cys33 in recombinant human soluble COMT was the residue most likely modified by 4-OHEN in vitro. These data suggest that inhibition of COMT methylation by 4-OHEN might reduce endogenous catechol estrogen clearance in vivo and further enhance toxicity.
Yao
Jiaqin
J
Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, 60612, USA.
Li
Yan
Y
Chang
Minsun
M
Wu
Huaping
H
Yang
Xiaofeng
X
Goodman
Julie E
JE
Liu
Xuemei
X
Liu
Hong
H
Mesecar
Andrew D
AD
Van Breemen
Richard B
RB
Yager
James D
JD
Bolton
Judy L
JL
D002394:Catechol O-Methyltransferase / Q000737:chemistry; D065098:Catechol O-Methyltransferase Inhibitors; D002851:Chromatography, High Pressure Liquid; D004855:Equilenin / Q000031:analogs & derivatives* / Q000737:chemistry*; D045166:Estradiol Congeners / Q000737:chemistry*; D002393:Estrogens, Catechol / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry; D008745:Methylation; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D065098:Catechol O-Methyltransferase Inhibitors; D045166:Estradiol Congeners; D002393:Estrogens, Catechol; D011994:Recombinant Proteins; C065250:4-hydroxy-equilenin; D002394:Catechol O-Methyltransferase; D004855:Equilenin