Selective inhibition of human recombinant cytochrome P450 1B1 by 2,4,3',5'-tetramethoxystilbene

YJ Chun, PhDa, SK Lee a, S Kim, PhD b, D. Kim c, FP Guengerich, PhD c

1College of Pharmacy, Chungang University, Seoul, Korea, 2 Natural Product Research Institute, Seoul National University, Seoul, Korea, 3 Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN

Human cytochrome P450 (CYP) 1 family (1A1, 1A2, or 1B1) is involved in the metabolic activation of a variety of procarcinogens. Especially, CYP1B1 enzyme shows a strong 17b-estradiol (E2) 4-hydroxylase activity. Recently the carcinogenic potentials of 4-hydroxyestradiol (4-OHE2), a major metabolite of E2 by CYP1B1 have been reported and metabolic activation of E2 to 4-OHE2 by CYP1B1 has been postulated to be a factor in mammary carcinogenesis. Analysis of series of trans-stilbene analogs yields one with strong CYP1B1 inhibition, 2,4,3',5'-tetramethoxystilbene (TMS), with all resveratrol phenols methylated and an O-methoxy group added to the 4-OH-substituted phenyl ring. TMS showed potent inhibition of the ethoxy resorufin O-deethylation (EROD) activity of CYP1B1 with an IC50 value of 6 nM (IC50 = 300 nM for CYP1A1 and IC50 = 3 uM for CYP1A2). TMS also strongly inhibited 4- and 2-hydroxylation of E2 by CYP1B1-expressing membranes or purified CYP1B1. TMS was a competitive inhibitor of P450 1B1 with a Ki of 3 nM. Using purified CYP1B1, the binding kinetic analysis was performed with TMS, yielding a Kd of 3 µM. The CYP1B1-catalyzed metabolic activation of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ) in an Escherichia coli lac (2-frameshift) mutagenesis test was inhibited with an IC50 of ~ 0.5 µM. Our results indicate that TMS is a selectively potent competitive inhibitor of CYP1B1 and warrants consideration as a good candidate for preventing estrogen-mediated mammary tumor formation in humans.

KEY WORDS: trans-stilbene, mammary tumor, MeIQ.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Prevention.