Inhibition of Purified and Membrane-Bound Flavin-Containing Monooxygenase 1 by (N,N-Dimethylamino)stilbene Carboxylates

Abstract

(E)-[2-(4-(Dimethylamino)phenyl)vinyl]benzenes bearing a nitrile or carboxyl group in the 2', 3', or 4' position were synthesized and tested for substrate activity with purified pig liver flavin-containing monooxygenase (FMO1). Although the nitrile derivatives were too insoluble to saturate the catalytic site at pH 7.4, they appeared to be substrates with K(m)'s somewhat above their maximum solubility (approximately equal to 0.1 mM) in the assay medium. Of the three carboxylic acid analogs, (E)-4-[2-(4(dimethylamino)phenyl)vinyl]benzoic acid had no detectable water solubility at pH 7.4, and measurements were restricted to (E)-3-[2-(4-(dimethylamino)phenyl)vinyl]benzoic acid (DS3CO) and (E)-2-[2-(4-(dimethylamino)phenyl)vinyl]benzoic acid (DS2CO). While DS3CO and DS2CO were substrates, they also inhibited FMO1 turnover. DS3CO was the more effective inhibitor, and at 2 mM it inhibited FMO1 and microsomal-catalyzed oxidation of methimazole (N-methyl-2-mercaptoimidazole) by 80-90%. Kinetic studies indicated that the aminostilbene carboxylates were noncompetitive with both the xenobiotic substrate, methimazole, and NADPH. However, inhibition constants calculated from double reciprocal plots of velocity vs NADPH were K(i)(comp) 130 and 150 microM for DS3CO and DS2CO, respectively, whereas the uncompetitive Ki's were 10-15 times higher, which suggests that inhibition of NADPH binding may be primarily responsible for inhibition of FMO1 by the aminostilbene carboxylates. This model is also consistent with inhibition of cyclohexanone monooxygenase, a bacterial analog of FMO. DS3CO and DS2CO were again noncompetitive with methimazole but primarily competitive with NADPH. The aminostilbene carboxylates had no detectable effects on activity of pig or rat liver NADPH-cytochrome P450 reductase, which suggests that they are not nonspecific flavoprotein antagonists.

Regulated O2 Activation in Flavin-Dependent Monooxygenases

“…13 Such a “bold” catalytic strategy is consistent with the biodegradative role of the liver FMOs, which hydroxylate literally hundreds of structurally variable nucleophiles including amines, thiols, and halogenated compounds. 2,9 The FMOs are constitutively expressed; the FAD-OOH forms and simply persists until intercepted by a substrate. Continuous delivery of substrates to the liver minimizes the steady release of H 2 O 2 from FAD-OOH that otherwise would occur.…”

“…Flavin-containing monooxygenases (FMOs) catalyze the oxygenation of diverse small organic molecules using O 2 , NADPH, and the flavin adenine dinucleotide (FAD) cofactor. , Genome sequencing has shown this family of enzymes to be widespread in bacteria, fungi, plants, and animals, though the mammalian FMOs are the only ones to have received significant prior attention. − Like the cytochrome P450s, the mammalian liver FMOs are involved in the degradation of xenobiotics. FMOs from other sources, by contrast, appear to be primarily involved in the biosynthesis of diverse and important natural products.…”

Development of a CD‐MEKC method for investigating the metabolism of tamoxifen by flavin‐containing monooxygenases and the inhibitory effects of methimazole, nicotine and DMXAA

“…reported that 2,3,7,8‐tetrachlorodibenzo‐ p ‐dioxin highly induced FMO2 and FMO3 in the mouse in an aryl hydrocarbon receptor‐dependent manner . Only a few potent competitive inhibitors of FMOs such as, indole‐3‐carbinol (I3C), ( N , N ‐dimethylamino) stilbene carboxylates and 3,3′‐diindolylmethane (DIM) have been reported . Most of the apparent FMO inhibition is due to alternate substrate competitive inhibition where a good nucleophile competes with the drug for FMO oxygenation.…”