Methoxyresorufin

Enhancement of 7-methoxyresorufin O-demethylation activity of human cytochrome P450 1A2 by molecular breeding.[Pubmed:15519301]

Arch Biochem Biophys. 2004 Dec 1;432(1):102-8.

Alkylresorufins are model substrates for cytochrome P450 (P450) 1A2. The ability of human P450 1A2 to catalyze 7-Methoxyresorufin O-demethylation was improved by screening of random mutant libraries (expressed in Escherichia coli) on the basis of 7-Methoxyresorufin O-demethylation. After three rounds of mutagenesis and screening, the triple mutant E163K/V193M/K170Q yielded a kcat > five times faster than wild type P450 1A2 in steady-state kinetic analysis using either isolated membrane fractions or purified, reconstituted enzymes. The enhanced catalytic activity was not attributed to changes in substrate affinity. The kinetic hydrogen isotope effect of the triple mutant did not change from wild type enzyme and suggests that C-H bond cleavage is rate-limiting in both enzymes. Homology modeling, based on an X-ray structure of rabbit P450 2C5, suggests that the locations of mutated residues are not close to the substrate binding site and therefore that structural elements outside of this site play roles in changing the catalytic activity. This approach has potential value in understanding P450 1A2 and generating engineered enzymes with enhanced catalytic activity.

Inhibition of methoxyresorufin demethylase activity by flavonoids in human liver microsomes.[Pubmed:9718089]

Life Sci. 1998;63(8):PL119-23.

Flavonoids are a class of dietary phytochemicals with anticarcinogenic properties. A series of ten structurally related flavonoids were evaluated for their effect on Methoxyresorufin O-demethylase (MROD) activity in human liver microsomes. All compounds inhibited this cytochrome P450 1A2 (CYP1A2) mediated activity. 3,5,7-Trihydoxyflavone (galangin) was the most potent inhibitor, followed by 3-hydroxyflavone and flavone. The relative inhibitory potency of flavonoids is related to their structures. The results suggest that flavonoids may modulate pharmacological and toxicological effects mediated by CYP1A2.

Arginine to lysine 108 substitution in recombinant CYP1A2 abolishes methoxyresorufin metabolism in lymphoblastoid cells.[Pubmed:12023936]

Br J Pharmacol. 2002 Jun;136(3):347-52.

1. Cytochrome P4501A2 (CYP1A2) activates a large number of procarcinogens to carcinogens. Phytochemicals such as flavones can inhibit CYP1A2 activity competitively, and hydroxylated derivatives of flavone (galangin) may be potent, selective inhibitors of CYP1A2 activity relative to CYP1A1 activity. Molecular modelling of the CYP1A2 interaction with hydroxylated derivatives of flavone suggests that a number of hydrophobic residues of the substrate-binding domain engage in hydrogen bonding with such inhibitors. 2. We have tested this model using site-directed mutagenesis of these residues in expression plasmids transfected into the human B-lymphoblastoid cell line, AHH-1 TK+/-. 3. Consistent with the molecular model's predicted placement in the active site, amino acid substitutions at the predicted residues abolished CYP1A2 enzymatic activity. 4. Transfected cell lines contained equal amounts of immunoreactive CYP1A2. 5. Our results support the molecular model's prediction of the critical amino acid residues present in the hydrophobic active site, residues that can hydrogen bond with CYP1A2 inhibitors and modify substrate binding and/or turnover.

Methoxyresorufin: an inappropriate substrate for CYP1A2 in the mouse.[Pubmed:9973187]

Biochem Pharmacol. 1998 Dec 15;56(12):1657-60.

Hepatic microsomes derived from Cypla2(-/-) knockout (KO) and parental strains of mice, C57BL/6N and 129Sv, were used to examine the specificity of Methoxyresorufin and acetanilide as substrates for CYP1A2 activity. In addition, animals from each group were exposed to CYP1-inducing compounds. As expected, microsomes from untreated 1a2 KO mice did not have immunodetectable CYP1A2 protein; however, Methoxyresorufin-O-demethylase (MROD, 25.5+/-6.1 pmol/min/mg protein) and acetanilide-4-hydroxylation (ACOH, 0.64+/-0.04 nmol/min/mg protein) activities were still present. Furthermore, induction of ethoxyresorufin-O-deethylase (EROD) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in 1a2 KO mice was accompanied by a greater than 70-fold increase in MROD activity. In contrast, ACOH was only induced 2-fold by TCDD. As with 1a2 KO mice, the parental strains exposed to TCDD or 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF) showed substantial EROD and MROD induction, whereas ACOH activity was induced to a lesser degree. PCB153 (2,2',4,4',5,5'-hexachlorobiphenyl) resulted in low levels of both EROD and MROD induction. Results indicate that both substrates are subject to metabolism by non-CYP1A2 sources, and the apparent contribution of CYP1A1 activity to Methoxyresorufin metabolism makes MROD unsuitable for differentiating CYP1A1 and CYP1A2 activities in the mouse.

Methoxyresorufin and benzyloxyresorufin: substrates preferentially metabolized by cytochromes P4501A2 and 2B, respectively, in the rat and mouse.[Pubmed:8373445]

Biochem Pharmacol. 1993 Sep 1;46(5):933-43.

The cytochrome P450 isozyme specificity for the O-dealkylation of Methoxyresorufin (MTR) and benzyloxyresorufin (BZR) in the rat and mouse was investigated. The induction of various alkoxyresorufin O-dealkylation activities was measured in male F344/NCr rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin or 3,4,5,3',4',5'-hexachlorobiphenyl. MTR and ethoxyresorufin (ETR) O-dealkylation activities were induced 30- and 80-fold, respectively, in the liver. ETR O-dealkylation activity was induced > 250-fold in the kidney, whereas the metabolism of MTR was induced only 30-fold in this extrahepatic tissue. Phenacetin, a fairly specific CYP1A2 inhibitor, caused concentration-dependent competitive inhibition of MTR O-dealkylation (ki approximately 20 microM at 0.5 microM substrate) in hepatic microsomes from 3,4,5,3',4',5'-hexachlorobiphenyl-treated rats. The corresponding ki for inhibition of ETR O-dealkylation by phenacetin was > or = 333 microM at a 0.5 microM substrate concentration. A monoclonal antibody displaying inhibitory activity against rat CYP1A1 inhibited ETR O-dealkylation activity, whereas it failed to inhibit MTR O-dealkylation activity. In contrast, a monoclonal antibody reactive with both CYP1A1 and CYP1A2 inhibited both O-dealkylation activities to an equal extent. Similar experiments, employing phenacetin or specific monoclonal antibodies, yielded comparable results when performed with mouse microsomes. The maximal induction of MTR O-dealkylation activity in mice was > 100-fold. The P450 isozyme specificity of BZR O-dealkylation was also examined in both rats and mice. Pregnenolone-alpha-carbonitrile, a strong inducer of CYP3A, only weakly induced BZR O-dealkylation activity. In addition, a monoclonal antibody that specifically inhibits CYP2B caused inhibition of BZR metabolism in microsomes from phenobarbital- or dexamethasone-pretreated rats. In B6C3F1 mice exposed to dietary Aroclor 1254, significant induction of hepatic MTR O-dealkylation activity was observed at concentrations lower than those required for the induction of ETR or BZR O-dealkylation. In summary, it would appear that MTR is a relatively specific substrate for CYP1A2 activity in rodents, while BZR appears to be relatively specific for CYP2B.