Coumarin Substrates for Cytochrome P450 2D6 Fluorescence Assays

Nakamura, Katsunori; Hanna, Imad; Cai, Hongliang; Nishimura, Yuki; Williams, Katherine M.; Guengerich, F. Peter

doi:10.1006/abio.2001.5098

Cited by 25 publications

(16 citation statements)

References 37 publications

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“…Second, CYP1A1 is efficient in steroid 16␣-hydroxylation (Schwarz et al, 2000), a reaction mediated in the mouse by cyp2d9. Consideration of the metabolism of the 7-alkoxycoumarins shows the nonoverlapping substrate preferences of CYP1A1 and CYP2D6; CYP1A1 metabolizes the neutral lipid-soluble 7-ethoxy-coumarin and 7-ethoxy-4-trifluoromethyl-coumarin (Penman et al, 1994), whereas CYP2D6 metabolizes a series of 4-aminomethyl-7-alkoxycoumarins (Nakamura et al, 2001) that are both basic and more water-soluble. The major finding in this article would not, therefore, have been predicted from first principles.…”

Section: Discussionmentioning

confidence: 99%

4-Hydroxylation of Debrisoquine by Human CYP1A1 and Its Inhibition by Quinidine and Quinine

Granvil

Krausz²,

Gelboin³

et al. 2002

J Pharmacol Exp Ther

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A panel of 15 recombinant cytochromes P450 expressed in human B-lymphoblastoid cells was used to study debrisoquine 4-hydroxylation. Both CYP2D6 and CYP1A1 carried out the reaction. The apparent K m (micromolar) and V max (picomoles per minute per picomole of P450) for CYP2D6 were 12.1 and 18.2 and for CYP1A1 were 23.1 and 15.2, respectively. CYP1A1 debrisoquine 4-hydroxylase was inhibited by the CYP1A1 inhibitor ␣-naphthoflavone and the CYP1A1 substrate 7-ethoxyresorufin. Additionally and surprisingly, this reaction was also inhibited by quinidine and quinine, with respective IC 50 values of 1.38 Ϯ 0.10 and 3.31 Ϯ 0.14 M, compared with those for CYP2D6 debrisoquine 4-hydroxylase of 0.018 Ϯ 0.05 and 3.75 Ϯ 2.07 M, respectively. Anti-CYP1A1 monoclonal antibody (mAb) 1-7-1 abolished CYP1A1 debrisoquine hydroxylase and anti-CYP2D6 mAb 50-1-3 eradicated CYP2D6 debrisoquine 4-hydroxylase. Three further CYP2D6-specific reactions were tested: dextromethorphan O-demethylation, bufuralol 1Ј-hydroxylation, and sparteine dehydrogenation. The CYP2D6 specificity, judged by the CYP2D6/CYP1A1 activity ratios was 18.5, 7.0, 6.0, and 1.6 for dextromethorphan, bufuralol, sparteine, and debrisoquine, respectively. Thus, debrisoquine is not a specific CYP2D6 substrate and quinidine is not a specific CYP2D6 inhibitor. These findings have significant implications for the conduct of in vitro drug metabolism inhibition studies and underscore the fallacy of "specific chemical inhibitors" of a supergene family of enzymes that have overlapping substrate specificities. The use of highly specific mAbs in such studies is mandated. It is unclear as yet whether these findings have implications for the relationship between CYP2D6 genotype and in vivo debrisoquine 4-hydroxylase activity.

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Section: Discussionmentioning

confidence: 99%

4-Hydroxylation of Debrisoquine by Human CYP1A1 and Its Inhibition by Quinidine and Quinine

Granvil

Krausz²,

Gelboin³

et al. 2002

J Pharmacol Exp Ther

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“…The genetic polymorphism has now been characterized extensively (29), and P450 2D6 is estimated to be involved in the oxidation of ϳ30% of the drugs used today (8,30). This protein has been the subject of investigations in this laboratory for some time (31); studies with recombinant P450 2D6 expressed in Escherichia coli have been done on the role of the N-terminal region of the protein in interactions with the NADPH-P450 reductase, the mechanism of P450 2D6 reactions supported by oxygen surrogates, and the role of Asp-301 in substrate binding and protein folding (32)(33)(34)(35). In the course of studies with the prototypic substrate bufuralol, we found that the rate of reduction of ferric P450 2D6 was enhanced by bufuralol and was not the rate-limiting step in the overall 1Ј-hydroxylation reaction (33).…”

Section: P450mentioning

confidence: 99%

Oxidation of Methoxyphenethylamines by Cytochrome P450 2D6

Guengerich

Miller²,

Hanna³

et al. 2002

Journal of Biological Chemistry

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Cytochrome P450 (P450) 2D6 is involved in the oxidation of a large fraction (ϳ30%) of drugs used by humans and also catalyzes the O-demethylation of the model substrates 3-and 4-methoxyphenethylamine followed by subsequent ring hydroxylation to dopamine. Burst kinetics were not observed; rate-limiting step(s) must occur prior to product formation. Rates of reduction of ferric P450 2D6 were stimulated by 3-or 4-methoxyphenethylamine or the inhibitor quinidine; reduction is not the most rate-limiting step. The non-competitive intramolecular deuterium isotope effect, an estimate of the intrinsic isotope effect, for 4-methoxyphenethylamine O-demethylation was 9.6. Intermolecular noncompetitive deuterium isotope effects of 3.1-3.8 were measured for k cat and k cat /K m for both O-demethylation reactions, implicating at least partially rate-limiting C-H bond breaking. Simulation of steady-state kinetic data yielded a catalytic mechanism dominated by the rates of (i) Fe 2؉ O 2 ؊ protonation (plus O-O bond scission) and (ii) C-H bond breaking, consistent with the appearance of the spectral intermediates in the steady state, attributed to iron-oxygen complexes. However, all the rates of individual steps (or rates of combined steps) are considerably higher than k cat , and the contributions of several steps must be considered in understanding rates of the P450 2D6 reactions.

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“…Higher throughput in the screening program could have been achieved by utilizing substrate analogs or derivatives allowing for colorimetric or similar rapid analysis methods (1,21,29,33). We reasoned, however, that enzymes evolved using these surrogate substrates might not show increased specificity for the industrially relevant avermectin substrate.…”

Section: Vol 73 2007 Directed Evolution Of Ema Cyps 4323mentioning

confidence: 99%

Biocatalytic Conversion of Avermectin to 4″-Oxo-Avermectin: Improvement of Cytochrome P450 Monooxygenase Specificity by Directed Evolution

Trefzer

Jungmann

Molnár

et al. 2007

Appl Environ Microbiol

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Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4؆-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4؆-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin.Emamectin benzoate is a potent semisynthetic insecticide used to control many agriculturally important pests. However, wider application of emamectin has been hindered by the high cost of its chemical synthesis, which involves regioselective oxidation of the 4Љ alcohol of the natural product avermectin, followed by the reductive amination of the resulting key intermediate 4Љ-oxo-avermectin (see Fig. 1). Achievement of high regioselectivity in the oxidation reaction requires the costly protection and deprotection of the very reactive allylic hydroxyl function at C-5 of the avermectin molecule.As an alternative to chemical synthesis, microbial conversion has been used successfully in other regioselective oxidation reactions to bypass complex protection and deprotection strategies. Many microbial oxidative bioconversion reactions are carried out by dehydrogenases (4) or oxidases (3, 11) while some use cytochrome P450 monooxygenases (CYPs) (12a, 12b, 15-17). The cytochrome P450 family is a large class of enzymes that catalyze hydroxylation, epoxidation, and demethylation reactions. Class II CYPs from eukaryotes accept electrons from NADPH via a NADPH-cytochrome P450 reductase, whereas class I CYPs in prokaryotes use distinct ferredoxins and ferredoxin reductases for electron transfer from NADH. Biotransformations using CYPs have to regenerate the cofactor, which can be achieved cost-effectively on an industrial scale by using metabolically active whole cells as biocatalysts. Examples of CYP-catalyzed biotransformations include the 11␤-hydroxylation of Reichstein S to hydrocortisone (2) and the microbial hydroxylation of zofimarin, a sordarin-related antibiotic (32).Recently, we have reported the isolation and characterization of 17 CYP enzymes, collectively named the Ema CYPs, capable of regioselectively oxidizing avermectin to 4Љ-oxo-avermectin (13,19,20). The Ema CYPs were isolated from closely related Streptomyces spec...

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Coumarin Substrates for Cytochrome P450 2D6 Fluorescence Assays

Cited by 25 publications

References 37 publications

4-Hydroxylation of Debrisoquine by Human CYP1A1 and Its Inhibition by Quinidine and Quinine

4-Hydroxylation of Debrisoquine by Human CYP1A1 and Its Inhibition by Quinidine and Quinine

Oxidation of Methoxyphenethylamines by Cytochrome P450 2D6

Biocatalytic Conversion of Avermectin to 4″-Oxo-Avermectin: Improvement of Cytochrome P450 Monooxygenase Specificity by Directed Evolution

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