Oxidation of Tienilic Acid by Human Yeast‐Expressed Cytochromes <i>P</i>‐450 2C8, 2C9, 2C18 and 2C19

Jean, P.; Lopez-Garcia, Pilar; Dansette, Patrick M.; Mansuy, Daniel; Goldstein, J L

doi:10.1111/j.1432-1033.1996.00797.x

Cited by 56 publications

(35 citation statements)

References 31 publications

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“…In addition to paclitaxel, 6 substrates hydroxylated by CYP2C8 (17beta-estradiol, 22) fluvastatin acid, 13,23,24) omeprazole, 25,26) rosiglitazone, 27) seratrodast 28) and tienilic acid, [29][30][31] Fig. 5b) were docked into the CYP2C8 model with Gold.…”

Section: Resultsmentioning

confidence: 99%

Characterization of the CYP2C8 Active Site by Homology Modeling

Tanaka

Kamiguchi

Okuda

et al. 2004

CHEMICAL & PHARMACEUTICAL BULLETIN

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Cytochrome P450s (CYPs) are membrane-associated heme proteins and hepatic microsomal enzymes participating in drug metabolism and detoxification. Among the human CYPs, CYP3A4 is the most important enzyme because of its participation in a wide range of drug metabolism and high level of expression in the human liver.1,2) On the other hand, in the CYP2C subfamily, CYP2C9 (e.g. fluoxetine, losartan, phenytoin, tolbutamide, torsemide, S-warfarin, and NSAIDs 3) ) and CYP2C19 (e.g. antidepressants, proton pump inhibitors, benzodiazepines, S-mephenytoin, and proguanil [4][5][6] ) are known to catalyze reactions for a lot of therapeutic agents. However, recently it has become clear that the contribution of CYP2C8 to drug metabolism has been underestimated, and CYP2C8 and CYP3A4 share some of their substrates (e.g. repaglinide, paclitaxel, morphine, carbamazepine, verapamil, zopiclone, and cerivastatin. 7-13) ). The crystal structure of mammalian CYP2C5 reported by Williams et al. 14) has enabled modeling of human CYPs. Thus, we have constructed CYP2C8, CYP2C9, and CYP2C19 models based on the CYP2C5 crystallographic coordinates, and compared them with the previously constructed CYP3A4 model 15) to elucidate the differences in their active sites. These models could be used to obtain helpful hints for avoiding undesirable drug-drug interactions. ExperimentalHomology Modeling The amino acid sequences of CYP2C8, CYP2C9, and CYP2C19 were taken from the ExPASy web site (http://tw.expasy.org/). The amino acid sequence alignment used for the homology modeling was obtained by ClustalW.16) According to the alignment (Fig. 1), homology models of CYP2C8, CYP2C9, and CYP2C19 were constructed based on the mammalian CYP2C5 crystal structure (PDB code:1DT6) using the homology module of Insight II (ver. 2000, Accelrys Inc., San Diego, California, U.S.A.). Using the Search/Generate-Loops function of Insight II, conformations of the insertion parts in the alignment were generated. After some manual adjustments to remove large steric hindrances, the whole structure was subjected to energy minimization over 1000 steps with the steepest descent minimizer and then 5000 steps with the conjugate gradient minimizer, to a maximum gradient of 0.1 kcal/mol Ϫ1 Å Ϫ1 , using the Discover-ESFF force field (ver. 980, Accelrys Inc., San Diego, California, U.S.A.). During the minimization procedure, the following conditions were adopted. The dielectric constant was set to 4*r, where r is the distance between two interacting atoms. The force constant of tethering constraints for the backbone of structurally conserved regions (SCRs, asterisks in Fig. 1) and heme was set to 40 kcal/Å 2 to prevent a large movement from the initial positions. Docking of Paclitaxel Paclitaxel, an anticancer drug from a natural compound, was docked into the CYP2C8 model using Gold (ver. 2.0, the To compare the features of the active sites of CYP2C8, CYP2C9, and CYP2C19, homology modeling was performed based on the crystallographic coordinates of mammalian CYP2C5. It was foun...

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Section: Resultsmentioning

confidence: 99%

Characterization of the CYP2C8 Active Site by Homology Modeling

Tanaka

Kamiguchi

Okuda

et al. 2004

CHEMICAL & PHARMACEUTICAL BULLETIN

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“…The reactive metabolites generated from these drugs further inactivate their metabolizing enzyme, CYP2C9 (Ló pez- Garcia et al, 1994;Jean et al, 1996;O'Donnell et al, 2003;Hutzler et al, 2009). The metabolic activation mechanism is known to be determined by the structure and substitution pattern on the thiophene ring.…”

Section: Discussionmentioning

confidence: 99%

“…In particular, CYP3A4 has been found to metabolize a variety of drugs and itself is inactivated by reactive intermediates (Zhou et al, 2005;Zhou, 2008). CYP2C9 was found to catalyze the metabolic activation of thiophene-containing drugs, such as suprofen and TA, and is inactivated by these drugs via mechanismbased inhibition (López- Garcia et al, 1994;Jean et al, 1996;O'Donnell et al, 2003;Hutzler et al, 2009). The 2,5-diaminothiophene derivative (compound 1) formed GSH conjugates by microsomal enzymes in the presence of NADPH, suggesting that it is an NADPH-dependent oxidative metabolic activation mediated by membrane-bound P450 or FMO enzymes.…”

Section: Downloaded Frommentioning

confidence: 99%

“…For instance, TA, a diuretic drug, and suprofen, an anti-inflammatory agent, are metabolically activated by CYP2C9. At the same time, both drugs inactivate CYP2C enzymes (López-Garcia et al, 1994;Jean et al, 1996;O'Donnell et al, 2003;Masubuchi and Horie, 2007;Hutzler et al, 2009). The metabolic activation of thiophene derivatives has been suggested to occur via sulfur oxidation or an epoxidation mechanism (Dansette et al, 1992(Dansette et al, , 2005Valadon et al, 1996;O'Donnell et al, 2003;Medower et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

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Novel Metabolic Bioactivation Mechanism for a Series of Anti-Inflammatory Agents (2,5-Diaminothiophene Derivatives) Mediated by Cytochrome P450 Enzymes

Yang²,

Shilliday³

et al. 2010

Drug Metab Dispos

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ABSTRACT:The thiophene moiety is considered a structural alert in molecular design in drug discovery, largely because several thiophene-containing drugs, including tienilic acid and suprofen, have been withdrawn from the market because of toxicities. Reactive thiophene intermediates, activated via sulfur oxidation or ring epoxidation, are possible culprits for these adverse side effects. In this work, the metabolic activation of an anti-inflammatory agent, 1-(3-carbamoyl-5-(2,3,5-trichlorobenzamido)thiophen-2-yl)urea), containing a 2,5-diaminothiophene structure, was studied in liver microsomes in the presence of glutathione or N-acetylcysteine as trapping agents. In addition, the glutathione conjugate was detected in bile from a bile duct-cannulated rat study. The structure of the glutathione conjugate was identified by mass spectrometry and 1 H NMR. The glutathione molecule was attached to the thiophene ring, replacing the existing proton. Metabolic phenotyping experiments, using chemical inhibitors or recombinant cytochromes P450 (P450), demonstrated that CYP3A4 was the major P450 enzyme responsible for the metabolic activation, followed by CYP1A2, 2Cs, and 2D6. A novel metabolic activation mechanism is proposed whereby the 2,5-diaminothiophene moiety undergoes oxidation to a 2,5-diimine thiophene reactive intermediate. This mechanism was used to support efforts to eliminate reactive metabolite generation via structural modification of ring substituents using structure-activity relationships. The disruption of formation of the 2,5-diimine reactive intermediate resulted in the elimination of glutathione conjugate formation both in vitro and in vivo and provided a rational approach to mitigating potential safety risks associated with this class of thiophenes in drug research and development.

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“…These results indicated that cynomolgus CYP2C18 is functional as a drug-metabolizing enzyme and has substrate selectivity. In humans, CYP2C18 metabolizes CYP2C9 substrates such as torsemide, phenytoin, diclofenac, tienilic acid and tolbutamide [7,8,11,12,24], indicating an overlapping functional property in CYP2C18 and CYP2C9. In cynomolgus macaques, CYP2C43 and CYP2C75, both highly homologous to human CYP2C9, also metabolize Smephenytoin [13,20], suggesting that CYP2C18 also shares a metabolic property in common with other CYP2Cs, CYP2C43 and CYP2C75.…”

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confidence: 99%

Identification and Characterization of CYP2C18 in the Cynomolgus Macaque (Macaca fascicularis)

Uno

Matsuno

Nakamura

et al. 2010

The Journal of Veterinary Medical Science

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ABSTRACT. The macaque is widely used for investigation of drug metabolism due to its evolutionary closeness to the human. However, the genetic backgrounds of drug-metabolizing enzymes have not been fully investigated; therefore, identification and characterization of drug-metabolizing enzyme genes are important for understanding drug metabolism in this species. In this study, we isolated and characterized a novel cytochrome P450 2C18 (CYP2C18) cDNA in cynomolgus macaques. This cDNA was highly homologous (96%) to human CYP2C18 cDNA. Cynomolgus CYP2C18 was preferentially expressed in the liver and kidney. Moreover, a metabolic assay using cynomolgus CYP2C18 protein heterologously expressed in Escherichia coli revealed its activity toward S-mephenytoin 4'-hydroxylation. These results suggest that cynomolgus CYP2C18 could function as a drug-metabolizing enzyme in the liver.KEY WORDS: cloning, CYP2C18, cytochrome P450, liver, monkey.J. Vet. Med. Sci. 72(2): 225-228, 2010 Cytochrome P450 (CYP) is a superfamily of some of the most important drug-metabolizing enzymes and consists of a large number of subfamilies [14]. In humans, the CYP2C subfamilies contain important enzymes that metabolize approximately 20% of all prescribed drugs [3]. In the CYP2C subfamily, CYP2C8, CYP2C9 and CYP2C19 are highly expressed in the liver, whereas protein expression of human CYP2C18 has not been seen at a detectable level in the liver [3]. CYP2C8, CYP2C9 and CYP2C19 exhibit metabolic activities toward clinically important drugs; the anticancer drug paclitaxel is metabolized by CYP2C8; the hypoglycemic agent tolbutamide, nonsteroidal anti-inflammatory drug diclofenac, anticonvulsant phenytoin and anticoagulant warfarin are metabolized by CYP2C9; and the antiulcer drug omeprazole and the anticonvulsant drug mephenytoin are metabolized by CYP2C19 [3]. All human CYP2Cs exhibit genetic polymorphisms. In particular, some genetic polymorphisms of CYP2C9 and CYP2C19 have been shown to result in toxicity or altered efficacy of drugs in humans.In cynomolgus macaques, which are used to evaluate drug effects and toxicities in preclinical studies of drug development, cDNAs for CYP2C20, CYP2C43, CYP2C75 and CYP2C76 have been identified [9,20]. CYP2C20 cDNA is highly homologous (95%) to human CYP2C8 cDNA, while CYP2C43 and CYP2C75 cDNAs are highly homologous (93-95%) to both human CYP2C9 and CYP2C19 cDNAs. CYP2C76 cDNA shows only 75-76% sequence identity to human CYP2Cs. CYP2C76 is not orthologous to any human CYPs [20] and is partly responsible for the difference of drug metabolism between cynomolgus macaques and humans [21]. Such species differences also could be explained by functional disparity of the orthologous CYPs between the two species, such as, if an ortholog to human CYP with low (or no) expression and drug-metabolizing activity, for example CYP2C18, shows abundant expression and substantial activity in cynomolgus macaques. To investigate this possibility, in this study, we isolated cynomolgus CYP2C18 cDNA encoding an ortholog of human C...

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Oxidation of Tienilic Acid by Human Yeast‐Expressed Cytochromes P‐450 2C8, 2C9, 2C18 and 2C19

Cited by 56 publications

References 31 publications

Characterization of the CYP2C8 Active Site by Homology Modeling

Characterization of the CYP2C8 Active Site by Homology Modeling

Novel Metabolic Bioactivation Mechanism for a Series of Anti-Inflammatory Agents (2,5-Diaminothiophene Derivatives) Mediated by Cytochrome P450 Enzymes

Identification and Characterization of CYP2C18 in the Cynomolgus Macaque (Macaca fascicularis)

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