The Experts below are selected from a list of 24 Experts worldwide ranked by ideXlab platform
Roland C Wolf - One of the best experts on this subject based on the ideXlab platform.
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why is quinidine an inhibitor of cytochrome p450 2d6 the role of key active site residues in quinidine binding
Journal of Biological Chemistry, 2005Co-Authors: Lesley A Mclaughlin, Carol A Kemp, Jack U Flanagan, Jeandidier Marechal, Gordon C K Roberts, Mark J I Paine, Michael J Sutcliffe, Clive J Ward, Roland C WolfAbstract:Abstract We have previously shown that Phe120, Glu216, and Asp301 in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolizing enzyme (Paine, M. J., McLaughlin, L. A., Flanagan, J. U., Kemp, C. A., Sutcliffe, M. J., Roberts, G. C., and Wolf, C. R. (2003) J. Biol. Chem. 278, 4021–4027 and Flanagan, J. U., Marechal, J.-D., Ward, R., Kemp, C. A., McLaughlin, L. A., Sutcliffe, M. J., Roberts, G. C., Paine, M. J., and Wolf, C. R. (2004) Biochem. J. 380, 353–360). We have now examined the effect of mutations of these residues on interactions of the enzyme with the prototypical CYP2D6 inhibitor, quinidine. Abolition of the negative charge at either or both residues 216 and 301 decreased quinidine inhibition of bufuralol 1′-hydroxylation and dextromethorphan O-demethylation by at least 100-fold. The apparent dissociation constants (Kd) for quinidine binding to the wild-type enzyme and the E216D and D301E mutants were 0.25–0.50 μm. The amide substitution of Glu216 or Asp301 resulted in 30–64-fold increases in the Kd for quinidine. The double mutant E216Q/D301Q showed the largest decrease in quinidine affinity, with a Kd of 65 μm. Alanine substitution of Phe120, Phe481,or Phe483 had only a minor effect on the inhibition of bufuralol 1′-hydroxylation and dextromethorphan O-demethylation and on binding. In contrast to the wild-type enzyme, a number of the mutants studied were found to be able to metabolize quinidine. E216F produced O-demethylated quinidine, and F120A and E216Q/D301Q produced both O-demethylated quinidine and 3-Hydroxyquinidine metabolites. Homology modeling and molecular docking were used to predict the modes of quinidine binding to the wild-type and mutant enzymes; these were able to rationalize the experimental observations.
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Why is quinidine an inhibitor of cytochrome P450 2D6? The role of key active site residues in quinidine binding
2005Co-Authors: Lesley A Mclaughlin, Carol A Kemp, Jack U Flanagan, Jeandidier Marechal, Gordon C K Roberts, Mark J I Paine, Michael J Sutcliffe, Clive J Ward, Roland C WolfAbstract:We have previously shown that residues Asp301, Glu216 and Phe120 in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolising enzyme. We have now examined the effect of mutations of these residues on interactions of the enzyme with the prototypical CYP2D6 inhibitor, quinidine. Abolition of the negative charge on either or both residues 216 and 301 decreased quinidine inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation by at least 100-fold. The apparent dissociation constants (Kd) for quinidine binding to wild type enzyme or to the Glu216Asp and Asp301Glu mutants were 0.25-0.50 μM. The amide substitutions of Glu216 or Asp301 resulted in 30 to 64-fold increases in Kd for quinidine. The double mutant Glu216Gln/Asp301Gln showed the largest decrease in quinidine affinity with a Kd of 65 μM. Changes in the mode of quinidine binding were indicated by changes in the optical difference spectra on binding. Alanine substitution of Phe120, Phe481 or Phe483 had only a minor effect on the inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation, and on binding. In contrast to the wild-type enzyme, a number of the mutants studied were found to be able to metabolise quinidine. CYP2D6 Asp301Gln and Asp301Asn produced small amounts of 3-Hydroxyquinidine, Asp301Ala and Asp301Phe produced O-demethylated quinidine, and Phe120Ala and Glu216Gln/Asp301Gln produced both these metabolites. Homology modelling and molecular docking were used to predict the modes of quinidine binding to wild type and mutant enzymes; these were able to rationalise the experimental observations
Lesley A Mclaughlin - One of the best experts on this subject based on the ideXlab platform.
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why is quinidine an inhibitor of cytochrome p450 2d6 the role of key active site residues in quinidine binding
Journal of Biological Chemistry, 2005Co-Authors: Lesley A Mclaughlin, Carol A Kemp, Jack U Flanagan, Jeandidier Marechal, Gordon C K Roberts, Mark J I Paine, Michael J Sutcliffe, Clive J Ward, Roland C WolfAbstract:Abstract We have previously shown that Phe120, Glu216, and Asp301 in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolizing enzyme (Paine, M. J., McLaughlin, L. A., Flanagan, J. U., Kemp, C. A., Sutcliffe, M. J., Roberts, G. C., and Wolf, C. R. (2003) J. Biol. Chem. 278, 4021–4027 and Flanagan, J. U., Marechal, J.-D., Ward, R., Kemp, C. A., McLaughlin, L. A., Sutcliffe, M. J., Roberts, G. C., Paine, M. J., and Wolf, C. R. (2004) Biochem. J. 380, 353–360). We have now examined the effect of mutations of these residues on interactions of the enzyme with the prototypical CYP2D6 inhibitor, quinidine. Abolition of the negative charge at either or both residues 216 and 301 decreased quinidine inhibition of bufuralol 1′-hydroxylation and dextromethorphan O-demethylation by at least 100-fold. The apparent dissociation constants (Kd) for quinidine binding to the wild-type enzyme and the E216D and D301E mutants were 0.25–0.50 μm. The amide substitution of Glu216 or Asp301 resulted in 30–64-fold increases in the Kd for quinidine. The double mutant E216Q/D301Q showed the largest decrease in quinidine affinity, with a Kd of 65 μm. Alanine substitution of Phe120, Phe481,or Phe483 had only a minor effect on the inhibition of bufuralol 1′-hydroxylation and dextromethorphan O-demethylation and on binding. In contrast to the wild-type enzyme, a number of the mutants studied were found to be able to metabolize quinidine. E216F produced O-demethylated quinidine, and F120A and E216Q/D301Q produced both O-demethylated quinidine and 3-Hydroxyquinidine metabolites. Homology modeling and molecular docking were used to predict the modes of quinidine binding to the wild-type and mutant enzymes; these were able to rationalize the experimental observations.
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Why is quinidine an inhibitor of cytochrome P450 2D6? The role of key active site residues in quinidine binding
2005Co-Authors: Lesley A Mclaughlin, Carol A Kemp, Jack U Flanagan, Jeandidier Marechal, Gordon C K Roberts, Mark J I Paine, Michael J Sutcliffe, Clive J Ward, Roland C WolfAbstract:We have previously shown that residues Asp301, Glu216 and Phe120 in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolising enzyme. We have now examined the effect of mutations of these residues on interactions of the enzyme with the prototypical CYP2D6 inhibitor, quinidine. Abolition of the negative charge on either or both residues 216 and 301 decreased quinidine inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation by at least 100-fold. The apparent dissociation constants (Kd) for quinidine binding to wild type enzyme or to the Glu216Asp and Asp301Glu mutants were 0.25-0.50 μM. The amide substitutions of Glu216 or Asp301 resulted in 30 to 64-fold increases in Kd for quinidine. The double mutant Glu216Gln/Asp301Gln showed the largest decrease in quinidine affinity with a Kd of 65 μM. Changes in the mode of quinidine binding were indicated by changes in the optical difference spectra on binding. Alanine substitution of Phe120, Phe481 or Phe483 had only a minor effect on the inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation, and on binding. In contrast to the wild-type enzyme, a number of the mutants studied were found to be able to metabolise quinidine. CYP2D6 Asp301Gln and Asp301Asn produced small amounts of 3-Hydroxyquinidine, Asp301Ala and Asp301Phe produced O-demethylated quinidine, and Phe120Ala and Glu216Gln/Asp301Gln produced both these metabolites. Homology modelling and molecular docking were used to predict the modes of quinidine binding to wild type and mutant enzymes; these were able to rationalise the experimental observations
Kim Brosen - One of the best experts on this subject based on the ideXlab platform.
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vitro metabolism of quinidine: the (3S)-3-hydroxylation of quinidine is a specific marker reaction for cytochrome P-4503A4 activity in human liver microsomes
1999Co-Authors: Torben Leo Nielsen, Birgitte Buur Rasmussen, Jean-pierre Flinois, Philippe Beaune, Kim BrosenAbstract:The aim of this study was to evaluate the (3S)-3-hydroxylation and the N-oxidation of quinidine as biomarkers for cytochrome P-450 (CYP)3A4 activity in human liver microsome prepara-tions. An HPLC method was developed to assay the metabo-lites (3S)-3-Hydroxyquinidine (3-OH-Q) and quinidine N-oxide (Q-N-OX) formed during incubation with microsomes from hu-man liver and from Saccharomyces cerevisiae strains express-ing 10 human CYPs. 3-OH-Q formation complied with Michae-lis-Menten kinetics (mean values of Vmax and Km: 74.4 nmol/ mg/h and 74.2 mM, respectively). Q-N-OX formation followed two-site kinetics with mean values of Vmax, Km and Vmax/Km for the low affinity isozyme of 15.9 nmol/mg/h, 76.1 mM and 0.03 ml/mg/h, respectively. 3-OH-Q and Q-N-OX formations were potently inhibited by ketoconazole, itraconazole, and tri
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determination of quinidine dihydroquinidine 3s 3 Hydroxyquinidine and quinidine n oxide in plasma and urine by high performance liquid chromatography
Journal of Chromatography B: Biomedical Sciences and Applications, 1994Co-Authors: Flemming Nielsen, Karin Kramer Nielsen, Kim BrosenAbstract:Abstract A specific and sensitive method for the quantitation of quinidine, (3 S )-3-Hydroxyquinidine, quinidine N-oxide, and dihydroquinidine in plasma and urine has been developed. The method is based on a single-step, liquid—liquid extraction procedure, followed by isocratic reversed-phase high-performance liquid chromatography, with fluorescence detection. After extraction from 250 μl plasma and 100 μl urine, the limit of determination is 10 n M and 25 n M , respectively. For the use as standards, commercially available quinidine was purified from dihydroquinidine; quinidine N-oxide was synthesized.
Clive J Ward - One of the best experts on this subject based on the ideXlab platform.
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why is quinidine an inhibitor of cytochrome p450 2d6 the role of key active site residues in quinidine binding
Journal of Biological Chemistry, 2005Co-Authors: Lesley A Mclaughlin, Carol A Kemp, Jack U Flanagan, Jeandidier Marechal, Gordon C K Roberts, Mark J I Paine, Michael J Sutcliffe, Clive J Ward, Roland C WolfAbstract:Abstract We have previously shown that Phe120, Glu216, and Asp301 in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolizing enzyme (Paine, M. J., McLaughlin, L. A., Flanagan, J. U., Kemp, C. A., Sutcliffe, M. J., Roberts, G. C., and Wolf, C. R. (2003) J. Biol. Chem. 278, 4021–4027 and Flanagan, J. U., Marechal, J.-D., Ward, R., Kemp, C. A., McLaughlin, L. A., Sutcliffe, M. J., Roberts, G. C., Paine, M. J., and Wolf, C. R. (2004) Biochem. J. 380, 353–360). We have now examined the effect of mutations of these residues on interactions of the enzyme with the prototypical CYP2D6 inhibitor, quinidine. Abolition of the negative charge at either or both residues 216 and 301 decreased quinidine inhibition of bufuralol 1′-hydroxylation and dextromethorphan O-demethylation by at least 100-fold. The apparent dissociation constants (Kd) for quinidine binding to the wild-type enzyme and the E216D and D301E mutants were 0.25–0.50 μm. The amide substitution of Glu216 or Asp301 resulted in 30–64-fold increases in the Kd for quinidine. The double mutant E216Q/D301Q showed the largest decrease in quinidine affinity, with a Kd of 65 μm. Alanine substitution of Phe120, Phe481,or Phe483 had only a minor effect on the inhibition of bufuralol 1′-hydroxylation and dextromethorphan O-demethylation and on binding. In contrast to the wild-type enzyme, a number of the mutants studied were found to be able to metabolize quinidine. E216F produced O-demethylated quinidine, and F120A and E216Q/D301Q produced both O-demethylated quinidine and 3-Hydroxyquinidine metabolites. Homology modeling and molecular docking were used to predict the modes of quinidine binding to the wild-type and mutant enzymes; these were able to rationalize the experimental observations.
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Why is quinidine an inhibitor of cytochrome P450 2D6? The role of key active site residues in quinidine binding
2005Co-Authors: Lesley A Mclaughlin, Carol A Kemp, Jack U Flanagan, Jeandidier Marechal, Gordon C K Roberts, Mark J I Paine, Michael J Sutcliffe, Clive J Ward, Roland C WolfAbstract:We have previously shown that residues Asp301, Glu216 and Phe120 in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolising enzyme. We have now examined the effect of mutations of these residues on interactions of the enzyme with the prototypical CYP2D6 inhibitor, quinidine. Abolition of the negative charge on either or both residues 216 and 301 decreased quinidine inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation by at least 100-fold. The apparent dissociation constants (Kd) for quinidine binding to wild type enzyme or to the Glu216Asp and Asp301Glu mutants were 0.25-0.50 μM. The amide substitutions of Glu216 or Asp301 resulted in 30 to 64-fold increases in Kd for quinidine. The double mutant Glu216Gln/Asp301Gln showed the largest decrease in quinidine affinity with a Kd of 65 μM. Changes in the mode of quinidine binding were indicated by changes in the optical difference spectra on binding. Alanine substitution of Phe120, Phe481 or Phe483 had only a minor effect on the inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation, and on binding. In contrast to the wild-type enzyme, a number of the mutants studied were found to be able to metabolise quinidine. CYP2D6 Asp301Gln and Asp301Asn produced small amounts of 3-Hydroxyquinidine, Asp301Ala and Asp301Phe produced O-demethylated quinidine, and Phe120Ala and Glu216Gln/Asp301Gln produced both these metabolites. Homology modelling and molecular docking were used to predict the modes of quinidine binding to wild type and mutant enzymes; these were able to rationalise the experimental observations
Jack U Flanagan - One of the best experts on this subject based on the ideXlab platform.
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why is quinidine an inhibitor of cytochrome p450 2d6 the role of key active site residues in quinidine binding
Journal of Biological Chemistry, 2005Co-Authors: Lesley A Mclaughlin, Carol A Kemp, Jack U Flanagan, Jeandidier Marechal, Gordon C K Roberts, Mark J I Paine, Michael J Sutcliffe, Clive J Ward, Roland C WolfAbstract:Abstract We have previously shown that Phe120, Glu216, and Asp301 in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolizing enzyme (Paine, M. J., McLaughlin, L. A., Flanagan, J. U., Kemp, C. A., Sutcliffe, M. J., Roberts, G. C., and Wolf, C. R. (2003) J. Biol. Chem. 278, 4021–4027 and Flanagan, J. U., Marechal, J.-D., Ward, R., Kemp, C. A., McLaughlin, L. A., Sutcliffe, M. J., Roberts, G. C., Paine, M. J., and Wolf, C. R. (2004) Biochem. J. 380, 353–360). We have now examined the effect of mutations of these residues on interactions of the enzyme with the prototypical CYP2D6 inhibitor, quinidine. Abolition of the negative charge at either or both residues 216 and 301 decreased quinidine inhibition of bufuralol 1′-hydroxylation and dextromethorphan O-demethylation by at least 100-fold. The apparent dissociation constants (Kd) for quinidine binding to the wild-type enzyme and the E216D and D301E mutants were 0.25–0.50 μm. The amide substitution of Glu216 or Asp301 resulted in 30–64-fold increases in the Kd for quinidine. The double mutant E216Q/D301Q showed the largest decrease in quinidine affinity, with a Kd of 65 μm. Alanine substitution of Phe120, Phe481,or Phe483 had only a minor effect on the inhibition of bufuralol 1′-hydroxylation and dextromethorphan O-demethylation and on binding. In contrast to the wild-type enzyme, a number of the mutants studied were found to be able to metabolize quinidine. E216F produced O-demethylated quinidine, and F120A and E216Q/D301Q produced both O-demethylated quinidine and 3-Hydroxyquinidine metabolites. Homology modeling and molecular docking were used to predict the modes of quinidine binding to the wild-type and mutant enzymes; these were able to rationalize the experimental observations.
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Why is quinidine an inhibitor of cytochrome P450 2D6? The role of key active site residues in quinidine binding
2005Co-Authors: Lesley A Mclaughlin, Carol A Kemp, Jack U Flanagan, Jeandidier Marechal, Gordon C K Roberts, Mark J I Paine, Michael J Sutcliffe, Clive J Ward, Roland C WolfAbstract:We have previously shown that residues Asp301, Glu216 and Phe120 in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolising enzyme. We have now examined the effect of mutations of these residues on interactions of the enzyme with the prototypical CYP2D6 inhibitor, quinidine. Abolition of the negative charge on either or both residues 216 and 301 decreased quinidine inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation by at least 100-fold. The apparent dissociation constants (Kd) for quinidine binding to wild type enzyme or to the Glu216Asp and Asp301Glu mutants were 0.25-0.50 μM. The amide substitutions of Glu216 or Asp301 resulted in 30 to 64-fold increases in Kd for quinidine. The double mutant Glu216Gln/Asp301Gln showed the largest decrease in quinidine affinity with a Kd of 65 μM. Changes in the mode of quinidine binding were indicated by changes in the optical difference spectra on binding. Alanine substitution of Phe120, Phe481 or Phe483 had only a minor effect on the inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation, and on binding. In contrast to the wild-type enzyme, a number of the mutants studied were found to be able to metabolise quinidine. CYP2D6 Asp301Gln and Asp301Asn produced small amounts of 3-Hydroxyquinidine, Asp301Ala and Asp301Phe produced O-demethylated quinidine, and Phe120Ala and Glu216Gln/Asp301Gln produced both these metabolites. Homology modelling and molecular docking were used to predict the modes of quinidine binding to wild type and mutant enzymes; these were able to rationalise the experimental observations
