Substituted Imidazole of 5-Fluoro-2-[4-[(2-phenyl-1<i>H</i>-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine Inactivates Cytochrome P450 2D6 by Protein Adduction

Nagy, Leslie D.; Mocny, Catherine S.; Diffenderfer, Laura E.; Hsi, David J.; Butler, Brendan F.; Arthur, Evan J.; Fletke, Kyle J.; Palamanda, Jairam; Nomeir, Amin A.; Furge, Laura Lowe

doi:10.1124/dmd.110.037630

Cited by 8 publications

(52 citation statements)

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“…We previously reported the metabolites of SCH 66712 formed by P450s 2D6, 2C9, and 2C19 (Nagy et al, 2011). In similar metabolism experiments with CYP3A4, mono-oxygenation of SCH 66712 at four different positions was apparent by the presence of four distinct m/z 355 ions in the mass spectral analysis (M+1).…”

Section: Methodsmentioning

confidence: 95%

“…Analysis of heme was as previously described with the following modifications (Nagy et al, 2011). Four reaction mixtures were used: controls containing no SCH 66712 and no NADPH, no SCH 66712 with NADPH (1 mM, final), or SCH 66712 (16 mM) without NADPH, and one experimental with SCH 66712 (16 mM) with NADPH (1 mM, final).…”

Section: Methodsmentioning

confidence: 99%

“…Metabolites of EMTPP formed by CYP3A4 were determined by LC-ESI-MS using the methods described previously with SCH 66712 with modification (Nagy et al, 2011). Briefly, CYP3A4 Supersomes were incubated for 45 minutes with EMTPP (100 mM) and NADPH (1 mM) in potassium phosphate (pH 7.4, 100 mM) and final volume of 100 ml.…”

Section: Methodsmentioning

confidence: 99%

“…A control sample not treated with TiCl 3 or HCl was placed on ice for 1 hour. All samples were then centrifuged, and the supernatant was analyzed by LC-ESI-MS as described previously (Nagy et al, 2011;Livezey et al, 2014).…”

Section: Methodsmentioning

confidence: 99%

“…1) (Hutzler et al, 2004;Nagy et al, 2011;Livezey et al, 2012). Amino acid sites of adduction on CYP2D6 are still unknown.…”

Section: Introductionmentioning

confidence: 99%

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Mechanism-Based Inactivation of Human Cytochrome P450 3A4 by Two Piperazine-Containing Compounds

et al. 2014

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Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of more than half of pharmaceutic drugs, and inactivation of CYP3A4 can lead to adverse drug-drug interactions. The substi-(EMTPP) have been previously identified as mechanism-based inactivators (MBI) of CYP2D6. The present study shows that both SCH 66712 and EMTPP are also MBIs of CYP3A4. Inhibition of CYP3A4 by SCH 66712 and EMTPP was determined to be concentration, time, and NADPH dependent. In addition, inactivation of CYP3A4 by SCH 66712 was shown to be unaffected by the presence of electrophile scavengers. SCH 66712 displays type I binding to CYP3A4 with a spectral binding constant (K s ) of 42.9 6 2.9 mM. The partition ratios for SCH 66712 and EMTPP were 11 and 94, respectively. Whole protein mass spectrum analysis revealed 1:1 binding stoichiometry of SCH 66712 and EMTPP to CYP3A4 and a mass increase consistent with adduction by the inactivators without addition of oxygen. Heme adduction was not apparent. Multiple monooxygenation products with each inactivator were observed; no other products were apparent. These are the first MBIs to be shown to be potent inactivators of both CYP2D6 and CYP3A4.

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Section: Methodsmentioning

confidence: 95%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…1) (Hutzler et al, 2004;Nagy et al, 2011;Livezey et al, 2012). Amino acid sites of adduction on CYP2D6 are still unknown.…”

Section: Introductionmentioning

confidence: 99%

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Mechanism-Based Inactivation of Human Cytochrome P450 3A4 by Two Piperazine-Containing Compounds

et al. 2014

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Human Cytochrome P450 Enzymes

Guengerich

2015

Cytochrome P450

124

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CYP2D6 Allelic Variants 34, 17-2, 17-3, and 53 and a Thr309Ala Mutant Display Altered Kinetics and NADPH Coupling in Metabolism of Bufuralol and Dextromethorphan and Altered Susceptibility to Inactivation by SCH 66712

et al. 2018

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Metabolic phenotype can be affected by multiple factors, including allelic variation and interactions with inhibitors. Human CYP2D6 is responsible for approximately 20% of cytochrome P450-mediated drug metabolism but consists of more than 100 known variants; several variants are commonly found in the population, whereas others are quite rare. Four CYP2D6 allelic variants-three with a series of mutations distal to the active site (*34, *17-2, *17-3) and one ultra-metabolizer with mutations near the active site (*53), along with reference *1 and an active site mutant of *1 (Thr309Ala)-were expressed, purified, and studied for interactions with the typical substrates dextromethorphan and bufuralol and the inactivator SCH 66712. We found that *34, *17-2, and *17-3 displayed reduced enzyme activity and NADPH coupling while producing the same metabolites as *1, suggesting a possible role for Arg296 in NADPH coupling. A higher-activity variant, *53, displayed similar NADPH coupling to *1 but was less susceptible to inactivation by SCH 66712. The Thr309Ala mutant showed similar activity to that of *1 but with greatly reduced NADPH coupling. Overall, these results suggest that kinetic and metabolic analysis of individual CYP2D6 variants is required to understand their possible contributions to variable drug response and the complexity of personalized medicine.

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Substituted Imidazole of 5-Fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine Inactivates Cytochrome P450 2D6 by Protein Adduction

Cited by 8 publications

References 31 publications

Mechanism-Based Inactivation of Human Cytochrome P450 3A4 by Two Piperazine-Containing Compounds

Mechanism-Based Inactivation of Human Cytochrome P450 3A4 by Two Piperazine-Containing Compounds

Human Cytochrome P450 Enzymes

CYP2D6 Allelic Variants 34, 17-2, 17-3, and 53 and a Thr309Ala Mutant Display Altered Kinetics and NADPH Coupling in Metabolism of Bufuralol and Dextromethorphan and Altered Susceptibility to Inactivation by SCH 66712

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Substituted Imidazole of 5-Fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine Inactivates Cytochrome P450 2D6 by Protein Adduction

Cited by 8 publications

References 31 publications

Mechanism-Based Inactivation of Human Cytochrome P450 3A4 by Two Piperazine-Containing Compounds

Mechanism-Based Inactivation of Human Cytochrome P450 3A4 by Two Piperazine-Containing Compounds

Human Cytochrome P450 Enzymes

CYP2D6 Allelic Variants *34, *17-2, *17-3, and *53 and a Thr309Ala Mutant Display Altered Kinetics and NADPH Coupling in Metabolism of Bufuralol and Dextromethorphan and Altered Susceptibility to Inactivation by SCH 66712

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CYP2D6 Allelic Variants 34, 17-2, 17-3, and 53 and a Thr309Ala Mutant Display Altered Kinetics and NADPH Coupling in Metabolism of Bufuralol and Dextromethorphan and Altered Susceptibility to Inactivation by SCH 66712