Substrate interaction with cytochrome P-450

Schenkman, John B.; Sligar, Stephen G.; Cinti, Dominick L.

doi:10.1016/0163-7258(81)90075-9

Cited by 195 publications

(97 citation statements)

References 86 publications

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“…The results showed that after dialysis the treated enzyme had only 16% of the activity of the control, indicating that the timedependent inhibition is an irreversible inactivation. The spectral binding constant (K s ) of CYP3cide for CYP3A4 was not possible to calculate from the observed difference spectra in which no significant spin shift was observed (Schenkman et al, 1981). Examination of the CO binding difference spectrum in inactivated enzyme versus control showed a substantial decrease in the 450-nm CO binding spectrum (Fig.…”

Section: Resultsmentioning

confidence: 99%

Selective Mechanism-Based Inactivation of CYP3A4 by CYP3cide (PF-04981517) and Its Utility as an In Vitro Tool for Delineating the Relative Roles of CYP3A4 versus CYP3A5 in the Metabolism of Drugs

et al. 2012

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CYP3cide (PF-4981517; 1-methyl-3-[1-methyl-5-(4-methylphenyl)-1H-pyrazol-4-yl]-4-[(3S)-3-piperidin-1-ylpyrrolidin-1-yl]-1H-pyrazolo[3,4-d]pyrimidine) is a potent, efficient, and specific time-dependent inactivator of human CYP3A4. When investigating its inhibitory properties, an extreme metabolic inactivation efficiency (k inact /K I ) of 3300 to 3800 ml ⅐ min ؊1 ⅐ mol ؊1 was observed using human liver microsomes from donors of nonfunctioning CYP3A5 (CYP3A5 *3/*3). This observed efficiency equated to an apparent K I between 420 and 480 nM with a maximal inactivation rate (k inact ) equal to 1.6 min ؊1 . Similar results were achieved with testosterone, another CYP3A substrate, and other sources of the CYP3A4 enzyme. To further illustrate the abilities of CYP3cide, its partition ratio of inactivation was determined with recombinant CYP3A4. These studies produced a partition ratio approaching unity, thus underscoring the inactivation capacity of CYP3cide. WhenCYP3cide was tested at a concentration and preincubation time to completely inhibit CYP3A4 in a library of genotyped polymorphic CYP3A5 microsomes, the correlation of the remaining midazolam 1-hydroxylase activity to CYP3A5 abundance was significant (R 2 value equal to 0.51, p value of <0.0001). The work presented here supports these findings by fully characterizing the inhibitory properties and exploring CYP3cide's mechanism of action. To aid the researcher, multiple commercially available sources of CYP3cide were established, and a protocol was developed to quantitatively determine CYP3A4 contribution to the metabolism of an investigational compound. Through the establishment of this protocol and the evidence provided here, we believe that CYP3cide is a very useful tool for understanding the relative roles of CYP3A4 versus CYP3A5 and the impact of CYP3A5 genetic polymorphism on a compound's pharmacokinetics.

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Section: Resultsmentioning

confidence: 99%

Selective Mechanism-Based Inactivation of CYP3A4 by CYP3cide (PF-04981517) and Its Utility as an In Vitro Tool for Delineating the Relative Roles of CYP3A4 versus CYP3A5 in the Metabolism of Drugs

et al. 2012

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“…Binding of substrates and xenobiotics to P450s can result in two types of characteristic spectral changes in the UV/VIS heme Soret spectrum, referred to as Type I and Type II (Schenkman et al, 1981;Isin and Guengerich, 2008). A classical Type I spectrum results from the spin-state shift of the Soret band at about 418 nm that represents the ferric (Fe III ) low-spin substrate free form tõ 390 nm for the ferric (Fe III ) high-spin form originating from the displacement of H 2 O as the sixth ligand when a substrate is bound in close proximity to the heme.…”

Section: Determination Of Spectral Dissociation Constantsmentioning

confidence: 99%

Ligand characterization of CYP4B1 isoforms modified for high-level expression inEscherichia coliand HepG2 cells

Roellecke

Jäger

Gyurov

et al. 2017

Protein Engineering, Design and Selection

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Human CYP4B1, a cytochrome P450 monooxygenase predominantly expressed in the lung, inefficiently metabolizes classical CYP4B1 substrates, such as the naturally occurring furan pro-toxin 4-ipomeanol (4-IPO). Highly active animal forms of the enzyme convert 4-IPO to reactive alkylating metabolite(s) that bind(s) to cellular macromolecules. By substitution of 13 amino acids, we restored the enzymatic activity of human CYP4B1 toward 4-IPO and this modified cDNA is potentially valuable as a suicide gene for adoptive T-cell therapies. In order to find novel pro-toxins, we tested numerous furan analogs in in vitro cell culture cytotoxicity assays by expressing the wildtype rabbit and variants of human CYP4B1 in human liver-derived HepG2 cells. To evaluate the CYP4B1 substrate specificities and furan analog catalysis, we optimized the N-terminal sequence of the CYP4B1 variants by modification/truncation and established their heterologous expression in Escherichia coli (yielding 70 and 800 nmol·l −1 of recombinant human and rabbit enzyme, respectively). Finally, spectral binding affinities and oxidative metabolism of the furan analogs by the purified recombinant CYP4B1 variants were analyzed: the naturally occurring perilla ketone was found to be the tightest binder to CYP4B1, but also the analog that was most extensively metabolized by oxidative processes to numerous non-reactive reaction products.

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“…More importantly, however, this finding indicates that ketoconazole inhibition is selective with some cytochrome P-450 enzymes being more affected than others. Presumably this reflects differences in affinities for binding to the cytochrome and formation of a sixth axial ferric coordinate complex between the imidazole nitrogen and the different haem proteins (Schenkman et al;Sheets & Mason, 1984. There has been previous speculation about this possibility (Sheets & Mason, 1984;Meredith et al, 1985), but this is the first direct evidence in humans of such specificity.…”

Section: Discussionmentioning

confidence: 99%

Effects of ketoconazole on the polymorphic 4‐hydroxylations of S‐ mephenytoin and debrisoquine.

Atiba

Blaschke

Wilkinson

1989

Brit J Clinical Pharma

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Studies were undertaken in 12 normal, male subjects to determine whether a metabolic interaction occurs between ketoconazole and mephenytoin. A single dose (400 mg) of ketoconazole produced a reduction in the 0-8 h urinary R/S ratio of mephenytoin following oral administration (100 mg) of racemic drug and after 28 daily doses the median value was further reduced to 42.9% of its baseline value. Within 7 days following discontinuation of ketoconazole the enantiomeric ratio had returned to its pre-study value. These findings are consistent with ketoconazole being a potent in vivo inhibitor of mephenytoin's 4-hydroxylation and confirm the ability of such an interaction to be predicted by in vitro studies with human liver microsomes. By contrast, ketoconazole had a much smaller effect on the 0-8 h urinary metabolic ratio of debrisoquine, indicating that ketoconazole has a selective inhibitory effect on different forms of cytochrome P-450.

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Substrate interaction with cytochrome P-450

Cited by 195 publications

References 86 publications

Selective Mechanism-Based Inactivation of CYP3A4 by CYP3cide (PF-04981517) and Its Utility as an In Vitro Tool for Delineating the Relative Roles of CYP3A4 versus CYP3A5 in the Metabolism of Drugs

Selective Mechanism-Based Inactivation of CYP3A4 by CYP3cide (PF-04981517) and Its Utility as an In Vitro Tool for Delineating the Relative Roles of CYP3A4 versus CYP3A5 in the Metabolism of Drugs

Ligand characterization of CYP4B1 isoforms modified for high-level expression inEscherichia coliand HepG2 cells

Effects of ketoconazole on the polymorphic 4‐hydroxylations of S‐ mephenytoin and debrisoquine.

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