Experimental Approaches to Analysis of Reactions of Cytochrome P450 Enzymes

Guengerich, F. Peter

doi:10.1002/9783527673261.ch08

Cited by 5 publications

(6 citation statements)

References 72 publications

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“…The output from these methods is easy to interrogate as they generate binding poses that can be inspected by the user and are rooted in the physical reality of explicitly modeling the binding event. Today, crystal structures are available for almost all CYP isoforms relevant to xenobiotic metabolism (Guengerich, ; Oostenbrink, ). Malleability of these enzymes, their complex interplay with water, and the hydrophobic character of their—in part—very large binding sites pose significant challenges to the application of structure‐based approaches, in particular docking.…”

Section: Methods For Site Of Metabolism Predictionmentioning

confidence: 99%

Computational methods and tools to predict cytochrome P450 metabolism for drug discovery

2019

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In this review, we present important, recent developments in the computational prediction of cytochrome P450 (CYP) metabolism in the context of drug discovery. We discuss in silico models for the various aspects of CYP metabolism prediction, including CYP substrate and inhibitor predictors, site of metabolism predictors (i.e., metabolically labile sites within potential substrates) and metabolite structure predictors. We summarize the different approaches taken by these models, such as rule‐based methods, machine learning, data mining, quantum chemical methods, molecular interaction fields, and docking. We highlight the scope and limitations of each method and discuss future implications for the field of metabolism prediction in drug discovery.

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Section: Methods For Site Of Metabolism Predictionmentioning

confidence: 99%

Computational methods and tools to predict cytochrome P450 metabolism for drug discovery

2019

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“…Studies with other human and experimental animal P450s have shown that steps 2, 4, 7, and 9 can all be rate-limiting in different reactions. 23 , 24 We utilized the approach of analyzing rates of individual reaction steps and measuring KIEs to kinetic models to determine which steps might be limiting, as has been done with several other P450s. 23 , 25 − 27 We conclude that major factors limiting the rate of lauric acid 12-hydroxylation are the rate of transfer of an electron from b 5 and the rate of C–H bond breaking.…”

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confidence: 99%

Kinetic Analysis of Lauric Acid Hydroxylation by Human Cytochrome P450 4A11

et al. 2014

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Cytochrome P450 (P450) 4A11 is the only functionally active subfamily 4A P450 in humans. P450 4A11 catalyzes mainly ω-hydroxylation of fatty acids in liver and kidney; this process is not a major degradative pathway, but at least one product, 20-hydroxyeicosatetraenoic acid, has important signaling properties. We studied catalysis by P450 4A11 and the issue of rate-limiting steps using lauric acid ω-hydroxylation, a prototypic substrate for this enzyme. Some individual reaction steps were studied using pre-steady-state kinetic approaches. Substrate and product binding and release were much faster than overall rates of catalysis. Reduction of ferric P450 4A11 (to ferrous) was rapid and not rate-limiting. Deuterium kinetic isotope effect (KIE) experiments yielded low but reproducible values (1.2–2) for 12-hydroxylation with 12-2H-substituted lauric acid. However, considerable “metabolic switching” to 11-hydroxylation was observed with [12-2H3]lauric acid. Analysis of switching results [Jones, J. P., et al. (1986) J. Am. Chem. Soc.108, 7074–7078] and the use of tritium KIE analysis with [12-3H]lauric acid [Northrop, D. B. (1987) Methods Enzymol.87, 607–625] both indicated a high intrinsic KIE (>10). Cytochrome b5 (b5) stimulated steady-state lauric acid ω-hydroxylation ∼2-fold; the apoprotein was ineffective, indicating that electron transfer is involved in the b5 enhancement. The rate of b5 reoxidation was increased in the presence of ferrous P450 mixed with O2. Collectively, the results indicate that both the transfer of an electron to the ferrous·O2 complex and C–H bond-breaking limit the rate of P450 4A11 ω-oxidation.

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“…Despite the vast amount of information available from CYP3A4 studies, there is still considerable lack of complete understanding of its allosteric properties and the detailed mechanism of drug-drug interactions. Hence, it is very hard to predict the effect of a new potential substrate, effector, or inhibitor, on the overall turnover of multiple pharmaceutics (24). Human cytochromes P450 are incorporated into the lipid membrane and are less stable or inactive in detergent solubilized systems, while experimental studies in lipid vesicles face other difficulties, typical for colloidal systems with restricted diffusion and phase heterogeneity.…”

Section: Introductionmentioning

confidence: 99%

“…Hence, it is very hard to predict the effect of a new potential substrate, effector, or inhibitor on the overall turnover of multiple pharmaceutics. 24 Human cytochromes P450 are incorporated into the lipid membrane and are less stable or inactive in detergent-solubilized systems, while experimental studies in lipid vesicles face other difficulties, typical for colloidal systems with restricted diffusion and phase heterogeneity. A useful alternative is provided by application of Nanodisc technology, which yields soluble homogeneous and functionally stable preparations of human cytochromes P450 incorporated into the nativelike lipid bilayer.…”

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confidence: 99%

Allosteric Interactions in Human Cytochrome P450 CYP3A4: The Role of Phenylalanine 213

et al. 2019

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The role of Phe213 in the allosteric mechanism of human cytochrome P450 CYP3A4 was studied using a combination of progesterone (PGS) and carbamazepine (CBZ) as probe substrates. We expressed, purified and incorporated in POPC Nanodiscs three mutants, F213A, F213S, and F213Y, and compared them with the wild-type CYP3A4 monitoring spectral titration, the rate of NADPH oxidation and steady-state product turnover rates with pure substrates and substrate mixtures. All mutants demonstrated higher activity with CBZ, lower activity with PGS, and reduced activation of CBZ epoxidation by PGS, most pronounced in F213A mutant. Using allatom molecular dynamics simulations we compared dynamics of WT CYP3A4 and the F213A mutant incorporated in the lipid bilayer and the effect of the presence of PGS molecule at the allosteric peripheral site and evaluated the critical role of Phe213 in mediating the heterotropic allosteric interactions in CYP3A4.

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Experimental Approaches to Analysis of Reactions of Cytochrome P450 Enzymes

Cited by 5 publications

References 72 publications

Computational methods and tools to predict cytochrome P450 metabolism for drug discovery

Computational methods and tools to predict cytochrome P450 metabolism for drug discovery

Kinetic Analysis of Lauric Acid Hydroxylation by Human Cytochrome P450 4A11

Allosteric Interactions in Human Cytochrome P450 CYP3A4: The Role of Phenylalanine 213

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