Catalytic selectivity of human cytochrome P450 enzymes: relevance to drug metabolism and toxicity

doi:10.1016/0378-4274(94)90156-2

Toxicology Letters

Volume 70, Issue 2, 1 February 1994, Pages 133-138

https://doi.org/10.1016/0378-4274(94)90156-2 Get rights and content

Abstract

About 30 different human cytochrome P450 (P450) enzymes have now been characterized in considerable detail. It is possible to elucidate their catalytic specificities towards drugs, steroids, carcinogens, and other potential substrates with in vitro assays. It is also possible to ascertain the levels of individual P450 enzymes in humans with the use of drugs, after appropriate assay validation. The ability to discern catalytic selectivity and levels of P450 enzymes within individuals offers considerable potential in drug development, prevention of undesirable drug-drug interactions, and understanding the etiology of diseases resulting from exposure to potentially toxic and carcinogenic chemicals.

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Development of an efficient transient expression system for Siraitia grosvenorii fruit and functional characterization of two NADPH-cytochrome P450 reductases
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Citation Excerpt :
A previous study have suggested that CPRs are indispensable for CYP450 expression (Sintupachee et al., 2014), and in various plant species, the coexpression patterns of CYP450s and CPRs have been estimated, including in S. miltiorrhiza (Guo et al., 2013), Papaver somniferum (Rosco et al., 1997) and A. thaliana (Christ et al., 2013). Generally, mammals harbour only one CPR (Guengerich, 1994), but one to four CPRs have been found in plants (Eberle et al., 2009; Shet et al., 1993), making it more difficult to determine the function of each CPR. Moreover, numerous plant CYP450s and CPRs have been previously identified and expressed in yeasts (Hamann and Møller, 2007; Jennewein et al., 2005; Urban et al., 1997) and have rarely been reported in plant-associated prokaryotic species.
Siraitia grosvenorii (Luo hanguo or monk fruit) is a valuable medicinal herb for which the market demand has increased dramatically worldwide. As promising natural sweeteners, mogrosides have received much attention from researchers because of their extremely high sweetness and lack of calories. Nevertheless, owing to the absence of genetic transformation methods, the molecular mechanisms underlying the regulation of mogroside biosynthesis have not yet been fully elucidated. Therefore, an effective method for gene function analysis needs to be developed for S. grosvenorii fruit. As a powerful approach, transient expression has become a versatile method to elucidate the biological functions of genes and proteins in various plant species. In this study, PBI121 with a β-glucuronidase (GUS) marker and tobacco rattle virus (TRV) were used as vectors for overexpression and silencing, respectively, of the SgCPR1 and SgCPR2 genes in S. grosvenorii fruit. The effectiveness of transient expression was validated by GUS staining in S. grosvenorii fruit, and the expression levels of SgCPR1 and SgCPR2 increased significantly after infiltration for 36 h. In addition, TRV-induced gene silencing suppressed the expression of SgCPR1 and SgCPR2 in S. grosvenorii fruit. More importantly, the production of the major secondary metabolites mogrol, mogroside IIE (MIIE) and mogroside III (MIII) was activated by the overexpression of SgCPR1 and SgCPR2 in S. grosvenorii fruit, with levels 1–2 times those in the control group. Moreover, the accumulation of mogrol, MIIE and MIII was decreased in the SgCPR1 and SgCPR2 gene silencing assays. Therefore, this transient expression approach was available for S. grosvenorii fruit, providing insight into the expression of the SgCPR1 and SgCPR2 genes involved in the mogroside biosynthesis pathway. Our study also suggests that this method has potential applications in the exploration of the molecular mechanisms, biochemical hypotheses and functional characteristics of S. grosvenorii genes.
Identification of human cytochrome P450 isozymes involved in the oxidative metabolism of carfentanil
2021, Toxicology Letters
Citation Excerpt :
M13 exhibited twice the μ-opioid receptor activity as carfentanil. It is well known that cytochrome P450 (CYP) is an enzyme family consisting of multiple isoforms (Gonzalez, 1992; Guengerich, 1994) and catalyzes the biotransformation of a variety of drugs and xenobiotics (Bachmann and Ghosh, 2001). In vitro data on the major CYP isoforms involved in a drug’s metabolism are useful in predicting its metabolic fate, genetic polymorphic regulation, and potential in vivo interactions with other therapeutic agents via inductive or inhibitory mechanisms in humans (FDA, 2020; Iwatsubo et al., 1996).
Carfentanil is an ultra-potent opioid with an analgesic potency 10,000 times that of morphine but has received little scientific investigation. In the present study, the human cytochrome P450 (CYP) isozymes catalyzing the oxidative metabolism of carfentanil were investigated. Using UHPLC-HRMS, Michaelis-Menten kinetics of formation for three major metabolites norcarfentanil (M1), pharmaceutical active metabolite 4-[(1-oxopropyl)phenylamino]-1-(2-hydroxyl-2-phenylethyl)-4-piperidinecarboxylic acid methyl ester (M11), and 4-[(1-oxopropyl)phenylamino]-1-(2-oxo-2-phenylethyl)-4-piperidinecarboxylic acid methyl ester (M15) were determined. Isozymes catalyzing the formation of the low abundant, highly active metabolite 1-[2-(2-hydroxylphenyl)ethyl]-4-[(1-oxopropyl)phenylamino]-4-piperidinecarboxylic acid methyl ester (M13) were also identified. Selective P450 inhibition studies with pooled human liver microsomes (HLMs) and recombinant CYP isozymes suggested that metabolites M1, M11, and M15 were predominantly formed by isozyme CYP3A5, followed by CYP3A4. Isozymes CYP2C8 and CYP2C9 also made contributions but to a much lesser extent. Highly potent metabolite M13 was predominantly formed by isozyme CYP2C9, followed by CYP2C8. These findings indicate that CYP3A5, CYP3A4, CYP2C8 and CYP2C9 play a major role in the transformation of carfentanil to M1 (norcarfentanil), M11, M13 and M15 through N-dealkylation of piperidine ring, hydroxylation of phenethyl group and ketone formation on phenethyl linker by human liver micrsomes.
Functional expression of two NADPH-cytochrome P450 reductases from Siraitia grosvenorii
2018, International Journal of Biological Macromolecules
Citation Excerpt :
Co-expression studies have shown that the expression levels of CPRs and P450s are correlated in a linear fashion [11]. Unlike yeast and mammals that only harbor one CPR [13,14], plants may contain one to four CPR paralogous genes [15,16]. Commonly, if a plant's CPR is functionally unknown, then an Arabidopsis CPR is used to verify the functions of the plant's P450s.
Cytochrome P450 reductase (CPR) is the redox partner of various P450s involved in primary and secondary metabolism. Here, we identified and characterized two paralogs of cytochrome P450 reductase from Siraitia grosvenorii. There were two full-length CPR isoforms in the S. grosvenorii fruit transcriptome dataset. They had the same open reading frames of 2, 124 bp, encoding 707 amino acids. A phylogenetic analysis characterized both SgCPR1 and SgCPR2 as Class II dicotyledonous CPRs. The recombinant proteins SgCPR1 and SgCPR2 could reduce cytochrome c and ferricyanide in a NADPH-dependent manner. The SgCPR1 and SgCPR2 transcripts were detected in all examined tissues of S. grosvenorii, and in fresh fruit, they had expression patterns similar to several key enzymes that require CPR as a partner during their biosynthesis. The expression levels of the SgCPRs were induced after a methyl jasmonate treatment. The extracts from yeast co-expressing SgCPR1/SgCPR2 and the cytochrome P450 enzyme CYP76AH1 produced ferruginol, indicating the positive effects of SgCPR1/SgCPR2 on the CYP76AH1 activity. A docking analysis confirmed the experimentally deduced functional activities of SgCPR1 and SgCPR2 for NADPH, FAD and FMN. Thus, SgCRP1 and SgCPR2 are both likely to participate in secondary metabolism, especially mogroside biosynthesis in S. grosvenorii.
Identification and expression patterns of UDP-glycosyltransferase (UGT) genes from insect pest Athetis lepigone (Lepidoptera: Noctuidae)
2017, Journal of Asia-Pacific Entomology
UDP-glucuronosyltransferase (UGT) genes, which belong to an ancient gene family and play very important roles in all organisms, encode extracellularly secreted proteins that are involved in the transfer of glycosyl residues from activated nucleotide sugars to acceptor hydrophobic molecules (aglycones). Athetis lepigone is an important polyphagous pest worldwide, and since 2011 it has become one of the major maize pests in North China. However, there have been no studies on pesticides for the effective control of this pest. In this study, we identified 23 putative UGT genes in A. lepigone by analysing previous antennal transcriptomic data. Phylogenetic analysis showed that all of the AlUGTs are distributed within 11 of 14 insect UGT sub-families. Tissue expression analysis revealed that > 70% of AlUGTs were primarily expressed in adult antennae, of which three (AlUGT33AD1, AlUGT40F6 and AlUGT40L4) and four (AlUGT33B18, AlUGT33F10, AlUGT40Q3 and AlUGT41D3) displayed male-biased and female-biased expression, respectively. Some AlUGTs, however, had higher expression levels in non-antennal tissues. Our study is the first to identify UGT genes in A. lepigone, which will help us to elucidate the diverse functions of these genes, and ultimately provide potential targets that will facilitate the development of efficient and environmentally friendly pesticides against A. lepigone.
Hepatic Drug Metabolism in Pediatric Patients
2017, Drug Metabolism in Diseases
For many drugs, clearance is the primary factor driving dosing regimens. In young children, hepatic metabolic clearance is influenced by maturational changes in the expression and activity of drug-metabolizing enzymes, as well as by changes in hepatic blood flow and perfusion, plasma protein binding, and active transport processes. The largest maturational changes in the different determinants of hepatic metabolic clearance are observed in the first months and years of life with largest interindividual variability in the youngest age ranges. Together, the physiochemical properties of a given drug and the interplay of the aforementioned factors in a developmental context determine the drug's total hepatic clearance. Knowledge on the maturational processes influencing hepatic drug clearance is limited. Centrally, an understanding of the ontogeny of drug-metabolizing enzymes is critical for sound clinical judgment regarding the use of certain drugs in young children.
Kinetic analysis of electron flux in cytochrome P450 reductases reveals differences in rate-determining steps in plant and mammalian enzymes
2015, Archives of Biochemistry and Biophysics
Herein, we compare the kinetic properties of CPR from Arabidopsis thaliana (ATR2), with CPR from Artemisia annua (aaCPR) and human CPR (hCPR). While all three CPR forms elicit comparable rates for cytochrome c³⁺ turnover, NADPH reduction of the FAD cofactor is ∼50-fold faster in aaCPR and ATR2 compared to hCPR, with a k_obs of ∼500 s⁻¹ (6 °C). Stopped-flow analysis of the isolated FAD-domains reveals that NADP⁺-FADH₂ charge-transfer complex formation is also significantly faster in the plant enzymes, but the rate of its decay is comparable for all three proteins. In hCPR, transfer of a hydride ion from NADPH to FAD is tightly coupled to subsequent FAD to FMN electron transfer, indicating that the former catalytic event is slow relative to the latter. In contrast, interflavin electron transfer is slower than NADPH hydride transfer in aaCPR and ATR2, occurring with an observed rate constant of ∼50 s⁻¹. Finally, the transfer of electrons from FMN to cytochrome c³⁺ is rapid (>10³ s⁻¹) in all three enzymes and does not limit catalytic turnover. In combination, the data reveal differences in rate-determining steps between plant CPR and their mammalian equivalent in mediating the flux of reducing equivalents from NADPH to external electron acceptors.

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MinireviewCatalytic selectivity of human cytochrome P450 enzymes: relevance to drug metabolism and toxicity

Abstract

J. Biol. Chem.

Lancet

Biochem. Pharmacol.

Lancet

The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature

DNA Cell Biol.

Characterization of human microsomal cytochrome P-450 enzymes

Annu. Rev. Pharmacol. Toxicol.

The molecular biology of cytochrome P450s

Pharmacol. Rev.

Oxidation of toxic and carcinogenic chemicals by human cytochrome P-450 enzymes

Chem. Res. Toxicol.

Separation of human liver tolbutamine hydroxylase and (S)-mephenytoin 4'-hydroxylase cytochrome P-450 enzymes

Mol. Pharmacol.

Minireview
Catalytic selectivity of human cytochrome P450 enzymes: relevance to drug metabolism and toxicity