scholarly journals Towards Accurate Genotype–Phenotype Correlations in the CYP2D6 Gene

2020 ◽  
Vol 10 (4) ◽  
pp. 158 ◽  
Author(s):  
Angel Pey
Keyword(s):  
Large Scale ◽  
Phenotypic Variability ◽  
Genetic Diseases ◽  
Individual Response ◽  
Cytochrome P450 Enzyme ◽  
Cyp2d6 Gene ◽  
Human Genes ◽  
P450 Enzyme ◽  
Simple Activity

Establishing accurate and large-scale genotype–phenotype correlations and predictions of individual response to pharmacological treatments are two of the holy grails of Personalized Medicine. These tasks are challenging and require an integrated knowledge of the complex processes that regulate gene expression and, ultimately, protein functionality in vivo, the effects of mutations/polymorphisms and the different sources of interindividual phenotypic variability. A remarkable example of our advances in these challenging tasks is the highly polymorphic CYP2D6 gene, which encodes a cytochrome P450 enzyme involved in the metabolization of many of the most marketed drugs (including SARS-Cov-2 therapies such as hydroxychloroquine). Since the introduction of simple activity scores (AS) over 10 years ago, its ability to establish genotype–phenotype correlations on the drug metabolizing capacity of this enzyme in human population has provided lessons that will help to improve this type of score for this, and likely many other human genes and proteins. Multidisciplinary research emerges as the best approach to incorporate additional concepts to refine and improve such functional/activity scores for the CYP2D6 gene, as well as for many other human genes associated with simple and complex genetic diseases.

Inhibition of aldosterone biosynthesis by staurosporine

2005 ◽  
Vol 386 (7) ◽  
pp. 663-669 ◽  
Author(s):  
Matthias Bureik ◽  
Alexander Mion ◽  
Christopher J. Kenyon ◽  
Rita Bernhardt
Keyword(s):  
Kinase Inhibitor ◽  
Therapeutic Potential ◽  
Hypotensive Effect ◽  
Cytochrome P450 Enzyme ◽  
Aldosterone Synthase ◽  
Kinase C ◽  
Steroid Hydroxylase ◽  
P450 Enzyme ◽  
Adrenodoxin Reductase

Abstract Staurosporine (STS) is a very potent broad-range kinase inhibitor, and its antiproliferative properties made it a lead compound for protein kinase C (PKC) inhibitors with therapeutic potential. Because STS also causes hypotension, we investigated in this study whether it directly interferes with the terminal steps of aldosterone biosynthesis; these are catalysed by a mitochondrial steroid hydroxylase system consisting of adrenodoxin reductase, adrenodoxin, and the cytochrome P450 enzyme hCYP11B2 (aldosterone synthase). Here we demonstrate that nanomolar concentrations of STS significantly reduced aldosterone synthase activity in transiently transfected COS-1 cells and in stably transfected V79MZh11B2 cells (IC50=11 nM). However, STS did not inhibit bovine aldosterone synthase in a reconstituted steroid hydroxylation assay. In transiently transfected COS-1 cells, the protein level of adrenodoxin (but not that of adrenodoxin reductase or of hCYP11B2) was significantly reduced after treatment with 2 nM STS. Finally, we show that STS treatment (1 μg/day) of mice reduced their aldosterone/renin ratio by almost 50% (p=0.015). To the best of our knowledge, this is the first report of a direct in vivo effect of STS on the renin-angiotensin-aldosterone system. We conclude (i) that the hypotensive effect of staurosporine is at least partly due to inhibition of aldosterone biosynthesis via adrenodoxin depletion, and (ii) that aldosterone biosynthesis can be regulated in vivo at the level of adrenodoxin availability.

scholarly journals Chlorogenic Acid as a Model Compound for Optimization of an In Vitro Gut Microbiome-Metabolism Model

Proceedings ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 31 ◽  
Author(s):  
Olivier Mortelé ◽  
Elias Iturrospe ◽  
Annelies Breynaert ◽  
Christine Lammens ◽  
Xavier Basil Britto ◽  
...  
Keyword(s):  
Chlorogenic Acid ◽  
Gut Microbiome ◽  
Model Compound ◽  
In Vitro Models ◽  
Cytochrome P450 Enzyme ◽  
Flight Mass Spectrometry ◽  
P450 Enzyme ◽  
Analytical Phase

It has been believed that the metabolism of xenobiotics occurred mainly by the cytochrome P450 enzyme system in the liver. However, recent data clearly suggest a significant role for the gut microbiota in the metabolism of xenobiotic compounds. This microbiotic biotransformation could lead to differences on activation, inactivation and possible toxicity of these compounds. In vitro models are generally used to study the colonic biotransformation as they allow easy dynamic and multiple sampling over time. However, to ensure this accurately mimics communities in vivo, the pre-analytical phase requires optimization. Chlorogenic acid, a polyphenolic compound abundantly present in the human diet, was used as a model compound to optimize a ready-to-use gut microbiome biotransformation platform. Samples of the in vitro gastrointestinal dialysis-model with colon stage were analyzed by liquid chromatography coupled to high resolution time-of-flight mass spectrometry. Complementary screening approaches were also employed to identify the biotransformation products.

scholarly journals Characterization of CYP71AX36 from Sunflower (Helianthus annuus L., Asteraceae)

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Maximilian Frey ◽  
Iris Klaiber ◽  
Jürgen Conrad ◽  
Aylin Bersch ◽  
Irini Pateraki ◽  
...  
Keyword(s):  
Helianthus Annuus ◽  
Chemical Shifts ◽  
Sesquiterpene Lactones ◽  
2D Nmr ◽  
Cytochrome P450 Enzyme ◽  
The Core ◽  
Pathway Reconstruction ◽  
P450 Enzyme ◽  
Sunflower Genome

Abstract Sesquiterpene lactones (STL) are a subclass of isoprenoids with many known bioactivities frequently found in the Asteraceae family. In recent years, remarkable progress has been made regarding the biochemistry of STL, and today the biosynthetic pathway of the core backbones of many STLs has been elucidated. Consequently, the focus has shifted to the discovery of the decorating enzymes that can modify the core skeleton with functional hydroxy groups. Using in vivo pathway reconstruction assays in heterologous organisms such as Saccharomyces cerevisiae and Nicotiana benthamiana, we have analyzed several cytochrome P450 enzyme genes of the CYP71AX subfamily from Helianthus annuus clustered in close proximity to one another on the sunflower genome. We show that one member of this subfamily, CYP71AX36, can catalyze the conversion of costunolide to 14-hydroxycostunolide. The catalytic activity of CYP71AX36 may be of use for the chemoenzymatic production of antileukemic 14-hydroxycostunolide derivatives and other STLs of pharmaceutical interest. We also describe the full 2D-NMR assignment of 14-hydroxycostunolide and provide all 13C chemical shifts of the carbon skeleton for the first time.

cyp7b1 catalyses the 7α-hydroxylation of dehydroepiandrosterone and25-hydroxycholesterol in rat prostate

2001 ◽  
Vol 355 (2) ◽  
pp. 509-515 ◽  
Author(s):  
Cécile MARTIN ◽  
Rhona BEAN ◽  
Ken ROSE ◽  
Fouad HABIB ◽  
Jonathan SECKL
Keyword(s):  
Prostate Gland ◽  
Northern Blotting ◽  
Sex Steroid ◽  
Cytochrome P450 Enzyme ◽  
Steroid Synthesis ◽  
Peripheral Tissues ◽  
Reverse Transcription Pcr ◽  
P450 Enzyme ◽  
Rat Prostate

Dehydroepiandrosterone (DHEA) is the most prominent circulating steroid in humans, and it is a precursor for sex-steroid synthesis in peripheral tissues, including the prostate. Recently, enzyme-mediated pre-receptor metabolism has been recognized as a key step in determining steroid action in vivo. Hydroxylation of 3β-steroids at the 7α-position has been reported in rat and human prostate to be a major inhibitory pathway to sex-steroid synthesis/action. However, the molecular identity of the enzyme responsible is so far unknown. We recently described a novel cytochrome P450 enzyme, cyp7b1, strongly expressed in the hippocampus of rodent brain, which catalyses the metabolism of DHEA, pregnenolone and 25-hydroxycholesterol to 7α-hydroxy products. In the light of this new enzyme, we have examined its possible role in 7α-hydroxylation conversion in rat prostate. NADPH-dependent 7α-hydroxylation was confirmed for 3β-hydroxysteroids including DHEA and androstenediol, as well as 25-hydroxycholesterol. Kinetic analysis yielded an apparent Km of 14±1µM for 7α-hydroxylation of DHEA in the prostate gland, a value similar to that recorded for recombinant cyp7b1 enzyme [13.6µM; Rose, Stapleton, Dott, Kieny, Best, Schwarz, Russell, Bjoorkheim, Seckl and Lathe (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4925-4930]. The Vmax value of the prostate was 46±2pmol/min per mg, and this activity was inhibited by clotrimazole, a P450-enzyme blocker. Moreover, RNA analysis (reverse-transcription PCR, Northern blotting and in situ hybridization) revealed a high expression of cyp7b1 mRNA in the rat prostate, restricted to the epithelium, suggesting that cyp7b1 catalyses oxysterol 7α-hydroxylation in the prostate gland.

scholarly journals Genomic analyses of glycine decarboxylase neurogenic mutations yield a large-scale prediction model for prenatal disease

PLoS Genetics ◽  
2021 ◽  
Vol 17 (2) ◽  
pp. e1009307
Author(s):  
Joseph Farris ◽  
Md Suhail Alam ◽  
Arpitha Mysore Rajashekara ◽  
Kasturi Haldar
Keyword(s):  
Neurological Disease ◽  
Large Scale ◽  
Systems Approach ◽  
Single Gene ◽  
Genetic Diseases ◽  
Pathogenic Mutation ◽  
Glycine Decarboxylase ◽  
Functional Consequences ◽  
Genome Level

Hundreds of mutations in a single gene result in rare diseases, but why mutations induce severe or attenuated states remains poorly understood. Defect in glycine decarboxylase (GLDC) causes Non-ketotic Hyperglycinemia (NKH), a neurological disease associated with elevation of plasma glycine. We unified a human multiparametric NKH mutation scale that separates severe from attenuated neurological disease with new in silico tools for murine and human genome level-analyses, gathered in vivo evidence from mice engineered with top-ranking attenuated and a highly pathogenic mutation, and integrated the data in a model of pre- and post-natal disease outcomes, relevant for over a hundred major and minor neurogenic mutations. Our findings suggest that highly severe neurogenic mutations predict fatal, prenatal disease that can be remedied by metabolic supplementation of dams, without amelioration of persistent plasma glycine. The work also provides a systems approach to identify functional consequences of mutations across hundreds of genetic diseases. Our studies provide a new framework for a large scale understanding of mutation functions and the prediction that severity of a neurogenic mutation is a direct measure of pre-natal disease in neurometabolic NKH mouse models. This framework can be extended to analyses of hundreds of monogenetic rare disorders where the underlying genes are known but understanding of the vast majority of mutations and why and how they cause disease, has yet to be realized.

Effects of 31 recombinant CYP2C19 variants on clomipramine metabolism in vitro

2021 ◽  
pp. 026988112110505
Author(s):  
Tian Lan ◽  
Ya-Qing Ma ◽  
Ya-Min Dang ◽  
Chen-Chen Wang ◽  
Ren-Ai Xu ◽  
...  
Keyword(s):  
High Performance ◽  
Intrinsic Clearance ◽  
Cytochrome P450 Enzyme ◽  
Liquid Chromatography Mass Spectrometry ◽  
Wild Type ◽  
Catalytic Activities ◽  
Enzyme Superfamily ◽  
P450 Enzyme

Background: CYP2C19 is an important member of the cytochrome P450 enzyme superfamily. We recently identified 31CYP2C19 alleles in the Han Chinese population; studying the effects of CYP2C19 on drug metabolism can help reduce adverse drug reactions and therapeutic failure. Aim: The aim of this study was to assess the catalytic activities of 31 allelic isoforms and their effects on the metabolism of clomipramine in vitro. Methods: The wild-type and 30 CYP2C19 variants were expressed in insect cells, and each variant was characterized using clomipramine as the substrate. Reactions were performed at 37°C with 5–150 μmol/L substrate for 30 min. By using ultra-high-performance liquid chromatography-mass spectrometry to detect the products, the kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of N-desmethyl clomipramine were determined. Results: Among the CYP2C19 variants tested, CYP2C19*29, L16F, and T130M showed extremely increased intrinsic clearance of clomipramine, CYP2C19*3C, and N277K showed similar intrinsic clearance (Vmax/Km) values with CYP2C19*1, while the intrinsic clearance values of other variants were significantly decreased (from 0.65% to 63.28%). In addition, CYP2C19*3 and 35FS could not be detected because they have no detectable enzyme activity. Conclusions: As the first report of 31 CYP2C19 alleles for clomipramine metabolism, our study could provide corresponding reference for clomipramine for further studies in vivo and offer valuable information relevant to the personalized medicine for CYP2C19-metabolized drug.

Fatty acid ω-hydroxylases of soybean: CYP86A gene expression and aliphatic suberin deposition

Botany ◽  
2020 ◽  
Vol 98 (6) ◽  
pp. 317-326
Author(s):  
Trish L.A. Tully ◽  
Pooja Kaushik ◽  
Jessica O’Connor ◽  
Mark A. Bernards
Keyword(s):  
Gene Expression ◽  
Oleic Acid ◽  
Fatty Acid ◽  
Hairy Root ◽  
Gene Expression Pattern ◽  
Physiological Role ◽  
Specific Gene ◽  
Cytochrome P450 Enzyme ◽  
P450 Enzyme

Suberin has been shown to play a role in protection against and response to stress, including defense against soil-borne pathogens. In soybean, oxidized fatty acids are the most predominant monomers contributing to root suberin, with 18-hydroxy-oleic acid being the most abundant. 18-Hydroxy-oleic acid is predicted to be synthesized by members of the CYP86A family of cytochrome P450 enzyme. Six putative CYP86A genes were identified through phylogenetic analysis of the soybean genome. Two of these, CYP86A37 and CYP86A38 show a root-specific gene expression pattern, and were further analyzed to assess their physiological role in suberin deposition using RNAi knockdown in a hairy root system. Soybean hairy roots were found to be nearly identical to soil-grown roots in terms of anatomy, suberin deposition patterns, and suberin chemistry. The RNAi knockdown of CYP86A37 and CYP86A38 yielded hairy root lines with reduced gene expression and a reduction in the oxidized monomers of suberin, most notably 18-hydroxy-oleic acid. Based on this evidence, CYP86A37 and CYP86A38 are predicted to function as fatty acid ω-hydroxylases in vivo.

Enzymic Cleavage Of In Vivo Formed Maillard-Type Compounds Involved In Haemostasis

1981 ◽  
Author(s):  
L Mester ◽  
L Szabados ◽  
M Mester
Keyword(s):  
Enzyme System ◽  
Basal Membrane ◽  
Enzyme Concentration ◽  
Cytochrome P450 Enzyme ◽  
Microsomal Preparation ◽  
Chemical Hydrolysis ◽  
P450 Enzyme ◽  
Liver Microsomal Preparation

Desoxyfructose derivatives of serotonin (Mester et al.,1975), of haemoglobin (Flückiger and Winterhalter, 1976), of poly-L-lysine (Mester et al., 1975) and of lysine rich histones (Kertesz-Crisba, 1977) are easily formed in vivo by a simple Maillard-type chemical reaction. Some of these compounds interfere with platelet functions (Mester et al.,1976) or contribute to the thickening of the basal membrane of blood vessels (Cerami et al., 1979).While the chemical synthesis of Maillard-type compounds proceeds readily even in vivo, the chemical cleavage of them needs sever conditions which certainly do not exist in vivo (Gottschalk, 1952). However, a slow liberation of serotonin from desoxyfructo-serotonin is observed in vivo, suggesting the existence of an enzyme system for the cleavage of Maillard-type sugar-amine derivatives. In vitro, using a sheep liver microsomal preparation rich in Cytochrome P450 enzyme, the liberation of serotonin is in linear correlation with the enzyme concentration. The cleavage of desoxyfructo-serotonin is activated by NADPH having its optimum at pH=7.4, excluding definitely the occurence of a chemical hydrolysis.Factors interfering with the enzyme system involved in the cleavage of Maillard-type compounds, may also interfere with haemostasis.

scholarly journals Endocannabinoid turnover in GtoPdb v.2021.3

2021 ◽  
Vol 2021 (3) ◽  
Author(s):  
Stephen P.H. Alexander ◽  
Patrick Doherty ◽  
Christopher J. Fowler ◽  
Jürg Gertsch ◽  
Mario Van der Stelt
Keyword(s):  
Cytochrome P450 ◽  
Phospholipase D ◽  
Cytochrome P450 Enzyme ◽  
Facilitated Diffusion ◽  
In Vitro Experiments ◽  
Diacylglycerol Lipase ◽  
P450 Enzyme ◽  
Key Enzyme

The principle endocannabinoids are 2-acylglycerol esters, such as 2-arachidonoylglycerol (2-AG), and N-acylethanolamines, such as anandamide (N-arachidonoylethanolamine, AEA). The glycerol esters and ethanolamides are synthesised and hydrolysed by parallel, independent pathways. Mechanisms for release and re-uptake of endocannabinoids are unclear, although potent and selective inhibitors of facilitated diffusion of endocannabinoids across cell membranes have been developed [28]. FABP5 (Q01469) has been suggested to act as a canonical intracellular endocannabinoid transporter in vivo [17]. For the generation of 2-arachidonoylglycerol, the key enzyme involved is diacylglycerol lipase (DAGL), whilst several routes for anandamide synthesis have been described, the best characterized of which involves N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD, [70]). A transacylation enzyme which forms N-acylphosphatidylethanolamines has been identified as a cytosolic enzyme, PLA2G4E (Q3MJ16) [62]. In vitro experiments indicate that the endocannabinoids are also substrates for oxidative metabolism via cyclooxygenase, lipoxygenase and cytochrome P450 enzyme activities [5, 23, 72].

scholarly journals Endocannabinoid turnover (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

2019 ◽  
Vol 2019 (4) ◽  
Author(s):  
Stephen P.H. Alexander ◽  
Patrick Doherty ◽  
Christopher J. Fowler ◽  
Jürg Gertsch ◽  
Mario Van der Stelt
Keyword(s):  
Cytochrome P450 ◽  
Phospholipase D ◽  
Cytochrome P450 Enzyme ◽  
Facilitated Diffusion ◽  
In Vitro Experiments ◽  
Diacylglycerol Lipase ◽  
P450 Enzyme ◽  
Key Enzyme

The principle endocannabinoids are 2-acylglycerol esters, such as 2-arachidonoylglycerol (2-AG), and N-acylethanolamines, such as anandamide (N-arachidonoylethanolamine, AEA). The glycerol esters and ethanolamides are synthesised and hydrolysed by parallel, independent pathways. Mechanisms for release and re-uptake of endocannabinoids are unclear, although potent and selective inhibitors of facilitated diffusion of endocannabinoids across cell membranes have been developed [19]. FABP5 (Q01469) has been suggested to act as a canonical intracellular endocannabinoid transporter in vivo [12]. For the generation of 2-arachidonoylglycerol, the key enzyme involved is diacylglycerol lipase (DAGL), whilst several routes for anandamide synthesis have been described, the best characterized of which involves N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD, [49]). A transacylation enzyme which forms N-acylphosphatidylethanolamines has recently been identified as a cytosolic enzyme, PLA2G4E (Q3MJ16) [43]. In vitro experiments indicate that the endocannabinoids are also substrates for oxidative metabolism via cyclooxygenase, lipoxygenase and cytochrome P450 enzyme activities [4, 16, 51].

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