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20 pages, 5757 KiB

Open AccessArticle

Bielectrode Strategy for Determination of CYP2E1 Catalytic Activity: Electrodes with Bactosomes and Voltammetric Determination of 6-Hydroxychlorzoxazone

by Alexey V. Kuzikov, Rami A. Masamrekh, Tatiana A. Filippova, Anastasiya M. Tumilovich, Natallia V. Strushkevich, Andrei A. Gilep, Yulia Yu. Khudoklinova and Victoria V. Shumyantseva

Biomedicines 2024, 12(1), 152; https://doi.org/10.3390/biomedicines12010152 - 11 Jan 2024

Cited by 3 | Viewed by 1466

Abstract

We describe a bielectrode system for evaluation of the electrocatalytic activity of cytochrome P450 2E1 (CYP2E1) towards chlorzoxazone. One electrode of the system was employed to immobilize Bactosomes with human CYP2E1, cytochrome P450 reductase (CPR), and cytochrome b₅ (cyt b₅). [...] Read more.

We describe a bielectrode system for evaluation of the electrocatalytic activity of cytochrome P450 2E1 (CYP2E1) towards chlorzoxazone. One electrode of the system was employed to immobilize Bactosomes with human CYP2E1, cytochrome P450 reductase (CPR), and cytochrome b₅ (cyt b₅). The second electrode was used to quantify CYP2E1-produced 6-hydroxychlorzoxazone by its direct electrochemical oxidation, registered using square-wave voltammetry. Using this system, we determined the steady-state kinetic parameters of chlorzoxazone hydroxylation by CYP2E1 of Bactosomes immobilized on the electrode: the maximal reaction rate (V_max) was 1.64 ± 0.08 min⁻¹, and the Michaelis constant (K_M) was 78 ± 9 μM. We studied the electrochemical characteristics of immobilized Bactosomes and have revealed that electron transfer from the electrode occurs both to the flavin prosthetic groups of CPR and the heme iron ions of CYP2E1 and cyt b₅. Additionally, it has been demonstrated that CPR has the capacity to activate CYP2E1 electrocatalytic activity towards chlorzoxazone, likely through intermolecular electron transfer from the electrochemically reduced form of CPR to the CYP2E1 heme iron ion. Full article

(This article belongs to the Special Issue Cytochrome P450 (CYP) in Health and Disease)

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19 pages, 18696 KiB

Open AccessArticle

Molecular Cloning, Heterologous Expression, Purification, and Evaluation of Protein–Ligand Interactions of CYP51 of Candida krusei Azole-Resistant Fungal Strain

by Tatsiana V. Tsybruk, Leonid A. Kaluzhskiy, Yuri V. Mezentsev, Tatyana N. Makarieva, Kseniya M. Tabakmaher, Natalia V. Ivanchina, Pavel S. Dmitrenok, Alexander V. Baranovsky, Andrei A. Gilep and Alexis S. Ivanov

Biomedicines 2023, 11(11), 2873; https://doi.org/10.3390/biomedicines11112873 - 24 Oct 2023

Cited by 1 | Viewed by 1607

Abstract

Due to the increasing prevalence of fungal diseases caused by fungi of the genus Candida and the development of pathogen resistance to available drugs, the need to find new effective antifungal agents has increased. Azole antifungals, which are inhibitors of sterol-14α-demethylase or CYP51, [...] Read more.

Due to the increasing prevalence of fungal diseases caused by fungi of the genus Candida and the development of pathogen resistance to available drugs, the need to find new effective antifungal agents has increased. Azole antifungals, which are inhibitors of sterol-14α-demethylase or CYP51, have been widely used in the treatment of fungal infections over the past two decades. Of special interest is the study of C. krusei CYP51, since this fungus exhibit resistance not only to azoles, but also to other antifungal drugs and there is no available information about the ligand-binding properties of CYP51 of this pathogen. We expressed recombinant C. krusei CYP51 in E. coli cells and obtained a highly purified protein. Application of the method of spectrophotometric titration allowed us to study the interaction of C. krusei CYP51 with various ligands. In the present work, the interaction of C. krusei CYP51 with azole inhibitors, and natural and synthesized steroid derivatives was evaluated. The obtained data indicate that the resistance of C. krusei to azoles is not due to the structural features of CYP51 of this microorganism, but rather to another mechanism. Promising ligands that demonstrated sufficiently strong binding in the micromolar range to C. krusei CYP51 were identified, including compounds 99 (Kd = 1.02 ± 0.14 µM) and Ch-4 (Kd = 6.95 ± 0.80 µM). The revealed structural features of the interaction of ligands with the active site of C. krusei CYP51 can be taken into account in the further development of new selective modulators of the activity of this enzyme. Full article

(This article belongs to the Special Issue Cytochrome P450 (CYP) in Health and Disease)

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10 pages, 269 KiB

Open AccessArticle

Association of CYP2C9*2 Allele with Sulphonylurea-Induced Hypoglycaemia in Type 2 Diabetes Mellitus Patients: A Pharmacogenetic Study in Pakistani Pashtun Population

by Asif Jan, Muhammad Saeed, Ramzi A. Mothana, Tahir Muhammad, Naveed Rahman, Abdullah R. Alanzi and Rani Akbar

Biomedicines 2023, 11(8), 2282; https://doi.org/10.3390/biomedicines11082282 - 16 Aug 2023

Cited by 3 | Viewed by 1811

Abstract

Polymorphism in cytochrome P450 (CYP) 2C9 enzyme is known to cause significant inter-individual differences in drug response and occurrence of adverse drug reactions. Different alleles of the CYP2C9 gene have been identified, but the notable alleles responsible for reduced enzyme activity are CYP2C9*2 [...] Read more.

Polymorphism in cytochrome P450 (CYP) 2C9 enzyme is known to cause significant inter-individual differences in drug response and occurrence of adverse drug reactions. Different alleles of the CYP2C9 gene have been identified, but the notable alleles responsible for reduced enzyme activity are CYP2C9*2 and CYP2C9*3. No pharmacogenetic data are available on CYP2C9*2 and CYP2C9*3 alleles in the Pakistani population. In Pakistan, pharmacogenetics, which examines the relationship between genetic factors and drug response, are in the early stages of development. We, for the first time, investigated the association between the CYP2C9 variant alleles CYP2C9*2 and CYP2C9*3 and the incidence of hypoglycaemia in patients with Type 2 diabetes mellitus (T2DM) receiving sulphonylurea medications. A total of n = 400 individuals of Pashtun ethnicity were recruited from 10 different districts of Khyber Pakhtunkhwa, Pakistan to participate in the study. The study participants were divided into two distinct groups: the case group (n = 200) and the control group (n = 200). The case group consisted of individuals with T2DM who were receiving sulphonylurea medications and experienced hypoglycaemia with it, whereas the control group included individuals with T2DM who were receiving sulphonylurea medication but did not experience sulphonylurea-induced hypoglycaemia (SIH). Blood samples were obtained from study participants following informed consent. DNA was isolated from whole blood samples using a Wiz-Prep DNA extraction kit. Following DNA isolation, CYP2C9 alleles were genotyped using MassARRAY sequencing platform at the Centre of Genomics at the Rehman Medical Institute (RMI). The frequency of CYP2C9*2 (low-activity allele) was more frequent in the diabetic patients with SIH compared to the control group (17.5% vs. 6.0%, p = 0.021). The frequency of its corresponding genotype CYP2C9*1/*2 was higher in cases compared to the control group (10% vs. 6% with p = 0.036); the same was true for genotype CYP2C9*2/*2 (7% vs. 3.5% with p = 0.028). Logistic regression analysis evidenced potential association of CYP2C9*2 allele and its genotypes with SIH. When adjusted for confounding factors such as age, weight, sex, mean daily dose of sulphonylurea, and triglyceride level, the association between the CYP2C9*2 allele and hypoglycaemia remained consistent. Confounding factors played no role in SIH (insignificant p-value) because both groups (cases and controls) were closely matched in term of age, weight, sex, mean daily dose of sulphonylurea, and triglyceride levels. Our study suggests that genetic information about a patient’s CYP2C9 gene/enzyme can potentially assist physicians in prescribing the most suitable and safest drug, based on their genetic make-up. Full article

(This article belongs to the Special Issue Cytochrome P450 (CYP) in Health and Disease)

11 pages, 1471 KiB

Open AccessArticle

Artemisia annua L. Extracts Irreversibly Inhibit the Activity of CYP2B6 and CYP3A4 Enzymes

by Martin Kondža, Marta Mandić, Ivona Ivančić, Sanda Vladimir-Knežević and Ivica Brizić

Biomedicines 2023, 11(1), 232; https://doi.org/10.3390/biomedicines11010232 - 16 Jan 2023

Cited by 2 | Viewed by 3605

Abstract

Artemisia annua L. has long been known for its medicinal properties and isolation of ingredients whose derivatives are used for therapeutic purposes. The CYP2B6 and CYP3A4 enzymes belong to a large family of cytochrome P450 enzymes. These enzymes are involved in the metabolism [...] Read more.

Artemisia annua L. has long been known for its medicinal properties and isolation of ingredients whose derivatives are used for therapeutic purposes. The CYP2B6 and CYP3A4 enzymes belong to a large family of cytochrome P450 enzymes. These enzymes are involved in the metabolism of drugs and other xeonobiotics. It is known that various compounds can induce or inhibit the activity of these enzymes. The aim of this study was to investigate the nature of the inhibitory effect of Artemisia annua extract on CYP2B6 and CYP3A4 enzymes, as well as the type of inhibition, the presence of reversible or pseudo-irreversible inhibition, and the possible heme destruction. The methanolic extract of Artemisia annua showed an inhibitory effect on CYP2B6 (by almost 90%) and CYP3A4 enzymes (by almost 70%). A significant decrease in heme concentration by 46.8% and 38.2% was observed in different assays. These results clearly indicate that the studied plant extracts significantly inhibited the activity of CYP2B6 and CYP3A4 enzymes. Moreover, they showed irreversible inhibition, which is even more important for possible interactions with drugs and dietary supplements. Full article

(This article belongs to the Special Issue Cytochrome P450 (CYP) in Health and Disease)

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18 pages, 3143 KiB

Open AccessArticle

Transcription Factors and ncRNAs Associated with CYP3A Expression in Human Liver and Small Intestine Assessed with Weighted Gene Co-Expression Network Analysis

by Huina Huang, Siqi Zhang, Xiaozhen Wen, Wolfgang Sadee, Danxin Wang, Siyao Yang and Liang Li

Biomedicines 2022, 10(12), 3061; https://doi.org/10.3390/biomedicines10123061 - 28 Nov 2022

Cited by 3 | Viewed by 2028

Abstract

CYP3A4, CYP3A5, and CYP3A7, which are located in a multigene locus (CYP3A), play crucial roles in drug metabolism. To understand the highly variable hepatic expression of CYP3As, regulatory network analyses have focused on transcription factors (TFs). Since long non-coding RNAs (lncRNAs) [...] Read more.

CYP3A4, CYP3A5, and CYP3A7, which are located in a multigene locus (CYP3A), play crucial roles in drug metabolism. To understand the highly variable hepatic expression of CYP3As, regulatory network analyses have focused on transcription factors (TFs). Since long non-coding RNAs (lncRNAs) likely contribute to such networks, we assessed the regulatory effects of both TFs and lncRNAs on CYP3A expression in the human liver and small intestine, main organs of CYP3A expression. Using weighted gene co-expression network analysis (WGCNA) of GTEx v8 RNA expression data and multiple stepwise regression analysis, we constructed TF-lncRNA-CYP3A co-expression networks. Multiple lncRNAs and TFs displayed robust associations with CYP3A expression that differed between liver and small intestines (LINC02499, HNF4A-AS1, AC027682.6, LOC102724153, and RP11-503C24.6), indicating that lncRNAs contribute to variance in CYP3A expression in both organs. Of these, HNF4A-AS1 had been experimentally demonstrated to affect CYP3A expression. Incorporating ncRNAs into CYP3A expression regulatory network revealed additional candidate TFs associated with CYP3A expression. These results serve as a guide for experimental studies on lncRNA-TF regulation of CYP3A expression in the liver and small intestines. Full article

(This article belongs to the Special Issue Cytochrome P450 (CYP) in Health and Disease)

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Review

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22 pages, 5226 KiB

Open AccessReview

Targeting Cytochrome P450 Enzymes in Ovarian Cancers: New Approaches to Tumor-Selective Intervention

by Yousef M. Al-saraireh, Fatemah O. F. O. Alshammari, Omar H. Abu-azzam, Sa’ed M. Al-dalain, Yahya M. Al-sarayra, Mansour Haddad, Hafiz Makeen, Aiman Al-Qtaitat, Mohammad Almermesh and Sameeh A. Al-sarayreh

Biomedicines 2023, 11(11), 2898; https://doi.org/10.3390/biomedicines11112898 - 26 Oct 2023

Cited by 4 | Viewed by 2187

Abstract

Over the past decade, there have been significant developments in treatment for ovarian cancer, yet the lack of targeted therapy with few side effects still represents a major issue. The cytochrome P450 (CYP) enzyme family plays a vital role in the tumorigenesis process [...] Read more.

Over the past decade, there have been significant developments in treatment for ovarian cancer, yet the lack of targeted therapy with few side effects still represents a major issue. The cytochrome P450 (CYP) enzyme family plays a vital role in the tumorigenesis process and metabolism of drugs and has a negative impact on therapy outcomes. Gaining more insight into CYP expression is crucial to understanding the pathophysiology of ovarian cancer since many isoforms are essential to the metabolism of xenobiotics and steroid hormones, which drive the disease’s development. To the best of our knowledge, no review articles have documented the intratumoral expression of CYPs and their implications in ovarian cancer. Therefore, the purpose of this review is to provide a clear understanding of differential CYP expression in ovarian cancer and its implications for the prognosis of ovarian cancer patients, together with the effects of CYP polymorphisms on chemotherapy metabolism. Finally, we discuss opportunities to exploit metabolic CYP expression for the development of novel therapeutic methods to treat ovarian cancer. Full article

(This article belongs to the Special Issue Cytochrome P450 (CYP) in Health and Disease)

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18 pages, 1888 KiB

Open AccessReview

Xenobiotic-Metabolizing Enzymes in Trematodes

by Viatcheslav Mordvinov and Maria Pakharukova

Biomedicines 2022, 10(12), 3039; https://doi.org/10.3390/biomedicines10123039 - 24 Nov 2022

Cited by 3 | Viewed by 2084

Abstract

Trematode infections occur worldwide causing considerable deterioration of human health and placing a substantial financial burden on the livestock industry. The hundreds of millions of people afflicted with trematode infections rely entirely on only two drugs (praziquantel and triclabendazole) for treatment. An understanding [...] Read more.

Trematode infections occur worldwide causing considerable deterioration of human health and placing a substantial financial burden on the livestock industry. The hundreds of millions of people afflicted with trematode infections rely entirely on only two drugs (praziquantel and triclabendazole) for treatment. An understanding of anthelmintic biotransformation pathways in parasites should clarify factors that can modulate therapeutic potency of anthelmintics currently in use and may lead to the discovery of synergistic compounds for combination treatments. Despite the pronounced epidemiological significance of trematodes, there is still no adequate understanding of the functionality of their metabolic systems, including xenobiotic-metabolizing enzymes. The review is focused on the structure and functional significance of the xenobiotic-metabolizing system in trematodes. Knowledge in this field can solve practical problems related to the search for new targets for antiparasitic therapy based on a focused action on certain elements of the parasite’s metabolic system. Knowledge of the functionality of this system is required to understand the adaptation of the biochemical processes of parasites residing in the host and mechanisms of drug resistance development, as well as to select a promising molecular target for the discovery and development of new anthelmintic drugs. Full article

(This article belongs to the Special Issue Cytochrome P450 (CYP) in Health and Disease)

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52 pages, 2907 KiB

Open AccessReview

The Role of CYP3A in Health and Disease

by Lyubov S. Klyushova, Maria L. Perepechaeva and Alevtina Y. Grishanova

Biomedicines 2022, 10(11), 2686; https://doi.org/10.3390/biomedicines10112686 - 24 Oct 2022

Cited by 27 | Viewed by 6137

Abstract

CYP3A is an enzyme subfamily in the cytochrome P450 (CYP) superfamily and includes isoforms CYP3A4, CYP3A5, CYP3A7, and CYP3A43. CYP3A enzymes are indiscriminate toward substrates and are unique in that these enzymes metabolize both endogenous compounds and diverse xenobiotics (including drugs); almost the [...] Read more.

CYP3A is an enzyme subfamily in the cytochrome P450 (CYP) superfamily and includes isoforms CYP3A4, CYP3A5, CYP3A7, and CYP3A43. CYP3A enzymes are indiscriminate toward substrates and are unique in that these enzymes metabolize both endogenous compounds and diverse xenobiotics (including drugs); almost the only common characteristic of these compounds is lipophilicity and a relatively large molecular weight. CYP3A enzymes are widely expressed in human organs and tissues, and consequences of these enzymes’ activities play a major role both in normal regulation of physiological levels of endogenous compounds and in various pathological conditions. This review addresses these aspects of regulation of CYP3A enzymes under physiological conditions and their involvement in the initiation and progression of diseases. Full article

(This article belongs to the Special Issue Cytochrome P450 (CYP) in Health and Disease)

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