RESUMEN

OBJECTIVES: Dose exposure is considered relevant for drug-associated falls in older adults, pointing to an importance of drug metabolism. Aim was to analyze individual factors altering drug metabolism such as enzyme saturation by drug exposure and pharmacogenetics in the context of drug-associated falls. DESIGN: Prospective population-based study (ActiFE-Ulm study). SETTING AND PARTICIPANTS: Community-dwelling older adults. METHODS: Focus was laid on the metabolism by polymorphic cytochrome P450 (CYP) enzymes CYP2C19, 2C9, and 2D6. Relevant variants of pharmacogenes were analyzed. Logistic binary regression analysis was used to calculate odds ratios (ORs) and 95% CIs for falls observed prospectively over a 1-year period with drug metabolism characteristics. RESULTS: In total, 1377 participants were included in the analysis. Although the phenotype predicted by the genotype was not, the use of drugs metabolized by CYP2C19 was associated with falls. Drugs not known as fall risk-increasing drugs (FRIDs; ie, non-FRIDs), but metabolized by CYP2C19, showed an OR of 1.46 (1.11-1.93) in adjusted analysis. Significant effect modification was observed for a reduced CYP2C19 activity phenotype with non-FRIDs metabolized by CYP2C19. CONCLUSIONS AND IMPLICATIONS: This study suggests an association between the occurrence of falls in older adults and the metabolic capacity of CYP2C19. Thus, an important step toward prevention of falls might be to personalize dosage and treatment length of the main drug classes known to be CYP2C19 substrates, such as many antidepressants, opioids, and sedatives, but also proton pump inhibitors in particular in poor and intermediate metabolizers.

RESUMEN

Vanillin (VAN) is a common flavoring agent that can cause liver damage when ingested in large amounts. Nevertheless, the precise processes responsible for its toxicity remain obscure. The present research aimed to examine the metabolic activation of VAN and establish a potential correlation between its reactive metabolites and its cytotoxicity. In rat liver microsomes incubated with VAN, reduced glutathione/N-acetylcysteine (GSH/NAC), and nicotinamide adenine dinucleotide phosphate (NADPH), two conjugates formed from GSH and one conjugate derived from NAC were identified. We also discovered one GSH conjugate in both the bile obtained from rats and the rat primary hepatocytes that were subjected to VAN exposure. Additionally, the NAC conjugate exerted in the urine of VAN-treated rats was observed. These results indicate that a quinone intermediate was produced from VAN both in vitro and in vivo. Next, we identified CYP3A as the main enzyme that initiated the bioactive pathway of VAN. After the activity of CYP3A was selectively inhibited by ketoconazole (KTZ), the generation of the GSH conjugate declined in hepatocytes exposed to VAN. Furthermore, the vulnerability to VAN-induced toxicity was alleviated by KTZ in hepatocytes. Thus, we propose that the cytotoxicity of VAN may derive from metabolic activation triggered by CYP3A.

Asunto(s)

Activación Metabólica , Benzaldehídos , Citocromo P-450 CYP3A , Hepatocitos , Microsomas Hepáticos , Ratas Sprague-Dawley , Animales , Benzaldehídos/metabolismo , Benzaldehídos/farmacología , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Ratas , Masculino , Citocromo P-450 CYP3A/metabolismo , Citocromo P-450 CYP3A/genética , Microsomas Hepáticos/metabolismo , Microsomas Hepáticos/efectos de los fármacos , Glutatión/metabolismo , Aromatizantes/metabolismo , Aromatizantes/química , Aromatizantes/toxicidad

RESUMEN

The recent re-emergence and the increasing popularity of nitazenes, a group of new synthetic opioids (NSO) that belong to the benzimidazole chemical class, has raised public health concerns. As a class of potential opioid analgesic agents whose development was discontinued in the 1960s due to their high potential for abuse, very little is known about their metabolism and physiologic disposition. In the current study, three nitazenes-butonitazene, isotonitazene and protonitaze were incubated in human liver microsomes (HLM), human S9 (HS9) fractions and recombinant cytochrome P450 enzymes. All three nitazenes were rapidly metabolized in both HLM and HS9 with over 95% depletion within 60 min. In HLM, butonitazene, isotonitazene and protonitazene had in vitro intrinsic clearance (CLint) (µL/min/mg protein) values of 309, 221 and 216 respectively compared to 150 of verapamil, the positive control. In HS9, CLint values were 217, 139, and 150 for butonitazene, isotonitazene and protonitazene respectively compared to only 35 for testosterone, the control probe substrate. Putative metabolite identified from this study include products of hydroxylation, desethylation, dealkylation, desethylation followed by dealkylation, and desethylation followed by hydroxylation. The metabolic phenotyping showed CYP2D6, CYP2B6 and CYP2C8 and the major hepatic enzymes responsible for the metabolism of nitazenes. Within 30 min of incubation, CYP2D6 depleted butonitazene (99%), isotonitazene (72%) and butonitazene (100%) significantly. The rapid metabolism of nitazenes may be an important factor in accurate and timely detections and quantitation of the unchanged drugs in human matrices following intoxication or in forensic analysis. The involvement of multiple polymorphic CYPs in their metabolism may play important roles in the susceptibility to intoxication and/or addiction, depending on the activity of the metabolites.

RESUMEN

During gliotoxin biosynthesis in fungi, the cytochrome P450 GliF enzyme catalyzes an unusual C-N ring-closure step while also an aromatic ring is hydroxylated in the same reaction cycle, which may have relevance to drug synthesis reactions in biotechnology. However, as the details of the reaction mechanism are still controversial, no applications have been developed yet. To resolve the mechanism of gliotoxin biosynthesis and gain insight into the steps leading to ring-closure, we ran a combination of molecular dynamics and density functional theory calculations on the structure and reactivity of P450 GliF and tested a range of possible reaction mechanisms, pathways and models. The calculations show that, rather than hydrogen atom transfer from the substrate to Compound I, an initial proton transfer transition state is followed by a fast electron transfer en route to the radical intermediate, and hence a non-synchronous hydrogen atom abstraction takes place. The radical intermediate then reacts by OH rebound to the aromatic ring to form a biradical in the substrate that, through ring-closure between the radical centers, gives gliotoxin products. Interestingly, the structure and energetics of the reaction mechanisms appear little affected by the addition of polar groups to the model and hence we predict that the reaction can be catalyzed by other P450 isozymes that also bind the same substrate. Alternative pathways, such as a pathway starting with an electrophilic attack on the arene to form an epoxide, are high in energy and are ruled out.

Asunto(s)

Sistema Enzimático del Citocromo P-450 , Gliotoxina , Oxidación-Reducción , Gliotoxina/biosíntesis , Gliotoxina/metabolismo , Gliotoxina/química , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/química , Simulación de Dinámica Molecular

RESUMEN

Infigratinib, an oral FGFR inhibitor for advanced cholangiocarcinoma, yielded two active metabolites, BHS697 and CQM157, with similar receptor affinity. Our study characterized P450s that are responsible for the metabolism of infigratinib to its two major active metabolites, BHS697 and CQM157. In vitro inhibition of P450s and UGTs by infigratinib, BHS697 or CQM157 was further investigated. The unbound apparent Km values for metabolism of infigratinib to BHS697 by HLM, human recombinant CYP2C8, CYP2C19, CYP2D6 and CYP3A4 enzymes are 4.47, 0.65, 2.50, 30.6 and 2.08 µM, while Vmax values are 90.0 pmol/min/mg protein, 0.13, 0.027, 0.81, and 0.56 pmol/min/pmol protein, respectively. The unbound apparent Km value for metabolism of infigratinib to CQM157 by HLM is 0.049 µM, while the Vmax value is 0.32 pmol/min/mg protein respectively. In HLM, infigratinib displayed moderate inhibition of CYP3A4 and CYP2C19 and weak or negligible inhibition of other P450 isoforms. BHS697 exhibited weak inhibition of CYP2B6, CYP2C9, CYP2C19 and CYP3A4, and no inhibition of CYP2C8 and CYP2D6. CQM157 moderately inhibited CYP2C9 and CYP3A4, and weakly or negligibly inhibited other P450 isoforms. Regarding UGTs, infigratinib moderately inhibited UGT1A4 and weakly inhibited UGT1A1, respectively. BHS697 weakly inhibited UGT1A1. In contrast, CQM157 moderately inhibited both UGT1A1 and UGT1A4. Our findings provide novel insights into the metabolism of and potential DDIs implicating infigratinib.

Asunto(s)

Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450 , Glucuronosiltransferasa , Humanos , Sistema Enzimático del Citocromo P-450/metabolismo , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Inhibidores Enzimáticos del Citocromo P-450/metabolismo , Glucuronosiltransferasa/metabolismo , Glucuronosiltransferasa/antagonistas & inhibidores , Microsomas Hepáticos/metabolismo , Microsomas Hepáticos/efectos de los fármacos , Pirimidinas/farmacología , Pirimidinas/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/metabolismo , Compuestos de Fenilurea

RESUMEN

The aim of this study was to investigate the frequency distribution of the cytochrome P450 (CYP450) enzymes, CYP2D6 and CYP2C19, and the form of tamoxifen metabolisation in premenopausal patients with breast cancer in the Han and Uygur ethnic groups of Xinjiang to guide rational clinical drug use. A total of 125 Han patients and 121 Uygur patients with premenopausal hormone-receptor-positive breast cancer treated at the Xinjiang Uygur Autonomous Region Cancer Hospital between 1 June 2011 and 1 December 2013 were selected. The common mutation sites in CYP450 were analysed using TaqMan® minor groove binder technology. Genetic testing was performed to determine other metabolic types of tamoxifen, and the genotypes and metabolic types were compared using a Chi-squared test. Between the Han and Uygur groups, there were significant differences in the frequencies of the CYP2D6 (*10/*10) and CYP2C19 (*1/*1) genotypes, with P-values of 0.002 and 0.015, respectively. Genotypes of CYP2D6 (*1/*1), CYP2D6 (*1/*5), CYP2D6 (*5/*5), CYP2D6 (*5/*10) and CYP2C19 (*3/*3) were expressed in the two patient groups, and the difference was not statistically significant (P > 0.05). In the Han patients, the proportions of extensive, intermediate and poor metabolisers of tamoxifen were 72, 24 and 4%, respectively, whereas those in the Uygur patients were 76.9, 17.4 and 5.7%, respectively, with no significant difference (P > 0.05). In conclusion, There were partial differences in the CYP2D6 and CYP2C19 gene polymorphisms of CYP450 between the Han and Uygur patients with premenopausal breast cancer, but there was no significant difference between the CYP2D6 and CYP2C19 phenotypes. Further research is needed to determine the relationship between the enzyme genetic differences of CYP450 and the pharmacokinetics and efficacy of tamoxifen. Although there were some differences in genotypes, these did not result in differences in the predicted tamoxifen metabolisation phenotype between the Han and Uygur patients with breast cancer. Therefore, the doses should be adjusted according to the individual genotype data.

RESUMEN

Mycotoxins are known environmental pollutants that may contaminate food and feed chains. Some mycotoxins are regulated in many countries to limit the trading of contaminated and harmful commodities. However, the so-called emerging mycotoxins are poorly understood and need to be investigated further. Fusaric acid is an emerging mycotoxin, noxious to plants and animals, but is known to be less toxic to plants when hydroxylated. The detoxification routes effective in animals have not been elucidated yet. In this context, this study integrated in silico and in vitro techniques to discover potential bioremediation routes to turn fusaric acid to its less toxic metabolites. The toxicodynamics of these forms in humans have also been addressed. An in silico screening process, followed by molecular docking and dynamics studies, identified CYP199A4 from the bacterium Rhodopseudomonas palustris HaA2 as a potential fusaric acid biotransforming enzyme. Its activity was confirmed in vitro. However, the effect of hydroxylation seemed to have a limited impact on the modelled toxicodynamics against human targets. This study represents a starting point to develop a hybrid in silico/in vitro pipeline to find bioremediation agents for other food, feed and environmental contaminants.

Asunto(s)

Ácido Fusárico , Micotoxinas , Animales , Humanos , Ácido Fusárico/toxicidad , Simulación del Acoplamiento Molecular , Micotoxinas/toxicidad , Alimentación Animal/análisis , Sistema Enzimático del Citocromo P-450

RESUMEN

The cytochrome P450 family of heme metalloenzymes (CYPs) catalyse important biological monooxygenation reactions. Mycobacterium marinum contains a gene encoding a CYP105Q4 enzyme of unknown function. Other members of the CYP105 CYP family have key roles in bacterial metabolism including the synthesis of secondary metabolites. We produced and purified the cytochrome P450 enzyme CYP105Q4 to enable its characterization. Several nitrogen-donor atom-containing ligands were found to bind to CYP105Q4 generating type II changes in the UV-vis absorbance spectrum. Based on the UV-vis absorbance spectra none of the potential substrate ligands we tested with CYP105Q4 were able to displace the sixth distal aqua ligand from the heme, though there was evidence for binding of oleic acid and amphotericin B. The crystal structure of CYP105Q4 in the substrate-free form was determined in an open conformation. A computational structural similarity search (Dali) was used to find the most closely related characterized relatives within the CYP105 family. The structure of CYP105Q4 enzyme was compared to the GfsF CYP enzyme from Streptomyces graminofaciens which is involved in the biosynthesis of a macrolide polyketide. This structural comparison to GfsF revealed conformational changes in the helices and loops near the entrance to the substrate access channel. A disordered B/C loop region, usually involved in substrate recognition, was also observed.

Asunto(s)

Mycobacterium marinum , Mycobacterium marinum/genética , Mycobacterium marinum/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Estructura Secundaria de Proteína , Macrólidos/química , Macrólidos/metabolismo , Hemo/química , Cristalografía por Rayos X

RESUMEN

Zearalenone (ZEN) is a mycoestrogen produced by Fusarium fungi. ZEN is a frequent contaminant in cereal-based products, representing significant health threat. The major reduced metabolites of ZEN are α-zearalenol (α-ZEL) and ß-zearalenol (ß-ZEL). Since the toxicokinetic interactions of ZEN/ZELs with cytochrome P450 enzymes (CYPs) and organic anion transporting polypeptides (OATPs) have been barely characterized, we examined these interactions applying in vitro models. ZEN and ZELs were relatively strong inhibitors of CYP3A4 and moderate inhibitors of CYP1A2 and CYP2C9. Both CYP1A2 and CYP3A4 decreased ZEN and ß-ZEL concentrations in depletion assays, while only CYP1A2 reduced α-ZEL levels. OATPs tested were strongly or moderately inhibited by ZEN and ZELs; however, these mycotoxins did not show higher cytotoxicity in OATP-overexpressing cells. Our results help the deeper understanding of the toxicokinetic/pharmacokinetic interactions of ZEN, α-ZEL, and ß-ZEL.

Asunto(s)

Micotoxinas , Transportadores de Anión Orgánico , Zearalenona , Zeranol/análogos & derivados , Zearalenona/toxicidad , Citocromo P-450 CYP1A2 , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450 , Péptidos

RESUMEN

AIMS: Dolutegravir increases serum creatinine by inhibiting its renal tubular secretion and elimination. We investigated determinants of early changes in serum creatinine in a southern African cohort starting first-line dolutegravir-based antiretroviral therapy (ART). METHODS: We conducted a secondary analysis of data from participants in a randomized controlled trial of dolutegravir, emtricitabine and tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide fumarate (TAF) (ADVANCE, NCT03122262). We assessed clinical, pharmacokinetic and genetic factors associated with change in serum creatinine from baseline to Week 4 using linear regression models adjusted for age, sex, baseline serum creatinine, HIV-1 RNA concentration, CD4 T-cell count, total body weight and co-trimoxazole use. RESULTS: We included 689 participants, of whom 470 had pharmacokinetic data and 315 had genetic data. Mean change in serum creatinine was 11.3 (SD 9.9) µmol.L-1. Factors that were positively associated with change in serum creatinine at Week 4 were increased log dolutegravir area under the 24-h concentration-time curve (change in creatinine coefficient [ß] = 2.78 µmol.L-1 [95% confidence interval (CI) 0.54, 5.01]), TDF use (ß = 2.30 [0.53, 4.06]), male sex (ß = 5.20 [2.92, 7.48]), baseline serum creatinine (ß = -0.22 [-0.31, -0.12]) and UGT1A1 rs929596 A→G polymorphism with a dominant model (ß = -2.33 [-4.49, -0.17]). The latter did not withstand correction for multiple testing. CONCLUSIONS: Multiple clinical and pharmacokinetic factors were associated with early change in serum creatinine in individuals initiating dolutegravir-based ART. UGT1A1 polymorphisms may play a role, but further research on genetic determinants is needed.

Asunto(s)

Creatinina , Infecciones por VIH , Compuestos Heterocíclicos con 3 Anillos , Oxazinas , Piperazinas , Piridonas , Humanos , Piridonas/farmacocinética , Oxazinas/farmacocinética , Oxazinas/uso terapéutico , Compuestos Heterocíclicos con 3 Anillos/farmacocinética , Compuestos Heterocíclicos con 3 Anillos/uso terapéutico , Compuestos Heterocíclicos con 3 Anillos/efectos adversos , Compuestos Heterocíclicos con 3 Anillos/administración & dosificación , Piperazinas/farmacocinética , Piperazinas/uso terapéutico , Masculino , Creatinina/sangre , Femenino , Infecciones por VIH/tratamiento farmacológico , Adulto , Sudáfrica , Persona de Mediana Edad , Glucuronosiltransferasa/genética , Fármacos Anti-VIH/farmacocinética , Fármacos Anti-VIH/uso terapéutico , Fármacos Anti-VIH/efectos adversos , VIH-1/genética , VIH-1/efectos de los fármacos , Inhibidores de Integrasa VIH/farmacocinética , Inhibidores de Integrasa VIH/uso terapéutico , Inhibidores de Integrasa VIH/efectos adversos , Tenofovir/farmacocinética , Tenofovir/uso terapéutico , Emtricitabina/uso terapéutico , Emtricitabina/farmacocinética , Polimorfismo de Nucleótido Simple

RESUMEN

Vector-borne infectious diseases are still an important global health problem. Malaria is the most important among them, mainly pediatric, life-threatening disease. Malaria and other vector-borne disorders caused by parasites, bacteria, and viruses have a strong impact on public health and significant economic costs. Most vector-borne diseases could be prevented by vector control, with attention to the ecological and biodiversity conservation aspects. Chemical control with pesticides and insecticides is widely used as a measure of prevention although increasing resistance to insecticides is a serious issue in vector control. Metabolic resistance is the most common mechanism and poses a big challenge. Insect enzyme systems, including monooxygenase CYP P450 enzymes, are employed by vectors mainly to metabolize insecticides thus causing resistance. The discovery and application of natural specific inhibitors/blockers of vector P450 enzymes as synergists for commonly used pesticides will contribute to the "greening" of insecticides. Besides vector CYPs, host CYP enzymes could also be exploited to fight against vector-borne diseases: using mostly their detoxifying properties and involvement in the immune response. Here, we review published research data on P450 enzymes from all players in vector-borne infections, that is, pathogens, vectors, and hosts, regarding the potential role of CYPs in disease. We discuss strategies on how to exploit cytochromes P450 in vector-borne disease control.

Asunto(s)

Enfermedades Transmisibles , Insecticidas , Malaria , Niño , Humanos , Insecticidas/farmacología , Resistencia a los Insecticidas , Malaria/prevención & control , Sistema Enzimático del Citocromo P-450/genética

RESUMEN

Ciprofol is a novel intravenous anesthetic agent. Its major glucuronide metabolite, M4, is found in plasma and urine. However, the specific isoforms of UDP-glucuronosyltransferases (UGTs) that metabolize ciprofol to M4 remain unknown. This study systematically characterized UGTs that contribute to the formation of M4 using human liver microsomes (HLM), human intestinal microsomes (HIM), and human recombinant UGTs. The inhibitory potential of ciprofol and M4 against major human UGTs and cytochrome P450 enzymes (P450s) was also explored. In vitro-in vivo extrapolation (IVIVE) and physiologically-based pharmacokinetic (PBPK) simulations were performed to predict potential in vivo drug-drug interactions (DDIs) caused by ciprofol. Glucuronidation of ciprofol followed Michaelis-Menten kinetics in both HLM and HIM with apparent Km values of 345 and 412 µM, Vmax values of 2214 and 444 nmol min-1·mg protein-1, respectively. The in vitro intrinsic clearances (CLint = Vmax/Km) for ciprofol glucuronidation by HLM and HIM were 6.4 and 1.1 µL min-1·mg protein-1, respectively. Human recombinant UGT studies revealed that UGT1A9 is the predominant isoform mediating M4 formation, followed by UGT1A7, with UGT1A8 playing a minor role. Ciprofol competitively inhibited CYP1A2 (Ki = 12 µM) and CYP2B6 (Ki = 4.7 µM), and noncompetitively inhibited CYP2C19 (Ki = 29 µM). No time-dependent inhibition by ciprofol was noted for CYP1A2, CYP2B6, or CYP2C19. In contrast, M4 showed limited or no inhibitory effects against selected P450s. Neither ciprofol nor M4 inhibited UGTs significantly. Initial IVIVE suggested potential ciprofol-mediated inhibition of CYP1A2, CYP2B6, and CYP2C19 inhibition in vivo. However, PBPK simulations showed no significant effect on phenacetin, bupropion, and S-mephenytoin exposure or peak plasma concentration. Our findings are pertinent for future DDI studies of ciprofol as either a perpetrator or victim drug.

Asunto(s)

Citocromo P-450 CYP1A2 , Microsomas Hepáticos , Humanos , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2C19/metabolismo , Microsomas Hepáticos/metabolismo , Glucuronosiltransferasa/metabolismo , Interacciones Farmacológicas , Cinética

RESUMEN

AIMS: The aim of this study is to demonstrate the use of PBPK modelling to explore the impact of ethnic differences on drug PK. METHODS: A PBPK model developed for lansoprazole was used to predict the clinical PK of lansoprazole in Japanese subjects by incorporating the physiological parameters of a Japanese population into the model. Further verification of the developed Japanese population with clinical studies involving eight other CYP substrates-omeprazole, ticlopidine, alprazolam, midazolam, nifedipine, cinacalcet, paroxetine and dextromethorphan-was also carried out. RESULTS: The PK of lansoprazole in both Caucasian and Japanese subjects was well predicted by the model as the observed data were within the 5th and 95th percentiles across all the clinical studies. In age- and sex-matched simulations in both the Caucasian and Japanese populations, the predicted PK (mean ± SD) of a single oral dose of 30-mg lansoprazole was higher in the Japanese population in all cases, with more than twofold higher AUC of 5.98 ± 6.43 mg/L.h (95% CI: 4.72, 7.24) vs. 2.46 ± 2.45 mg/L.h (95% CI: 1.98, 2.94) in one scenario. In addition, in two out of the nine clinical DDIs of lansoprazole and the additional CYP substrates simulated using the Japanese population, the predicted DDI in Japanese was more than 1.25-fold that in Caucasians, indicating an increased DDI liability. CONCLUSIONS: By accounting for various physiological parameters that characterize a population in a PBPK model, the impact of the different identified interethnic differences on the drug's PK can be explored, which can inform the adoption of drugs from one region to another.

RESUMEN

INTRODUCTION: Acute promyelocytic leukemia (APL) is the most malignant form of acute myeloid leukemia (AML) with short survival without treatment. All trans retinoic acid (ATRA) is a vitamin A metabolite and plays an important role in the treatment of APL. Hypercalcemia is a rare side effect of ATRA. CASE REPORT: A 67-year-old female patient was investigated due to widespread bruising and pancytopenia. The patient was diagnosed with APL and remission was achieved by administering idarubicin together with ATRA in the induction treatment. The patient has hypocalcemia due to acquired hypoparathyroidism, and it was observed that the calcium level increased with the initiation of fluconazole 200 mg/day for antifungal prophylaxis together with ATRA in the consolidation treatment. It was observed that the calcium value reached 13 mg/dL by increasing the fluconazole to 400 mg/day treatment dose due to oral mucositis. MANAGEMENT AND OUTCOME: The development of hypercalcemia has been reported in previous case reports when ATRA is used together with voriconazole, fosfluconazole, itraconazole, and posaconazole, which inhibit cytochrome P450 enzymes. In this case, it is the first in the literature that a patient with hypocalcemia due to acquired hypoparathyroidism developed hypercalcemia after fluconazole and ATRA were used together. DISCUSSION: Since hypercalcemia may develop while azole drugs are administered during ATRA treatment, it is important to monitor calcium levels to prevent complications of hypercalcemia.

RESUMEN

Bacterial cytochrome P450 enzymes catalyze various and often intriguing tailoring reactions during the biosynthesis of natural products. In contrast to the majority of membrane-bound P450 enzymes from eukaryotes, bacterial P450 enzymes are soluble proteins and therefore represent excellent candidates for in vitro biochemical investigations. In particular, cyclodipeptide synthase-associated cytochrome P450 enzymes have recently gained attention due to the broad spectrum of reactions they catalyze, i.e. hydroxylation, aromatization, intramolecular C-C bond formation, dimerization, and nucleobase addition. The latter reaction has been described during the biosynthesis of guanitrypmycins, guatrypmethines and guatyromycines in various Streptomyces strains, where the nucleobases guanine and hypoxanthine are coupled to cyclodipeptides via C-C, C-N, and C-O bonds. In this chapter, we provide an overview of cytochrome P450 enzymes involved in the C-C coupling of cyclodipeptides with nucleobases and describe the protocols used for the successful characterization of these enzymes in our laboratory. The procedure includes cloning of the respective genes into expression vectors and subsequent overproduction of the corresponding proteins in E. coli as well as heterologous expression in Streptomyces. We describe the purification and in vitro biochemical characterization of the enzymes and protocols to isolate the produced compounds for structure elucidation.

Asunto(s)

Escherichia coli , Streptomyces , Escherichia coli/genética , Escherichia coli/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Streptomyces/metabolismo , Catálisis

RESUMEN

P450 fatty acid decarboxylases are able to utilize hydrogen peroxide as the sole cofactor to decarboxylate free fatty acids to produce α-olefins with abundant applications as drop-in biofuels and important chemical precursors. In this chapter, we review diverse approaches for discovery, characterization, engineering, and applications of P450 fatty acid decarboxylases. Information gained from structural data has been advancing our understandings of the unique mechanisms underlying alkene production, and providing important insights for exploring new activities. To build an efficient olefin-producing system, various engineering strategies have been proposed and applied to this unusual P450 catalytic system. Furthermore, we highlight a select number of applied examples of P450 fatty acid decarboxylases in enzyme cascades and metabolic engineering.

Asunto(s)

Carboxiliasas , Ácidos Grasos , Ácidos Grasos/metabolismo , Alquenos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Catálisis

RESUMEN

Phomactin diterpenoids possess a unique bicyclo[9.3.1]pentadecane skeleton with multiple oxidative modifications, and are good platelet-activating factor (PAF) antagonists that can inhibit PAF-induced platelet aggregation. In this study, we identified the gene cluster (phm) responsible for the biosynthesis of phomactins from a marine fungus, Phoma sp. ATCC 74077. Despite the complexity of their structures, phomactin biosynthesis only requires two enzymes: a type I diterpene cyclase PhmA and a P450 monooxygenase PhmC. PhmA was found to catalyze the formation of the phomactatriene, while PhmC sequentially catalyzes the oxidation of multiple sites, leading to the generation of structurally diverse phomactins. The rearrangement mechanism of the diterpene scaffold was investigated through isotope labeling experiments. Additionally, we obtained the crystal complex of PhmA with its substrate analogue FGGPP and elucidated the novel metal-ion-binding mode and enzymatic mechanism of PhmA through site-directed mutagenesis. This study provides the first insight into the biosynthesis of phomactins, laying the foundation for the efficient production of phomactin natural products using synthetic biology approaches.

Asunto(s)

Diterpenos , Factor de Activación Plaquetaria , Hongos

RESUMEN

The members of the bacterial cytochrome P450 (CYP) monooxygenase family CYP125, catalyze the oxidation of steroid derivatives including cholesterol and phytosterols, as the initial activating step in their catabolism. However, several bacterial species contain multiple genes encoding CYP125 enzymes and other CYP enzymes which catalyze cholesterol/cholest-4-en-3-one hydroxylation. An important question is why these bacterium have more than one enzyme with overlapping substrate ranges capable of catalyzing the terminal oxidation of the alkyl chain of these sterols. To further understand the role of these enzymes we investigated CYP125A6 and CYP125A7 from Mycobacterium marinum with various cholesterol analogues. These have modifications on the A and B rings of the steroid and we assessed the substrate binding and catalytic activity of these with each enzyme. CYP125A7 gave similar results to those reported for the CYP125A1 enzyme from M. tuberculosis. Differences in the substrate binding and catalytic activity with the cholesterol analogues were observed with CYP125A6. For example, while cholesteryl sulfate could bind to both enzymes it was only oxidized by CYP125A6 and not by CYP125A7. CYP125A6 generated higher levels of metabolites with the majority of C-3 and C-7 substituted cholesterol analogues such 7-ketocholesterol. However, 5α-cholestan-3ß-ol was only oxidized by CYP125A7 enzyme. The cholest-4-en-3-one and 7-ketocholesterol-bound forms of the CYP125A6 and CYP125A7 enzymes were modelled using AlphaFold. The structural models highlighted differences in the binding modes of the steroid derivatives within the same enzyme. Significant changes in the binding mode of the steroids between these CYP125 enzymes and other bacterial cholesterol oxidizing enzymes, CYP142A3 and CYP124A1, were also seen. Despite this, all these models predicted the selectivity for terminal methyl hydroxylation, in agreement with the experimental data.

Asunto(s)

Mycobacterium marinum , Mycobacterium tuberculosis , Oxidación-Reducción , Sistema Enzimático del Citocromo P-450/metabolismo , Esteroides , Esteroles

RESUMEN

As substrates of CYP2C8, CYP3A4/5 and CYP2D6, chloroquine's (CQ) and hydroxychloroquine's (HCQ) efficacy and safety may be affected by variants in the genes encoding these enzymes. This paper aims to assimilate the current evidence on the pharmacogenomics of CQ/HCQ and to identify risk phenotypes affecting the safety or efficacy of these drugs. It has been found that some CYP3A5, CYP2D6 and CYP2C8 genetic variants may affect the safety or effectiveness of CQ/HCQ. The phenotypes predictively representing ultra-rapid and poor metabolizers have been considered high-risk phenotypes. After considering these high-risk phenotypes in different ethnic groups, it is predicted that a considerable proportion of patients taking CQ/HCQ may be at risk of either therapeutic failure or severe toxicities.

Asunto(s)

Cloroquina , Hidroxicloroquina , Humanos , Hidroxicloroquina/efectos adversos , Cloroquina/efectos adversos , Citocromo P-450 CYP2C8 , Citocromo P-450 CYP2D6/genética , Farmacogenética

RESUMEN

INTRODUCTION: Cytochrome P450 enzymes (P450s) are recognized as the most versatile catalysts worldwide, playing vital roles in numerous biological metabolism and biosynthesis processes across all kingdoms of life. Despite the vast number of P450 genes available in databases (over 300,000), only a small fraction of them (less than 0.2%) have undergone functional characterization. OBJECTIVES: To provide a convenient platform with abundant information on P450s and their corresponding reactions, we introduce the P450Rdb database, a manually curated resource compiles literature-supported reactions catalyzed by P450s. METHODS: All the P450s and Reactions were manually curated from the literature and known databases. Subsequently, the P450 reactions organized and categorized according to their chemical reaction type and site. The website was developed using HTML and PHP languages, with the MySQL server utilized for data storage. RESULTS: The current version of P450Rdb catalogs over 1,600 reactions, involving more than 590 P450s across a diverse range of over 200 species. Additionally, it offers a user-friendly interface with comprehensive information, enabling easy querying, browsing, and analysis of P450s and their corresponding reactions. P450Rdb is free available at http://www.cellknowledge.com.cn/p450rdb/. CONCLUSIONS: We believe that this database will significantly promote structural and functional research on P450s, thereby fostering advancements in the fields of natural product synthesis, pharmaceutical engineering, biotechnological applications, agricultural and crop improvement, and the chemical industry.