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Fa Yi Xue Za Zhi Aug 2021Objective To study the metabolic transformation pathways of 4F-MDMB-BUTINACA in vivo by establishing zebrafish models. Methods Six adult zebrafish were randomly divided...
Objective To study the metabolic transformation pathways of 4F-MDMB-BUTINACA in vivo by establishing zebrafish models. Methods Six adult zebrafish were randomly divided into blank control group and experimental group, with three fish in each group. After the zebrafish in the experimental group were exposed to 1 μg/mL 4F-MDMB-BUTINACA for 24 h, they were transferred to clean water and cleaned three times, then pretreated for instrumental analysis. The zebrafish in blank control group were not exposed to 4F-MDMB-BUTINACA. Mass spectrometry and structural analysis of 4F-MDMB-BUTINACA and its metabolites were conducted by liquid chromatography-high resolution mass spectrometry and Mass Frontier software. Results A total of twenty-six metabolites of 4F-MDMB-BUTINACA were identified in zebrafish, including eighteen phase Ⅰ metabolites and eight phase Ⅱ metabolites. The main metabolic pathways of phase Ⅰ metabolites of 4F-MDMB-BUTINACA in zebrafish were ester hydrolysis, N-dealkylation, oxidative defluorination and hydroxylation, while the main metabolic pathway of phase Ⅱ metabolites was glucuronidation. Conclusion Metabolite Md24 (ester hydrolysis) and Md25 (ester hydrolysis combined with dehydrogenation) would be recommended to be potentially good biomarkers for abuse of 4F-MDMB-BUTINACA.
Topics: Animals; Cannabinoids; Chromatography, Liquid; Illicit Drugs; Microsomes, Liver; Zebrafish
PubMed: 34726001
DOI: 10.12116/j.issn.1004-5619.2021.310401 -
Molecular Cell Aug 2020Protein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation channel formed by the Sec61 complex. Unrelated small-molecule...
Protein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation channel formed by the Sec61 complex. Unrelated small-molecule natural products and synthetic compounds inhibit Sec61 with differential effects for different substrates or for Sec61 from different organisms, making this a promising target for therapeutic intervention. To understand the mode of inhibition and provide insight into the molecular mechanism of this dynamic translocon, we determined the structure of mammalian Sec61 inhibited by the Mycobacterium ulcerans exotoxin mycolactone via electron cryo-microscopy. Unexpectedly, the conformation of inhibited Sec61 is optimal for substrate engagement, with mycolactone wedging open the cytosolic side of the lateral gate. The inability of mycolactone-inhibited Sec61 to effectively transport substrate proteins implies that signal peptides and transmembrane domains pass through the site occupied by mycolactone. This provides a foundation for understanding the molecular mechanism of Sec61 inhibitors and reveals novel features of translocon function and dynamics.
Topics: Animals; Binding Sites; Cell-Free System; Dogs; Gene Expression; HCT116 Cells; HEK293 Cells; Humans; Macrolides; Microsomes; Molecular Dynamics Simulation; Mutation; Mycobacterium ulcerans; Pancreas; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Transport; Ribosomes; SEC Translocation Channels; Structural Homology, Protein; Substrate Specificity
PubMed: 32692975
DOI: 10.1016/j.molcel.2020.06.013 -
Molecules (Basel, Switzerland) Jan 2020The importance of the gut microbiota in drug metabolism, especially in that of nonabsorbable drugs, has become known. The aim of this study was to explore the...
The importance of the gut microbiota in drug metabolism, especially in that of nonabsorbable drugs, has become known. The aim of this study was to explore the metabolites of triptolide by the gut microbiota. With high-performance liquid chromatography coupled with tandem mass spectrometry and ion trap time-of-flight multistage mass spectrometry (LC-MS/MS and LC/MS-IT-TOF), four metabolites of triptolide (M1, M2, M3, and M4) were found in the intestinal contents of rats. M1 and M2, were isomeric monocarbonyl-hydroxyl-substituted metabolites with molecular weights of 390. M3 and M4 were isomeric dehydrogenated metabolites with molecular weights of 356. Among the four metabolites, the dehydrogenated metabolites (M3 and M4) were reported in the gut microbiota for the first time. The metabolic behaviors of triptolide in the gut microbiota and liver microsomes of rats were further compared. The monocarbonyl-hydroxyl-substituted metabolites (M1 and M2) were generated in both systems, and another monohydroxylated metabolite (M5) was found only in the liver microsomes. The combined results suggested that the metabolism of triptolide in the gut microbiota was specific, with two characteristic, dehydrogenated metabolites. This investigation might provide a theoretical basis for the elucidation of the metabolism mechanism of triptolide and guide its proper application in clinical administration.
Topics: Animals; Chromatography, High Pressure Liquid; Diterpenes; Epoxy Compounds; Gastrointestinal Microbiome; Immunosuppressive Agents; Male; Microsomes, Liver; Phenanthrenes; Rats; Rats, Sprague-Dawley; Tandem Mass Spectrometry
PubMed: 32019202
DOI: 10.3390/molecules25030606 -
Biochimica Et Biophysica Acta.... Jul 2018ERAD is an important process of protein quality control that eliminates misfolded or unassembled proteins from ER. Before undergoing proteasome degradation, the...
ERAD is an important process of protein quality control that eliminates misfolded or unassembled proteins from ER. Before undergoing proteasome degradation, the misfolded proteins are dislocated from ER membrane into cytosol, which requires the AAA ATPase p97/VCP and its cofactor, the NPL4-UFD1 dimer. Here, we performed a CRISPR-based screen and identify many candidates for ERAD regulation. We further confirmed four proteins, FBOX2, TRIM6, UFL1 and WDR20, are novel regulators for ERAD. Then the molecular mechanism for WDR20 in ERAD is further characterized. Depletion of WDR20 inhibits the degradation of TCRα, a typical ERAD substrate, while WDR20 overexpression reduces TCRα protein level. WDR20 associates with TCRα and central regulators of the ERAD system, p97, GP78 and HRD1. A portion of WDR20 localizes to the ER-containing microsomal membrane. WDR20 expression increases TCRα ubiquitination, and HRD1 E3 ligase is essential for the process. WDR20 seems to serve as an adaptor protein to mediate the interaction between p97 and TCRα. Our study provides novel candidates and reveals an unexpected role of WDR20 in ERAD regulation.
Topics: Adenosine Triphosphatases; CRISPR-Cas Systems; Carrier Proteins; Cell Line, Tumor; Endoplasmic Reticulum-Associated Degradation; HEK293 Cells; Humans; Microsomes; Nuclear Proteins; Proteasome Endopeptidase Complex; Receptors, Antigen, T-Cell, alpha-beta; Ubiquitination
PubMed: 29655804
DOI: 10.1016/j.bbamcr.2018.04.006 -
Drug Metabolism and Pharmacokinetics Feb 2018More than 20% of clinically used drugs are glucuronidated by a microsomal enzyme UDP-glucuronosyltransferase (UGT). Inhibition or induction of UGT can result in an... (Review)
Review
Species differences in drug glucuronidation: Humanized UDP-glucuronosyltransferase 1 mice and their application for predicting drug glucuronidation and drug-induced toxicity in humans.
More than 20% of clinically used drugs are glucuronidated by a microsomal enzyme UDP-glucuronosyltransferase (UGT). Inhibition or induction of UGT can result in an increase or decrease in blood drug concentration. To avoid drug-drug interactions and adverse drug reactions in individuals, therefore, it is important to understand whether UGTs are involved in metabolism of drugs and drug candidates. While most of glucuronides are inactive metabolites, acyl-glucuronides that are formed from compounds with a carboxylic acid group can be highly toxic. Animals such as mice and rats are widely used to predict drug metabolism and drug-induced toxicity in humans. However, there are marked species differences in the expression and function of drug-metabolizing enzymes including UGTs. To overcome the species differences, mice in which certain drug-metabolizing enzymes are humanized have been recently developed. Humanized UGT1 (hUGT1) mice were created in 2010 by crossing Ugt1-null mice with human UGT1 transgenic mice in a C57BL/6 background. hUGT1 mice can be promising tools to predict human drug glucuronidation and acyl-glucuronide-associated toxicity. In this review article, studies of drug metabolism and toxicity in the hUGT1 mice are summarized. We further discuss research and strategic directions to advance the understanding of drug glucuronidation in humans.
Topics: Animals; Drug-Related Side Effects and Adverse Reactions; Forecasting; Glucuronides; Glucuronosyltransferase; Humans; Metabolic Clearance Rate; Mice; Mice, Transgenic; Microsomes, Liver; Species Specificity
PubMed: 29079228
DOI: 10.1016/j.dmpk.2017.10.002 -
BMC Plant Biology Jan 2021Simmondsia chinensis (jojoba) is the only plant known to store wax esters instead of triacylglycerols in its seeds. Wax esters are composed of very-long-chain...
BACKGROUND
Simmondsia chinensis (jojoba) is the only plant known to store wax esters instead of triacylglycerols in its seeds. Wax esters are composed of very-long-chain monounsaturated fatty acids and fatty alcohols and constitute up to 60% of the jojoba seed weight. During jojoba germination, the first step of wax ester mobilization is catalyzed by lipases. To date, none of the jojoba lipase-encoding genes have been cloned and characterized. In this study, we monitored mobilization of storage reserves during germination of jojoba seeds and performed detailed characterization of the jojoba lipases using microsomal fractions isolated from germinating seeds.
RESULTS
During 26 days of germination, we observed a 60-70% decrease in wax ester content in the seeds, which was accompanied by the reduction of oleosin amounts and increase in glucose content. The activity of jojoba lipases in the seed microsomal fractions increased in the first 50 days of germination. The enzymes showed higher activity towards triacylglycerols than towards wax esters. The maximum lipase activity was observed at 60 °C and pH around 7 for triacylglycerols and 6.5-8 for wax esters. The enzyme efficiently hydrolyzed various wax esters containing saturated and unsaturated acyl and alcohol moieties. We also demonstrated that jojoba lipases possess wax ester-synthesizing activity when free fatty alcohols and different acyl donors, including triacylglycerols and free fatty acids, are used as substrates. For esterification reactions, the enzyme utilized both saturated and unsaturated fatty alcohols, with the preference towards long chain and very long chain compounds.
CONCLUSIONS
In in vitro assays, jojoba lipases catalyzed hydrolysis of triacylglycerols and different wax esters in a broad range of temperatures. In addition, the enzymes had the ability to synthesize wax esters in the backward reaction. Our data suggest that jojoba lipases may be more similar to other plant lipases than previously assumed.
Topics: Caryophyllales; Esters; Germination; Hydrolysis; Lipase; Lipids; Microsomes; Orlistat; Plant Proteins; Seeds; Substrate Specificity; Temperature; Triglycerides; Waxes
PubMed: 33468064
DOI: 10.1186/s12870-020-02823-4 -
Pharmaceutical Biology Dec 2019Omeprazole and astragaloside IV (AS-IV) are widely used for the treatment of gastric ulcers in China clinics. This study investigates the effects of AS-IV on the...
Omeprazole and astragaloside IV (AS-IV) are widely used for the treatment of gastric ulcers in China clinics. This study investigates the effects of AS-IV on the pharmacokinetics of omeprazole in rats. The pharmacokinetics of orally administered omeprazole (2 mg/kg), with or without AS-IV (100 mg/kg/day for 7 days) pretreatment, were investigated in male Sprague-Dawley rats (two groups of six animals each) using LC-MS/MS. A Caco-2 cell transwell model and rat liver microsome incubation systems were also used to support the pharmacokinetic data and investigate its potential mechanism. The results indicated that co-administration of AS-IV could decrease the systemic exposure of omeprazole significantly ( < 0.05), including AUC (717.20 ± 177.63 vs. 1166.25 ± 186.65 ng h/mL) and (272.35 ± 25.81 vs. 366.34 ± 32.57 ng/mL). The of omeprazole also decreased significantly (1.78 ± 0.15 vs. 2.23 ± 0.27 h, < 0.05). The efflux ratio of omeprazole across the Caco-2 cell transwell model increased significantly from 1.73 to 2.67 ( < 0.05), and the metabolic stability of omeprazole was decreased from 42.6 ± 7.8 to 26.2 ± 5.1 min with the pretreatment of AS-IV ( < 0.05). AS-IV could decrease the systemic exposure of omeprazole in rats when AS-IV and omeprazole were co-administered, and it might exert these effects through decreasing the absorption of omeprazole by inducing , or through accelerating the metabolism of omeprazole in rat liver by inducing the activity of CYP3A4.
Topics: Animals; Caco-2 Cells; Humans; Male; Microsomes, Liver; Omeprazole; Rats; Rats, Sprague-Dawley; Saponins; Triterpenes
PubMed: 31290355
DOI: 10.1080/13880209.2019.1636828 -
Neurobiology of Disease Mar 2018α-synuclein (αS) is a small protein that self-aggregates into α-helical oligomer species and subsequently into larger insoluble amyloid fibrils that accumulate in...
α-synuclein (αS) is a small protein that self-aggregates into α-helical oligomer species and subsequently into larger insoluble amyloid fibrils that accumulate in intraneuronal inclusions during the development of Parkinson's disease. Toxicity of αS oligomers and fibrils has been long debated and more recent data are suggesting that both species can induce neurodegeneration. However while most of these data are based on differences in structure between oligomer and aggregates, often preassembled in vitro, the in vivo situation might be more complex and subcellular locations where αS species accumulate, rather than their conformation, might contribute to enhanced toxicity. In line with this observation, we have shown that αS oligomers and aggregates are associated with the endoplasmic reticulum/microsomes (ER/M) membrane in vivo and how accumulation of soluble αS oligomers at the ER/M level precedes neuronal degeneration in a mouse model of α-synucleinopathies. In this paper we took a further step, investigating the biochemical and functional features of αS species associated with the ER/M membrane. We found that by comparison with non-microsomal associated αS (P10), the ER/M-associated αS pool is a unique population of oligomers and aggregates with specific biochemical traits such as increased aggregation, N- and C-terminal truncations and phosphorylation at serine 129. Moreover, when administered to murine primary neurons, ER/M-associated αS species isolated from diseased A53T human αS transgenic mice induced neuronal changes in a time- and dose-dependent manner. In fact the addition of small amounts of ER/M-associated αS species from diseased mice to primary cultures induced the formation of beads-like structures or strings of fibrous αS aggregates along the neurites, occasionally covering the entire process or localizing at the soma level. By comparison treatment with P10 fractions from the same diseased mice resulted in the formation of scarce and small puncta only when administered at high amount. Moreover, increasing the amount of P100/M fractions obtained from diseased and, more surprisingly, from presymptomatic mice induced a significant level of neuronal death that was prevented when neurons were treated with ER/M fractions immunodepleted of αS high molecular weight (HMW) species. These data provide the first evidence of the existence of two different populations of αS HMW species in vivo, putting the spotlight on the association to ER/M membrane as a necessary step for the acquisition of αS toxic features.
Topics: Animals; Apoptosis; Cell Line, Tumor; Cerebral Cortex; Disease Models, Animal; Endoplasmic Reticulum; Humans; Mice, Transgenic; Microsomes; Molecular Weight; Nerve Degeneration; Neurodegenerative Diseases; Neurons; Primary Cell Culture; Protein Aggregation, Pathological; alpha-Synuclein
PubMed: 29246724
DOI: 10.1016/j.nbd.2017.12.004 -
International Journal of Medical... 2020Acetaminophen (APAP) and roxithromycin (ROX) are often used in combination in clinical practice. To evaluate their drug-drug interactions (DDIs) and the hepatotoxicity...
Acetaminophen (APAP) and roxithromycin (ROX) are often used in combination in clinical practice. To evaluate their drug-drug interactions (DDIs) and the hepatotoxicity of co-administration, rats were randomly separated into four groups: Control, APAP (50 mg/kg), ROX (5.5 mg/kg) and APAP-ROX (50 mg/kg and 5.5 mg/kg, respectively). The pharmacokinetic parameters between APAP and ROX were assayed by HPLC, and a cocktail method was used to evaluate the activities of cytochrome (CYP) 450. The liver microsome CYP2E1 protein was detected using Western blot. The levels of plasma parameters, mRNA levels of inflammatory factors (TNF-α, INF-γ, VCAM-1, CXCL-1 and STAT-3) and antioxidant factors (Nrf-2, GSTA, GCLC-1, HO-1 and NQO1) were determined using real-time PCR, along with the observation on histopathological changes in the liver tissue. APAP and ROX co-treatment significantly increased CYP2E1 activity, decreased CYP2D6 activity and prolonged APAP and ROX clearance. Co-treatment increased mRNA expressions of TNF-α, NQO1 and MDA while decreasing GPX and SOD levels. Histopathological evidence showed the changes of liver tissues in terms of structure, size and tight arrangement. This study confirmed that a combination of APAP and ROX inhibited APAP metabolism and that the peak concentration of ROX was delayed. The resulting high level of CYP2E1 may induce oxidative stress and cause liver damage.
Topics: Acetaminophen; Alanine Transaminase; Animals; Antioxidants; Chromatography, High Pressure Liquid; Chromatography, Liquid; Cytochrome P-450 CYP2E1; Cytochrome P-450 Enzyme System; Drug Interactions; Female; Microsomes, Liver; NF-E2-Related Factor 2; Oxidation-Reduction; Oxidative Stress; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Roxithromycin
PubMed: 32132876
DOI: 10.7150/ijms.38527 -
British Journal of Pharmacology Oct 2019Extracellular vesicles (EVs) are constitutively shed from cells and released by various stimuli. Their protein and RNA cargo are modified by the stimulus, and in disease...
BACKGROUND AND PURPOSE
Extracellular vesicles (EVs) are constitutively shed from cells and released by various stimuli. Their protein and RNA cargo are modified by the stimulus, and in disease conditions can carry pathological cargo involved in disease progression. Neutral sphingomyelinase 2 (nSMase2) is a major regulator in at least one of several independent routes of EV biogenesis, and its inhibition is a promising new therapeutic approach for neurological disorders. Unfortunately, known inhibitors exhibit μM potency, poor physicochemical properties, and/or limited brain penetration. Here, we sought to identify a drug-like inhibitor of nSMase2.
EXPERIMENTAL APPROACH
We conducted a human nSMase2 high throughput screen (>365,000 compounds). Selected hits were optimized focusing on potency, selectivity, metabolic stability, pharmacokinetics, and ability to inhibit EV release in vitro and in vivo.
KEY RESULTS
We identified phenyl(R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-carbamate (PDDC), a potent (pIC = 6.57) and selective non-competitive inhibitor of nSMase2. PDDC was metabolically stable, with excellent oral bioavailability (%F = 88) and brain penetration (AUC /AUC = 0.60). PDDC dose-dependently (pEC = 5.5) inhibited release of astrocyte-derived extracellular vesicles (ADEV). In an in vivo inflammatory brain injury model, PDDC robustly inhibited ADEV release and the associated peripheral immunological response. A closely related inactive PDDC analogue was ineffective.
CONCLUSION AND IMPLICATIONS
PDDC is a structurally novel, potent, orally available, and brain penetrant inhibitor of nSMase2. PDDC inhibited release of ADEVs in tissue culture and in vivo. PDDC is actively being tested in animal models of neurological disease and, along with closely related analogues, is being considered for clinical translation.
Topics: Animals; Astrocytes; Brain; Cells, Cultured; Dose-Response Relationship, Drug; Extracellular Vesicles; High-Throughput Screening Assays; Humans; Male; Mice; Microsomes, Liver; Molecular Structure; Rats; Rats, Sprague-Dawley; Structure-Activity Relationship
PubMed: 31273753
DOI: 10.1111/bph.14789