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Life Science Alliance Feb 2023We recently developed a "Build and Retrieve" cryo-electron microscopy (cryo-EM) methodology, which is capable of simultaneously producing near-atomic resolution cryo-EM...
We recently developed a "Build and Retrieve" cryo-electron microscopy (cryo-EM) methodology, which is capable of simultaneously producing near-atomic resolution cryo-EM maps for several individual proteins from a heterogeneous, multiprotein sample. Here we report the use of "Build and Retrieve" to define the composition of a raw human brain microsomal lysate. From this sample, we simultaneously identify and solve cryo-EM structures of five different brain enzymes whose functions affect neurotransmitter recycling, iron metabolism, glycolysis, axonal development, energy homeostasis, and retinoic acid biosynthesis. Interestingly, malfunction of these important proteins has been directly linked to several neurodegenerative disorders, such as Alzheimer's, Huntington's, and Parkinson's diseases. Our work underscores the importance of cryo-EM in facilitating tissue and organ proteomics at the atomic level.
Topics: Humans; Cryoelectron Microscopy; Electrons; Microsomes; Brain; Proteomics
PubMed: 36450447
DOI: 10.26508/lsa.202201724 -
Journal of Lipid Research Jun 2019New fluorogenic ceramidase substrates derived from the -acyl modification of our previously reported probes (RBM14) are reported. While none of the new probes were...
New fluorogenic ceramidase substrates derived from the -acyl modification of our previously reported probes (RBM14) are reported. While none of the new probes were superior to the known RBM14C12 as acid ceramidase substrates, the corresponding nervonic acid amide (RBM14C24:1) is an efficient and selective substrate for the recombinant human neutral ceramidase, both in cell lysates and in intact cells. A second generation of substrates, incorporating the natural 2-(-acylamino)-1,3-diol-4-ene framework (compounds RBM15) is also reported. Among them, the corresponding fatty acyl amides with an unsaturated -acyl chain can be used as substrates to determine alkaline ceramidase (ACER)1 and ACER2 activities. In particular, compound RBM15C18:1 has emerged as the best fluorogenic probe reported so far to measure ACER1 and ACER2 activities in a 96-well plate format.
Topics: Alkaline Ceramidase; Cell Line; Ceramides; HT29 Cells; Humans; Intracellular Signaling Peptides and Proteins; Magnetic Resonance Spectroscopy; Microsomes; Molecular Structure; RNA-Binding Proteins; Sphingolipids; Umbelliferones
PubMed: 30926626
DOI: 10.1194/jlr.D092759 -
Molecules (Basel, Switzerland) Sep 2022Human cytochrome P450 enzymes (CYPs) are heme-containing monooxygenases. This superfamily of drug-metabolizing enzymes is responsible for the metabolism of most drugs...
Human cytochrome P450 enzymes (CYPs) are heme-containing monooxygenases. This superfamily of drug-metabolizing enzymes is responsible for the metabolism of most drugs and other xenobiotics. The inhibition of CYPs may lead to drug-drug interactions and impair the biotransformation of drugs. CYP inducers may decrease the bioavailability and increase the clearance of drugs. Based on the freely available databases ChEMBL and PubChem, we have collected over 70,000 records containing the structures of inhibitors and inducers together with the IC50 values for the inhibitors of the five major human CYPs: 1A2, 3A4, 2D6, 2C9, and 2C19. Based on the collected data, we developed (Q)SAR models for predicting inhibitors and inducers of these CYPs using GUSAR and PASS software. The developed (Q)SAR models could be applied for assessment of the interaction of novel drug-like substances with the major human CYPs. The created (Q)SAR models demonstrated reasonable accuracy of prediction. They have been implemented in the web application P450-Analyzer that is freely available via the Internet.
Topics: Cytochrome P-450 Enzyme System; Drug Interactions; Heme; Humans; Microsomes, Liver; Protein Isoforms; Xenobiotics
PubMed: 36144612
DOI: 10.3390/molecules27185875 -
Biological & Pharmaceutical Bulletin 2019Predicting human pharmacokinetics (PK) such as clearance (CL) and volume of distribution (Vd) is a critical component of drug discovery. These predictions are mainly... (Review)
Review
Utility of Chimeric Mice with Humanized Liver for Predicting Human Pharmacokinetics in Drug Discovery: Comparison with in Vitro-in Vivo Extrapolation and Allometric Scaling.
Predicting human pharmacokinetics (PK) such as clearance (CL) and volume of distribution (Vd) is a critical component of drug discovery. These predictions are mainly performed by in vitro-in vivo extrapolation (IVIVE) using human biological samples, such as hepatic microsomes and hepatocytes. However, some issues with this process have arisen, such as inconsistencies between in vitro and in vivo findings; the integration of predicted CYP, non-CYP and transporter-mediated human PK; and the difficulty of evaluating very metabolically stable compounds. Various approaches to solving these issues have been reported. Allometric scaling using experimental animals has also often been used. However, this method has also shown many problems due to interspecies differences, albeit that various correction methods have been proposed. Another approach involves the production of chimeric mice with humanized liver via the transplantation of human hepatocytes into mice. The livers of these mice are repopulated mostly with human hepatocytes and express human drug-metabolizing enzymes and drug transporters, suggesting that these mice are useful for solving the issues of IVIVE and allometric scaling, and more reliably predicting human PK. In this review, we summarize human PK prediction methods using IVIVE, allometric scaling and chimeric mice with humanized liver, and discuss the utility of predicting human PK in drug discovery by comparing these chimeric mice with IVIVE and allometric scaling.
Topics: Animals; Drug Discovery; Humans; Liver; Mice; Mice, Transgenic; Microsomes, Liver; Pharmaceutical Preparations; Pharmacokinetics
PubMed: 30828063
DOI: 10.1248/bpb.b18-00754 -
Scientific Reports Dec 2017Metapristone is the primary metabolite of the abortifacient mifepristone (RU486), and is being developed as a safe and effective cancer metastatic chemopreventive agent...
Metapristone is the primary metabolite of the abortifacient mifepristone (RU486), and is being developed as a safe and effective cancer metastatic chemopreventive agent for both sexes. Here, we systematically investigated the sex-related pharmacokinetics of metapristone in both rats and dogs, and explored the related mechanisms of actions. Administration of metapristone to rats and dogs showed that plasma concentrations of metapristone (AUC, C ) were significantly higher in female dogs and rats than in males. The sex-related differences in pharmacokinetics become more significant after ten consecutive days of oral administration. Female liver microsomes metabolized metapristone significantly slower than the male ones. The results from P450 reaction phenotyping using recombinant cDNA-expressed human CYPs in conjunction with specific CYP inhibitors suggested that CYP1A2 and CYP3A4 are the predominant CYPs involved in the metapristone metabolism, which were further confirmed by the enhanced protein levels of CYP1A2 and CYP3A4 induced by 1-week oral administration of metapristone to rats. The highest tissue concentration of metapristone was found in the liver. The study demonstrates, for the first time, the sex-related pharmacokinetics of metapristone, and reveals that activities of liver microsomal CYP1A2 and CYP3A4 as well as the renal clearance are primarily responsible for the sex-related pharmacokinetics.
Topics: Animals; Cell Proliferation; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP3A; Dogs; Female; Hormone Antagonists; Male; Microsomes, Liver; Mifepristone; Rats; Rats, Sprague-Dawley; Sex Factors; Tissue Distribution
PubMed: 29215040
DOI: 10.1038/s41598-017-17225-0 -
Analytical and Bioanalytical Chemistry Sep 2023Synthetic cathinones, one of the most prevalent categories of new psychoactive substances, have been posing a serious threat to public health. Methylmethcathinones...
Synthetic cathinones, one of the most prevalent categories of new psychoactive substances, have been posing a serious threat to public health. Methylmethcathinones (MMCs), notably 3-MMC, have seen an alarming increase in their use in the last decade. The metabolism and toxicology of a large majority of synthetic cathinones, including 3-MMC and 2-MMC, remain unknown. Traditionally, male-derived liver materials have been used as in vitro metabolic incubations to investigate the metabolism of xenobiotics, including MMCs. Therefore, little is known about the metabolism in female-derived in vitro models and the potential sex-specific differences in biotransformation. In this study, the metabolism of 2-MMC, 3-MMC, and 4-MMC was investigated using female rat and human liver microsomal incubations, as well as male rat and human liver microsomal incubations. A total of 25 phase I metabolites of MMCs were detected and tentatively identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Seven sex-specific metabolites were detected exclusively using pooled male rat liver microsomal incubations. In addition, the metabolites generated from the sex-dependent in vitro metabolic incubations that were present in both male and female rat liver microsomal incubations showed differences in relative abundance. Yet, neither sex-specific metabolites nor significant differences in relative abundance were observed from pooled human liver microsomal incubations. This is the first study to report the phase I metabolic pathways of MMCs using in vitro metabolic incubations for both male and female liver microsomes, and the relative abundance of the metabolites observed from each sex.
Topics: Rats; Male; Humans; Female; Animals; Chromatography, Liquid; Tandem Mass Spectrometry; Alkaloids; Liver; Microsomes, Liver
PubMed: 37452840
DOI: 10.1007/s00216-023-04815-3 -
Pharmacology Research & Perspectives Feb 2022The bioavailability of drugs is often related to intestinal metabolism and transport mechanisms. In previous studies, pharmaceutical excipients were recognized as inert...
The pharmaceutical excipient PEG400 affect the absorption of baicalein in Caco-2 monolayer model by interacting with UDP-glucuronosyltransferases and efflux transport proteins.
The bioavailability of drugs is often related to intestinal metabolism and transport mechanisms. In previous studies, pharmaceutical excipients were recognized as inert substances in clinical safety evaluations. However, a large number of studies have shown that pharmaceutical excipients regulate the metabolism and transport of drugs in the body and improve the bioavailability. The pharmaceutical excipient polyethylene glycol 400 (PEG400) as a good solubilizer and surfactant has the potential to improve the bioavailability of drugs. The combined action of UDP-glucuronosyltransferases (UGTs) and efflux transport proteins is responsible for the intestinal disposition and poor bioavailability of baicalein. Our aim is to study the effect of PEG400 on the absorption of baicalein on the Caco-2 monolayer, and confirm the interaction of PEG400 with UGTs (UGT1A8 and UGT1A9) and efflux transports. We initially found that baicalein in the Caco-2 monolayer would be metabolized into glucuronide conjugates BG and B6G under the action of UGT1A8 and UGT1A9 on the endoplasmic reticulum membrane, and then mainly excreted to different sides by acting of MRP and BCRP. The addition of PEG400 significantly accelerated the metabolism of B in Caco-2 cells and increased the penetration of BG and B6G. Furthermore, PEG400 also significantly decreased the efflux ratio of BG and B6G, which was the evidence of the interaction with the efflux transporters. In the in vitro intestinal microsome regeneration system, low concentration PEG400 decreased the K value of UGT1A8 and UGT1A9 (key enzymes that mediate the production of BG and B6G); high concentration PEG400 enhanced the V value of UGT1A8 and UGT1A9. In conclusion, our results determined that PEG400 interacted with some UGTs and efflux transporters, which were the main factors affecting the absorption of baicalein.
Topics: Antioxidants; Biological Availability; Biological Transport; Caco-2 Cells; Excipients; Flavanones; Glucuronosyltransferase; Humans; Intestinal Absorption; Membrane Transport Proteins; Microsomes; Polyethylene Glycols; UDP-Glucuronosyltransferase 1A9
PubMed: 35148019
DOI: 10.1002/prp2.928 -
Drug Design, Development and Therapy 2020The purpose of this study was to examine the effects of voriconazole on the pharmacokinetics of vonoprazan.
PURPOSE
The purpose of this study was to examine the effects of voriconazole on the pharmacokinetics of vonoprazan.
METHODS
Fifteen Sprague-Dawley rats were randomly divided into three groups: five rats in each group, including control group, single-dose group (a single dose of 30 mg/kg of voriconazole), and multiple-dose group (multiple doses of 30 mg/(kg•day) per dose of voriconazole). Each group of rats was given an oral dose of 10 mg/kg vonoprazan 30 min after the administration of voriconazole or vehicle. After the oral administration of vonoprazan, 50 µL of blood was collected into 1.5-mL heparinized tubes via the caudal vein. The concentration of vonoprazan in plasma was quantified by ultra-performance liquid chromatography/tandem mass spectrometry. Both in vitro effects of voriconazole on vonoprazan and the mechanism of the observed inhibition were studied in rat liver microsomes.
RESULTS
When orally administered, voriconazole increased the area under the plasma concentration-time curve (AUC), prolonged the elimination half-life (t), and decreased the clearance (CL) of vonoprazan; there was no significant difference between the single-dose and multiple-dose groups. Voriconazole inhibited the metabolism of vonoprazan at an IC50 of 2.93 μM and showed mixed inhibition. The results of the in vivo experiments were consistent with those of the in vitro experiments.
CONCLUSION
Our findings provide the evidence of drug-drug interactions between voriconazole and vonoprazan that could occur with pre-administration of voriconazole. Thus, clinicians should pay attention to the resulting changes in pharmacokinetic parameters and accordingly, adjust the dose of vonoprazan in clinical settings.
Topics: Administration, Oral; Animals; Chromatography, High Pressure Liquid; Male; Microsomes, Liver; Pyrroles; Rats; Rats, Sprague-Dawley; Sulfonamides; Tandem Mass Spectrometry; Voriconazole
PubMed: 32581516
DOI: 10.2147/DDDT.S255427 -
Fa Yi Xue Za Zhi Aug 2021Synthetic cannabinoids are currently a class of new psychoactive substances with the largest variety and most abused. Metabolite identification research can provide... (Review)
Review
Synthetic cannabinoids are currently a class of new psychoactive substances with the largest variety and most abused. Metabolite identification research can provide basic data for monitoring synthetic cannabinoids abuse, which is the current research hotspot. The main trend of structural modification of synthetic cannabinoid is to replace the fluorine atom on pentyl indole or indazole cyclopentyl with hydrogen atom, which greatly improves the biological activity of the compound. The main metabolic reactions include hydroxylation, fluoropentyl oxidative, ester hydrolyze, amide hydrolysis. Liquid chromatography-high resolution mass spectrometry has become the preferred choice for the structural identification of metabolites. This review mainly summarizes research on metabolism software prediction and human hepatocyte model, human liver microsomes model, rat in vivo model, zebrafish model and fungus model in metabolite identification based on the structure and classification of synthetic cannabinoids.
Topics: Animals; Caenorhabditis elegans; Cannabinoids; Chromatography, Liquid; Microsomes, Liver; Rats; Zebrafish
PubMed: 34725997
DOI: 10.12116/j.issn.1004-5619.2021.310602 -
Scientific Reports Feb 2020Artificial giant vesicles have proven highly useful as membrane models in a large variety of biophysical and biochemical studies. They feature accessibility for...
Artificial giant vesicles have proven highly useful as membrane models in a large variety of biophysical and biochemical studies. They feature accessibility for manipulation and detection, but lack the compositional complexity needed to reconstitute complicated cellular processes. For the plasma membrane (PM), this gap was bridged by the establishment of giant PM vesicles (GPMVs). These native membranes have facilitated studies of protein and lipid diffusion, protein interactions, electrophysiology, fluorescence analysis of lateral domain formation and protein and lipid partitioning as well as mechanical membrane properties and remodeling. The endoplasmic reticulum (ER) is key to a plethora of biological processes in any eukaryotic cell. However, its intracellular location and dynamic and intricate tubular morphology makes it experimentally even less accessible than the PM. A model membrane, which will allow the afore-mentioned types of studies on GPMVs to be performed on ER membranes outside the cell, is therefore genuinely needed. Here, we introduce the formation of giant ER vesicles, termed GERVs, as a new tool for biochemistry and biophysics. To obtain GERVs, we have isolated ER membranes from Saccharomyces cerevisiae and fused them by exploiting the atlastin-like fusion protein Sey1p. We demonstrate the production of GERVs and their utility for further studies.
Topics: Apyrase; Cell Membrane; Cytological Techniques; Diffusion; Endoplasmic Reticulum; Lipid Metabolism; Microscopy, Confocal; Microsomes; Reproducibility of Results; SEC Translocation Channels; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 32080222
DOI: 10.1038/s41598-020-59700-1