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Scientific Reports Dec 2020Cytochrome P450 2J2 (CYP2J2) is responsible for the epoxidation of endogenous arachidonic acid, and is involved in the metabolism of exogenous drugs. To date, no crystal...
Cytochrome P450 2J2 (CYP2J2) is responsible for the epoxidation of endogenous arachidonic acid, and is involved in the metabolism of exogenous drugs. To date, no crystal structure of CYP2J2 is available, and the proposed structural basis for the substrate recognition and specificity in CYP2J2 varies with the structural models developed using different computational protocols. In this study, we developed a new structural model of CYP2J2, and explored its sensitivity to substrate binding by molecular dynamics simulations of the interactions with chemically similar fluorescent probes. Our results showed that the induced-fit binding of these probes led to the preferred active poses ready for the catalysis by CYP2J2. Divergent conformational dynamics of CYP2J2 due to the binding of each probe were observed. However, a stable hydrophobic clamp composed of residues I127, F310, A311, V380, and I487 was identified to restrict any substrate access to the active site of CYP2J2. Molecular docking of a series of compounds including amiodarone, astemizole, danazol, ebastine, ketoconazole, terfenadine, terfenadone, and arachidonic acid to CYP2J2 confirmed the role of those residues in determining substrate binding and specificity of CYP2J2. In addition to the flexibility of CYP2J2, the present work also identified other factors such as electrostatic potential in the vicinity of the active site, and substrate strain energy and property that have implications for the interpretation of CYP2J2 metabolism.
Topics: Arachidonic Acid; Astemizole; Butyrophenones; Catalytic Domain; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Humans; Hydrophobic and Hydrophilic Interactions; Kinetics; Molecular Docking Simulation; Oxidation-Reduction; Piperidines; Protein Binding; Substrate Specificity
PubMed: 33335233
DOI: 10.1038/s41598-020-79284-0 -
Oxidative Medicine and Cellular... 20206,8-Diprenylorobol is a phytochemical derived from the roots of Fisch. 6,8-Diprenylorobol exhibits several biological activities, but the effects of 6,8-diprenylorobol...
6,8-Diprenylorobol is a phytochemical derived from the roots of Fisch. 6,8-Diprenylorobol exhibits several biological activities, but the effects of 6,8-diprenylorobol on cancers have been hardly investigated. This study is aimed at elucidating the anticancer effect and working mechanism of 6,8-diprenylorobol in HepG2 and Huh-7, two kinds of human hepatocellular carcinoma (HCC) cell lines. WST-1, cell counting, and colony formation assays and morphological change analysis showed that 6,8-diprenylorobol treatment decreased the cell viability and proliferation rate. Cell cycle analysis indicated that 6,8-diprenylorobol treatment increased the population of the G1/0 stage. Annexin V/PI double staining and TUNEL analysis showed that 6,8-diprenylorobol treatment increased the apoptotic cell population and DNA fragmentation. Western blot analysis showed that 6,8-diprenylorobol treatment increased the expression of cleaved PARP1, cleaved caspase-3, FOXO3, Bax, Bim, p21, and p27 but decreased the expression of Bcl2 and BclXL. Interestingly, 6,8-diprenylorobol inhibited CYP2J2-mediated astemizole -demethylation and ebastine hydroxylase activities with values of 9.46 and 2.61 M, respectively. CYP2J2 siRNA transfection enhanced the anticancer effect of 6,8-diprenylorobol in HepG2 and Huh-7 cells through the downregulation of CYP2J2 protein expression and upregulation of FOXO3. Taken together, this study proposes that 6,8-diprenylorobol treatment may be a useful therapeutic option against HCC by targeting CYP2J2 and FOXO3.
Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Hepatocellular; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Forkhead Box Protein O3; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Neoplasm Proteins
PubMed: 33312341
DOI: 10.1155/2020/8887251 -
Expert Opinion on Drug Safety Jan 2021: This study aimed to measure the association of various H1-antihistamines (H1A) with Torsade de Pointes (TdP), and present a comprehensive overview of H1A-induced...
: This study aimed to measure the association of various H1-antihistamines (H1A) with Torsade de Pointes (TdP), and present a comprehensive overview of H1A-induced TdP cases reported to the Food and Drug Administration Adverse Event Reporting System (FAERS). : All H1A-induced TdP cases (n = 406) were retrieved from the FAERS database using the preferred term 'Torsade de Pointes' of MedDRA version-22 from 1990 to 2019. Four data-mining algorithms were used for disproportionality analysis: Reporting Odds Ratio (ROR); Proportional Reporting Ratio (PRR), Empirical Bayes Geometric Mean (EBGM), and Information Content (IC). H1A with >3 TdP cases were included. : A total of 12 signals (Astemizole, cetirizine, chlorpheniramine, clemastine, desloratadine, diphenhydramine, hydroxyzine, loratadine, meclizine, promethazine, terfenadine, and trimeprazine) were identified including six new signals (cetirizine, chlorpheniramine, clemastine, desloratadine, loratadine, and meclizine). The number of risk factors (p = 0.031) and concomitant QT-prolonging drugs (p = <0.001) were significantly lower among new signals vs old signals. Moreover, new signals were strongly associated with QT-prolongation, cardiac reactions, and electrolyte abnormalities as compared with old signals. : Our study found the increased torsadogenic potential of new signals compared with previously known old signals, hence necessitating clinical studies to determine the actual torsadogenic potential of newly identified signals.
Topics: Adolescent; Adult; Adverse Drug Reaction Reporting Systems; Aged; Algorithms; Child; Child, Preschool; Data Mining; Databases, Factual; Female; Histamine H1 Antagonists; Humans; Infant; Long QT Syndrome; Male; Middle Aged; Pharmacovigilance; Risk Factors; Torsades de Pointes; United States; United States Food and Drug Administration; Young Adult
PubMed: 33141610
DOI: 10.1080/14740338.2021.1846717 -
Oncogene Jan 2021Ether-à-go-go-1 (EAG1), one of the potassium channels, is involved in various physiological processes and plays an important role in the tumorigenesis of many kinds of...
Ether-à-go-go-1 (EAG1), one of the potassium channels, is involved in various physiological processes and plays an important role in the tumorigenesis of many kinds of cancer. EAG1 is highly expressed in hepatocarcinoma cells and is closely related to clinical prognosis, but the molecular mechanism remains elusive. In this study, we verified that EAG1 promotes the proliferation of hepatocellular carcinoma (HCC) both in vitro and in vivo. It promotes cell cycle progression by inhibiting the ubiquitination of SKP2. In addition, EAG1 promotes the migration and invasion of HCC by promoting cell pseudopod formation. Furthermore, in a high-pressure plasmid-injected mouse liver orthotopic carcinoma model, astemizole, an EAG family blocker, can significantly inhibit the formation of liver cancer. Meanwhile, liver-specific EAG1 knockout mice show resistance to hepatocarcinogenesis. This research demonstrated that EAG1 plays an important role in the progression of HCC, and could be a potential therapeutic target for HCC.
Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Ether-A-Go-Go Potassium Channels; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; Mice; Neoplasm Metastasis; Neoplasm Transplantation; Prognosis; S-Phase Kinase-Associated Proteins; Ubiquitination; Up-Regulation
PubMed: 33097858
DOI: 10.1038/s41388-020-01522-6 -
Drug Metabolism and Disposition: the... Nov 2020Cytochrome P450 2J2 (CYP2J2) enzyme attracts more attention because it not only metabolizes clinical drugs but also mediates the biotransformation of important...
Cytochrome P450 2J2 (CYP2J2) enzyme attracts more attention because it not only metabolizes clinical drugs but also mediates the biotransformation of important endogenous substances and the regulation of physiologic function. Although CYP2J2 is very important, few animal models are available to study its function In particular, a gene knockout (KO) rat model for drug metabolism and pharmacokinetics is not available. In this report, the CRISPR/Cas9 technology was used to delete rat , the orthologous genes of in humans. The KO rats were viable and fertile and showed no off-target effect. Compared with wild-type (WT) rats, the mRNA and protein expression of in liver, small intestine, and heart of KO rats were completely absent. At the same time, mRNA expression and protein expression were significantly decreased in these tissues. Further and metabolic studies of astemizole, a typical substrate of CYP2J, indicated that CYP2J was functionally inactive in KO rats. The heart function indexes of WT and KO rats were also measured and compared. The myocardial enzymes, including creatine kinase-muscle brain type (CK-MB), creatine kinase (CK), and CK-MB/CK ratio, of KO rats increased by nearly 140%, 80%, and 60%, respectively. In conclusion, this study successfully developed a new KO rat model, which is a useful tool to study the function of CYP2J in drug metabolism and cardiovascular disease. SIGNIFICANCE STATEMENT: Human CYP2J2 is involved not only in clinical drug metabolism but also in the biotransformation of important endogenous substances. Therefore, it is very important to construct new animal models to study its function . This study successfully developed a new CYP2J knockout rat model by using CRISPR/Cas9 technology. This rat model provides a useful tool to study the role of CYP2J in drug metabolism and diseases.
Topics: Animals; Astemizole; Biotransformation; CRISPR-Cas Systems; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Drug Evaluation, Preclinical; Feasibility Studies; Female; Gene Knockdown Techniques; Male; Models, Animal; Rats; Rats, Transgenic
PubMed: 32878767
DOI: 10.1124/dmd.120.000114 -
Biomedicines Jul 2020Adrenocortical carcinoma (ACC) presents a high risk of relapse and metastases with outcomes not improving despite extensive research and new targeted therapies. We...
Adrenocortical carcinoma (ACC) presents a high risk of relapse and metastases with outcomes not improving despite extensive research and new targeted therapies. We recently showed that the Hedgehog receptor Patched is expressed in ACC, where it strongly contributes to doxorubicin efflux and treatment resistance. Here, we report the identification of a new inhibitor of Patched drug efflux, the anti-histaminergic drug astemizole. We show that astemizole enhances the cytotoxic, proapoptotic, antiproliferative and anticlonogenic effects of doxorubicin on ACC cells at concentrations of astemizole or doxorubicin that are not effective by themselves. Our results suggest that a low concentration of astemizole sensitizes ACC cells to doxorubicin, which is a component of the standard treatment for ACC composed of etoposide, doxorubicin, cisplatin and mitotane (EDPM). Patched uses the proton motive force to efflux drugs. This makes its function specific to cancer cells, thereby avoiding toxicity issues that are commonly observed with inhibitors of ABC multidrug transporters. Our data provide strong evidence that the use of astemizole or a derivative in combination with EDPM could be a promising therapeutic option for ACC by increasing the treatment effectiveness at lower doses of EDPM, which would reduce the severe side effects of this regimen.
PubMed: 32751066
DOI: 10.3390/biomedicines8080251 -
Journal of Pharmacological and... Sep 2020Screening compounds for activity on the hERG channel using patch clamp is a crucial part of safety testing. Automated patch clamp (APC) is becoming widely accepted as an...
Reliable identification of cardiac liability in drug discovery using automated patch clamp: Benchmarking best practices and calibration standards for improved proarrhythmic assessment.
INTRODUCTION
Screening compounds for activity on the hERG channel using patch clamp is a crucial part of safety testing. Automated patch clamp (APC) is becoming widely accepted as an alternative to manual patch clamp in order to increase throughput whilst maintaining data quality. In order to standardize APC experiments, we have investigated the effects on IC values under different conditions using several devices across multiple sites.
METHODS
APC instruments SyncroPatch 384i, SyncroPatch 384PE and Patchliner, were used to record hERG expressed in HEK or CHO cells. Up to 27 CiPA compounds were used to investigate effects of voltage protocol, incubation time, labware and time between compound preparation and experiment on IC values.
RESULTS
All IC values of 21 compounds recorded on the SyncroPatch 384PE correlated well with IC values from the literature (Kramer et al., 2013) regardless of voltage protocol or labware, when compounds were used immediately after preparation, but potency of astemizole decreased if prepared in Teflon or polypropylene (PP) compound plates 2-3 h prior to experiments. Slow acting compounds such as dofetilide, astemizole, and terfenadine required extended incubation times of at least 6 min to reach steady state and therefore, stable IC values.
DISCUSSION
Assessing the influence of different experimental conditions on hERG assay reliability, we conclude that either the step-ramp protocol recommended by CiPA or a standard 2-s step-pulse protocol can be used to record hERG; a minimum incubation time of 5 min should be used and although glass, Teflon, PP or polystyrene (PS) compound plates can be used for experiments, caution should be taken if using Teflon, PS or PP vessels as some adsorption can occur if experiments are not performed immediately after preparation. Our recommendations are not limited to the APC devices described in this report, but could also be extended to other APC devices.
Topics: Animals; Arrhythmias, Cardiac; Astemizole; Benchmarking; CHO Cells; Calibration; Cardiovascular Agents; Cell Line; Cricetulus; Drug Discovery; Drug Evaluation, Preclinical; ERG1 Potassium Channel; HEK293 Cells; Heart; Humans; Patch-Clamp Techniques; Phenethylamines; Polypropylenes; Polytetrafluoroethylene; Reference Standards; Reproducibility of Results; Sulfonamides; Terfenadine
PubMed: 32565325
DOI: 10.1016/j.vascn.2020.106884 -
Biophysical Journal May 2020High-throughput in vitro drug assays have been impacted by recent advances in human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) technology and by...
High-throughput in vitro drug assays have been impacted by recent advances in human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) technology and by contact-free all-optical systems simultaneously measuring action potentials (APs) and Ca transients (CaTrs). Parallel computational advances have shown that in silico simulations can predict drug effects with high accuracy. We combine these in vitro and in silico technologies and demonstrate the utility of high-throughput experimental data to refine in silico hiPSC-CM populations and to predict and explain drug action mechanisms. Optically obtained hiPSC-CM APs and CaTrs were used from spontaneous activity and under optical pacing in control and drug conditions at multiple doses. An updated version of the Paci2018 model was developed to refine the description of hiPSC-CM spontaneous electrical activity; a population of in silico hiPSC-CMs was constructed and calibrated using simultaneously recorded APs and CaTrs. We tested in silico five drugs (astemizole, dofetilide, ibutilide, bepridil, and diltiazem) and compared the outcomes to in vitro optical recordings. Our simulations showed that physiologically accurate population of models can be obtained by integrating AP and CaTr control records. Thus, constructed population of models correctly predicted the drug effects and occurrence of adverse episodes, even though the population was optimized only based on control data and in vitro drug testing data were not deployed during its calibration. Furthermore, the in silico investigation yielded mechanistic insights; e.g., through simulations, bepridil's more proarrhythmic action in adult cardiomyocytes compared to hiPSC-CMs could be traced to the different expression of ion currents in the two. Therefore, our work 1) supports the utility of all-optical electrophysiology in providing high-content data to refine experimentally calibrated populations of in silico hiPSC-CMs, 2) offers insights into certain limitations when translating results obtained in hiPSC-CMs to humans, and 3) shows the strength of combining high-throughput in vitro and population in silico approaches.
Topics: Action Potentials; Adult; Computer Simulation; Drug Evaluation; Humans; Induced Pluripotent Stem Cells; Myocytes, Cardiac
PubMed: 32298635
DOI: 10.1016/j.bpj.2020.03.018 -
Biofabrication Feb 2020Current practices in drug development have led to therapeutic compounds being approved for widespread use in humans, only to be later withdrawn due to unanticipated...
Current practices in drug development have led to therapeutic compounds being approved for widespread use in humans, only to be later withdrawn due to unanticipated toxicity. These occurrences are largely the result of erroneous data generated by in vivo and in vitro preclinical models that do not accurately recapitulate human physiology. Herein, a human primary cell- and stem cell-derived 3D organoid technology is employed to screen a panel of drugs that were recalled from market by the FDA. The platform is comprised of multiple tissue organoid types that remain viable for at least 28 days, in vitro. For many of these compounds, the 3D organoid system was able to demonstrate toxicity. Furthermore, organoids exposed to non-toxic compounds remained viable at clinically relevant doses. Additional experiments were performed on integrated multi-organoid systems containing liver, cardiac, lung, vascular, testis, colon, and brain. These integrated systems proved to maintain viability and expressed functional biomarkers, long-term. Examples are provided that demonstrate how multi-organoid 'body-on-a-chip' systems may be used to model the interdependent metabolism and downstream effects of drugs across multiple tissues in a single platform. Such 3D in vitro systems represent a more physiologically relevant model for drug screening and will likely reduce the cost and failure rate associated with the approval of new drugs.
Topics: Astemizole; Capecitabine; Cell Culture Techniques; Cell Survival; Cells, Cultured; Heart Rate; Humans; Lab-On-A-Chip Devices; Liver; Myocardium; Organoids; Pharmaceutical Preparations; Spheroids, Cellular
PubMed: 32101533
DOI: 10.1088/1758-5090/ab6d36 -
Genes Jan 2020Retinoblastoma is the most common pediatric intraocular malignant tumor. Unfortunately, low cure rates and low life expectancy are observed in low-income countries....
Retinoblastoma is the most common pediatric intraocular malignant tumor. Unfortunately, low cure rates and low life expectancy are observed in low-income countries. Thus, alternative therapies are needed for patients who do not respond to current treatments or those with advanced cases of the disease. (Eag1) is a voltage-gated potassium channel involved in cancer. expression is upregulated by the human papilloma virus (HPV) oncogene E7, suggesting that retinoblastoma protein (pRb) may regulate . Astemizole is an antihistamine that is suggested to be repurposed for cancer treatment; it targets proteins implicated in cancer, including histamine receptors, ATP binding cassette transporters, and Eag channels. Here, we investigated Eag1 regulation using pRb and Eag1 expression in human retinoblastoma. The effect of astemizole on the cell proliferation of primary human retinoblastoma cultures was also studied. HeLa cervical cancer cells (HPV-positive and expressing Eag1) were transfected with . mRNA expression was studied using qPCR, and protein expression was assessed using western blotting and immunochemistry. Cell proliferation was evaluated with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. transfection down-regulated Eag1 mRNA and protein expression. The human retinoblastoma samples displayed heterogeneous Eag1 mRNA and protein expression. Astemizole decreased cell proliferation in primary retinoblastoma cultures. Our results suggest that Eag1 mRNA and protein expression was regulated by pRb in vitro, and that human retinoblastoma tissues had heterogeneous Eag1 mRNA and protein expression. Furthermore, our results propose that the multitarget drug astemizole may have clinical relevance in patients with retinoblastoma, for instance, in those who do not respond to current treatments.
Topics: Astemizole; Cell Line, Tumor; Cell Proliferation; Child, Preschool; Ether-A-Go-Go Potassium Channels; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Infant; Male; Oncogenes; RNA, Messenger; Retinal Neoplasms; Retinoblastoma; Retinoblastoma Protein; Transfection
PubMed: 31973216
DOI: 10.3390/genes11020119