scholarly journals Establishment of a stable SARS-CoV-2 replicon system for application in high-throughput screening.

2021 ◽  
Author(s):  
Tomohisa Tanaka ◽  
Akatsuki Saito ◽  
Tatsuya Suzuki ◽  
Yoichi Miyamoto ◽  
Kazuo Takayama ◽  
...  
Keyword(s):  
Viral Replication ◽  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Luciferase Activity ◽  
Fusion Gene ◽  
Viral Proteins ◽  
Renilla Luciferase ◽  
Interferon Β ◽  
Subgenomic Replicon

Experiments with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are limited by the need for biosafety level 3 (BSL3) conditions. A SARS-CoV-2 replicon system rather than an in vitro infection system is suitable for antiviral screening since it can be handled under BSL2 conditions and does not produce infectious particles. However, the reported replicon systems are cumbersome because of the need for transient transfection in each assay. In this study, we constructed a bacterial artificial chromosome vector (the replicon-BAC vector) including the SARS-CoV-2 replicon and a fusion gene encoding Renilla luciferase and neomycin phosphotransferase II, examined the antiviral effects of several known compounds, and then established a cell line stably harboring the replicon-BAC vector. Several cell lines transiently transfected with the replicon-BAC vector produced subgenomic replicon RNAs (sgRNAs) and viral proteins, and exhibited luciferase activity. In the transient replicon system, treatment with remdesivir or interferon-β but not with camostat or favipiravir suppressed the production of viral agents and luciferase, indicating that luciferase activity corresponds to viral replication. VeroE6/Rep3, a stable replicon cell line based on VeroE6 cells, was successfully established and continuously produced viral proteins, sgRNAs and luciferase, and their production was suppressed by treatment with remdesivir or interferon-β. Molnupiravir, a novel coronavirus RdRp inhibitor, inhibited viral replication more potently in VeroE6/Rep3 cells than in VeroE6-based transient replicon cells. In summary, our stable replicon system will be a powerful tool for the identification of SARS-CoV-2 antivirals through high-throughput screening.

scholarly journals Potential High-Throughput Assay for Screening Inhibitors of West Nile Virus Replication

2003 ◽  
Vol 77 (23) ◽  
pp. 12901-12906 ◽  
Author(s):  
Michael K. Lo ◽  
Mark Tilgner ◽  
Pei-Yong Shi
Keyword(s):  
West Nile Virus ◽  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Health Priorities ◽  
Stable Cell Line ◽  
West Nile ◽  
Subgenomic Replicon ◽  
Genes Encoding ◽  
High Throughput Assay

ABSTRACT Prevention and treatment of infection by West Nile virus (WNV) and other flaviviruses are public health priorities. We describe a reporting cell line that can be used for high-throughput screening of inhibitors against all targets involved in WNV replication. Dual reporter genes, encoding Renilla luciferase (Rluc) and neomycin phosphotransferase (Neo), were engineered into a WNV subgenomic replicon, resulting in Rluc/NeoRep. Geneticin selection of BHK-21 cells transfected with Rluc/NeoRep yielded a stable cell line that contains persistently replicating replicons. Incubation of the reporting cells with known WNV inhibitors decreased Rluc activity, as well as the replicon RNA level. The efficacies of the inhibitors, as measured by the depression of Rluc activity in the reporting cells, are comparable to those derived from authentic viral infection assays. Therefore, the WNV reporting cell line can be used as a high-throughput assay for anti-WNV drug discovery. A similar approach should be applicable to development of genetics-based antiviral assays for other flaviviruses.

scholarly journals Identification of a juvenile-hormone signaling inhibitor via high-throughput screening of a chemical library

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Takumi Kayukawa ◽  
Kenjiro Furuta ◽  
Keisuke Nagamine ◽  
Tetsuro Shinoda ◽  
Kiyoaki Yonesu ◽  
...  
Keyword(s):  
Juvenile Hormone ◽  
Cell Line ◽  
Signaling Pathway ◽  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Ex Vivo ◽  
Agricultural Field ◽  
Chemical Library ◽  
Field Development

Abstract Insecticide resistance has recently become a serious problem in the agricultural field. Development of insecticides with new mechanisms of action is essential to overcome this limitation. Juvenile hormone (JH) is an insect-specific hormone that plays key roles in maintaining the larval stage of insects. Hence, JH signaling pathway is considered a suitable target in the development of novel insecticides; however, only a few JH signaling inhibitors (JHSIs) have been reported, and no practical JHSIs have been developed. Here, we established a high-throughput screening (HTS) system for exploration of novel JHSIs using a Bombyx mori cell line (BmN_JF&AR cells) and carried out a large-scale screening in this cell line using a chemical library. The four-step HTS yielded 69 compounds as candidate JHSIs. Topical application of JHSI48 to B. mori larvae caused precocious metamorphosis. In ex vivo culture of the epidermis, JHSI48 suppressed the expression of the Krüppel homolog 1 gene, which is directly activated by JH-liganded receptor. Moreover, JHSI48 caused a parallel rightward shift in the JH response curve, suggesting that JHSI48 possesses a competitive antagonist-like activity. Thus, large-scale HTS using chemical libraries may have applications in development of future insecticides targeting the JH signaling pathway.

scholarly journals Phenothiazine Neuroleptics Signal to the Human Insulin Promoter as Revealed by a Novel High-Throughput Screen

2010 ◽  
Vol 15 (6) ◽  
pp. 663-670 ◽  
Author(s):  
Alice Kiselyuk ◽  
Suzette Farber-Katz ◽  
Tom Cohen ◽  
Seung-Hee Lee ◽  
Ifat Geron ◽  
...  
Keyword(s):  
Cell Line ◽  
Human Insulin ◽  
High Throughput ◽  
High Throughput Screening ◽  
Promoter Activity ◽  
Central Element ◽  
Bhlh Transcription Factor ◽  
Β Cell ◽  
Insulin Promoter ◽  
Short Term Exposure

A number of diabetogenic stimuli interact to influence insulin promoter activity, making it an attractive target for both mechanistic studies and therapeutic interventions. High-throughput screening (HTS) for insulin promoter modulators has the potential to reveal novel inputs into the control of that central element of the pancreatic β-cell. A cell line from human islets in which the expression of insulin and other β-cell-restricted genes are modulated by an inducible form of the bHLH transcription factor E47 was developed. This cell line, T6PNE, was adapted for HTS by transduction with a vector expressing green fluorescent protein under the control of the human insulin promoter. The resulting cell line was screened against a library of known drugs for those that increase insulin promoter activity. Members of the phenothiazine class of neuroleptics increased insulin gene expression upon short-term exposure. Chronic treatment, however, resulted in suppression of insulin promoter activity, consistent with the effect of phenothiazines observed clinically to induce diabetes in chronically treated patients. In addition to providing insights into previously unrecognized targets and mechanisms of action of phenothiazines, the novel cell line described here provides a broadly applicable platform for mining new molecular drug targets and central regulators of β-cell differentiated function.

scholarly journals Use of Leishmania donovani Field Isolates Expressing the Luciferase Reporter Gene in In Vitro Drug Screening

2005 ◽  
Vol 49 (9) ◽  
pp. 3776-3783 ◽  
Author(s):  
Ashutosh ◽  
Suman Gupta ◽  
Ramesh ◽  
Shyam Sundar ◽  
Neena Goyal
Keyword(s):  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
Leishmania Donovani ◽  
Luciferase Activity ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Field Isolates

ABSTRACT Currently available primary screens for the selection of candidate antileishmanial compounds are not ideal. These techniques are time-consuming, laborious, and difficult to scale and require macrophages, which limit their use for high-throughput screening. We have developed Leishmania donovani field isolates that constitutively express the firefly luciferase reporter gene (luc) as a part of an episomal vector. An excellent correlation between parasite number and luciferase activity was observed. luc expression was stable, even in the absence of drug selection, for 4 weeks. The transfectants were infective to macrophages, and intracellular amastigotes exhibited luciferase activity. The suitability of these recombinant field isolates for in vitro screening of antileishmanial drugs was established. The luciferase-expressing sodium stibogluconate-resistant cell lines offer a model for the screening of compounds for resistance. The system is in routine use at the Central Drug Research Institute, Lucknow, India, for high-throughput screening of newly synthesized compounds.

scholarly journals A High-Throughput Screening Platform Targeting PDLIM5 for Pulmonary Hypertension

2016 ◽  
Vol 21 (4) ◽  
pp. 333-341 ◽  
Author(s):  
Han Cheng ◽  
Tianji Chen ◽  
Merve Tor ◽  
Deborah Park ◽  
Qiyuan Zhou ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Drug Discovery ◽  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Pdz Domain ◽  
Luciferase Reporter ◽  
Targeted Drug ◽  
Screening Platform ◽  
Targeted Drug Discovery

Pulmonary arterial hypertension is a complex disease with multiple etiologic factors. PDLIM5, a member of the Enigma subfamily of PDZ and LIM domain protein family, contains an N-terminal PDZ domain and three LIM domains at its C-terminus. We have previously shown that overexpression of PDLIM5 prevents hypoxia-induced pulmonary hypertension (PH), and deletion of PDLIM5 in smooth muscle cells enhances hypoxia-induced PH in vivo. These results suggest that PDLIM5 may be a novel therapeutic target of PH. In this study, we aim to establish a high-throughput screening platform for PDLIM5-targeted drug discovery. We generated a stable mink lung epithelial cell line (MLEC) containing a transforming growth factor–β/Smad luciferase reporter with lentivirus-mediated suppression of PDLIM5 (MLEC-shPDLIM5) and measured levels of Smad2/3 and pSmad2/3. We found that in MLEC, suppression of PDLIM5 decreased Smad-dependent luciferase activity, Smad3, and pSmad3. We used MLEC-shPDLIM5 and a control cell line (MLEC-shCTL) to screen the Prestwick library (1200 compounds) and identified and validated paclitaxel as a PDLIM5 inhibitor in MLEC. Furthermore, we showed that paclitaxel inhibited Smad2 expression and Smad3 phosphorylation in A549 cells. Our study suggests that this system is robust and suitable for PDLIM5-targeted drug discovery.

scholarly journals Identification of Novel Human High-Density Lipoprotein Receptor Up-regulators Using a Cell-Based High-Throughput Screening Assay

2007 ◽  
Vol 12 (2) ◽  
pp. 211-219 ◽  
Author(s):  
Yuan Yang ◽  
Zhongbing Zhang ◽  
Wei Jiang ◽  
Lei Gao ◽  
Guiyu Zhao ◽  
...  
Keyword(s):  
High Density Lipoprotein ◽  
Cell Line ◽  
High Throughput ◽  
Hepg2 Cells ◽  
High Throughput Screening ◽  
Density Lipoprotein ◽  
High Density ◽  
Luciferase Reporter ◽  
Stable Cell Line ◽  
Active Compounds

Scavenger receptor class B type I (SR-BI) is the high-affinity high-density lipoprotein (HDL) receptor, and CLA-1 is the human homologue of the murine SR-BI. CLA-1/SR-BI receptor has been suggested as a new preventative and/or therapeutic target for atherosclerosis due to its pivotal role in overall HDL cholesterol (HDL-C) metabolism and its antiatherogenic activity in vivo. To search for active compounds that can increase CLA-1 transcription, a novel cell-based assay was developed for application in high-throughput screening (HTS). Human hepatoma HepG2 cells were transfected with a CLA-1-promoter-luciferase reporter gene construct, and the stable transfected cell line was selected and named CLAp-LUC HepG2. With rosiglitazone as a positive control, this stable cell line was used to establish a specific CLA-1 gene expression assay in a 96-well microplate format. The evaluating parameter Z' value of 0.64 showed that this cell-based HTS assay was robust and reliable. Screening of 6000 microbial secondary metabolite crude extracts identified 8 positive strains. Between 2 identified CLA-1 up-regulators produced by actinomycete strain 04-4776, 4776B may stimulate not only the expression of CLA-1 on the transcriptional and translational levels but also the activity of CLA-1 to uptake the HDL-C in HepG2 cells. The active compounds originated from this HTS assay may be developed to drug candidates or lead compounds for new antiatherosclerosis agents.

scholarly journals An Isogenic Cell Line Panel for Sequence-based Screening of Targeted Anti-cancer Drugs

2021 ◽  
Author(s):  
Ashley L Cook ◽  
Nicolas Wyhs ◽  
Surojit B Sur ◽  
Blair Ptak ◽  
Maria Popoli ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Screening ◽  
Growth Inhibitor ◽  
Suppressor Gene ◽  
Loss Of Function ◽  
Cancer Pathways ◽  
Isogenic Cell Line ◽  
Cell Line Panel

We describe the creation and characterization of an isogenic cell line panel representing common cancer pathways, with multiple features optimized for high-throughput screening. More than 1,800 cell lines from three normal human cells were generated using CRISPR-technologies. Surprisingly, we discovered most of these lines did not result in complete gene inactivation, despite integration of sgRNA at the desired genomic site. However, a subset of the lines harbored true, biallelic disruptions of the targeted tumor suppressor gene, yielding a final panel of 100 well-characterize lines covering 19 pathways frequently subject to loss of function in cancers. This panel included genetic markers optimized for sequence-based ratiometric assays for drug-based screening assays. To illustrate the potential utility of this panel, we developed a multiplexed high-throughput screen that identified Wee1 inhibitor MK-1775 as a selective growth inhibitor of cells with inactivation of TP53. These cell lines and screening approach should prove useful for researchers studying a variety of cellular and biochemical phenomena.

scholarly journals Development and utilization of human decidualization reporter cell line uncovers new modulators of female fertility

2019 ◽  
Vol 116 (39) ◽  
pp. 19541-19551
Author(s):  
Meade Haller ◽  
Yan Yin ◽  
Liang Ma
Keyword(s):  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Patient Specific ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
A Genome ◽  
Decidual Cells

Failure of embryo implantation accounts for a significant percentage of female infertility. Exquisitely coordinated molecular programs govern the interaction between the competent blastocyst and the receptive uterus. Decidualization, the rapid proliferation and differentiation of endometrial stromal cells into decidual cells, is required for implantation. Decidualization defects can cause poor placentation, intrauterine growth restriction, and early parturition leading to preterm birth. Decidualization has not yet been systematically studied at the genetic level due to the lack of a suitable high-throughput screening tool. Herein we describe the generation of an immortalized human endometrial stromal cell line that uses yellow fluorescent protein under the control of the prolactin promoter as a quantifiable visual readout of the decidualization response (hESC-PRLY cells). Using this cell line, we performed a genome-wide siRNA library screen, as well as a screen of 910 small molecules, to identify more than 4,000 previously unrecognized genetic and chemical modulators of decidualization. Ontology analysis revealed several groups of decidualization modulators, including many previously unappreciated transcription factors, sensory receptors, growth factors, and kinases. Expression studies of hits revealed that the majority of decidualization modulators are acutely sensitive to ovarian hormone exposure. Gradient treatment of exogenous factors was used to identify EC50 values of small-molecule hits, as well as verify several growth factor hits identified by the siRNA screen. The high-throughput decidualization reporter cell line and the findings described herein will aid in the development of patient-specific treatments for decidualization-based recurrent pregnancy loss, subfertility, and infertility.

scholarly journals Development of a Cell-Based High-Throughput Screening Assay to Identify Porcine Host Defense Peptide-Inducing Compounds

2018 ◽  
Vol 2018 ◽  
pp. 1-13 ◽  
Author(s):  
Zhuo Deng ◽  
Jing Wang ◽  
Wentao Lyu ◽  
Xuwen Wieneke ◽  
Robert Matts ◽  
...  
Keyword(s):  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Hdac Inhibitors ◽  
Luciferase Reporter ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
A Cell

Novel alternatives to antibiotics are needed for the swine industry, given increasing restrictions on subtherapeutic use of antibiotics. Augmenting the synthesis of endogenous host defense peptides (HDPs) has emerged as a promising antibiotic-alternative approach to disease control and prevention. To facilitate the identification of HDP inducers for swine use, we developed a stable luciferase reporter cell line, IPEC-J2/PBD3-luc, through permanent integration of a luciferase reporter gene driven by a 1.1 kb porcine β-defensin 3 (PBD3) gene promoter in porcine IPEC-J2 intestinal epithelial cells. Such a stable reporter cell line was employed in a high-throughput screening of 148 epigenetic compounds and 584 natural products, resulting in the identification of 41 unique hits with a minimum strictly standardized mean difference (SSMD) value of 3.0. Among them, 13 compounds were further confirmed to give at least a 5-fold increase in the luciferase activity in the stable reporter cell line, with 12 being histone deacetylase (HDAC) inhibitors. Eight compounds were subsequently observed to be comparable to sodium butyrate in inducing PBD3 mRNA expression in parental IPEC-J2 cells in the low micromolar range. Six HDAC inhibitors including suberoylanilide hydroxamine (SAHA), HC toxin, apicidin, panobinostat, SB939, and LAQ824 were additionally found to be highly effective HDP inducers in a porcine 3D4/31 macrophage cell line. Besides PBD3, other HDP genes such as PBD2 and cathelicidins (PG1–5) were concentration-dependently induced by those compounds in both IPEC-J2 and 3D4/31 cells. Furthermore, the antibacterial activities of 3D4/31 cells were augmented following 24 h exposure to HDAC inhibitors. In conclusion, a cell-based high-throughput screening assay was developed for the discovery of porcine HDP inducers, and newly identified HDP-inducing compounds may have potential to be developed as alternatives to antibiotics for applications in swine and possibly other animal species.

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