Faculty Opinions recommendation of High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split Renilla luciferase.

2004 ◽  
Author(s):  
Sjef Smeekens
Keyword(s):  
High Throughput ◽  
Nuclear Transport ◽  
Noninvasive Imaging ◽  
Renilla Luciferase

scholarly journals A Dual Luciferase Multiplexed High-Throughput Screening Platform for Protein-Protein Interactions

2003 ◽  
Vol 8 (6) ◽  
pp. 676-684 ◽  
Author(s):  
Bart W. Nieuwenhuijsen ◽  
Youping Huang ◽  
Yuren Wang ◽  
Fernando Ramirez ◽  
Gary Kalgaonkar ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Small Molecule ◽  
High Throughput ◽  
Protein Interactions ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Rgs Proteins ◽  
Protein Protein Interactions ◽  
Luciferase Reporter Gene ◽  
Small Molecule Modulators

To study the biology of regulators of G-protein signaling (RGS) proteins and to facilitate the identification of small molecule modulators of RGS proteins, the authors recently developed an advanced yeast 2-hybrid (YTH) assay format for GαZand RGS-Z1. Moreover, they describe the development of a multiplexed luciferase-based assay that has been successfully adapted to screen large numbers of small molecule modulators of protein-protein interactions. They generated and evaluated 2 different luciferase reporter gene systems for YTH interactions, a Gal4 responsive firefly luciferase reporter gene and a Gal4 responsive Renilla luciferase reporter gene. Both the firefly and Renilla luciferase reporter genes demonstrated a 40-to 50-fold increase in luminescence in strains expressing interacting YTH fusion proteins versus negative control strains. Because the firefly and Renilla luciferase proteins have different substrate specificity, the assays were multiplexed. The multiplexed luciferase-based YTH platform adds speed, sensitivity, simplicity, quantification, and efficiency to YTH high-throughput applications and therefore greatly facilitates the identification of small molecule modulators of protein-protein interactions as tools or potential leads for drug discovery efforts.

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.

High-Throughput, Noninvasive Imaging of Root Systems

2012 ◽  
pp. 177-187 ◽  
Author(s):  
Anjali S. Iyer-Pascuzzi ◽  
Paul R. Zurek ◽  
Philip N. Benfey
Keyword(s):  
High Throughput ◽  
Noninvasive Imaging ◽  
Root Systems

scholarly journals Identifying Initiation and Elongation Inhibitors of Dengue Virus RNA Polymerase in a High-Throughput Lead-Finding Campaign

2014 ◽  
Vol 20 (1) ◽  
pp. 153-163 ◽  
Author(s):  
Thomas M. Smith ◽  
Siew Pheng Lim ◽  
Kimberley Yue ◽  
Scott A. Busby ◽  
Rishi Arora ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Rna Polymerase ◽  
High Throughput ◽  
Rna Synthesis ◽  
De Novo ◽  
Antiviral Drug ◽  
Viral Rna ◽  
Renilla Luciferase ◽  
Viral Pathogen ◽  
Open Conformation

Dengue virus (DENV) is the most significant mosquito-borne viral pathogen in the world and is the cause of dengue fever. The DENV RNA-dependent RNA polymerase (RdRp) is conserved among the four viral serotypes and is an attractive target for antiviral drug development. During initiation of viral RNA synthesis, the polymerase switches from a “closed” to “open” conformation to accommodate the viral RNA template. Inhibitors that lock the “closed” or block the “open” conformation would prevent viral RNA synthesis. Herein, we describe a screening campaign that employed two biochemical assays to identify inhibitors of RdRp initiation and elongation. Using a DENV subgenomic RNA template that promotes RdRp de novo initiation, the first assay measures cytosine nucleotide analogue (Atto-CTP) incorporation. Liberated Atto fluorophore allows for quantification of RdRp activity via fluorescence. The second assay uses the same RNA template but is label free and directly detects RdRp-mediated liberation of pyrophosphates of native ribonucleotides via liquid chromatography–mass spectrometry. The ability of inhibitors to bind and stabilize a “closed” conformation of the DENV RdRp was further assessed in a differential scanning fluorimetry assay. Last, active compounds were evaluated in a renilla luciferase–based DENV replicon cell-based assay to monitor cellular efficacy. All assays described herein are medium to high throughput, are robust and reproducible, and allow identification of inhibitors of the open and closed forms of DENV RNA polymerase.

scholarly journals Development of a High-Throughput Screening Strategy for Upregulators of the OPG/RANKL Ratio with the Potential for Antiosteoporosis Effects

2016 ◽  
Vol 21 (7) ◽  
pp. 738-748 ◽  
Author(s):  
Shiqiang Gong ◽  
Xiaowan Han ◽  
Xuehong Li ◽  
Jun Yang ◽  
Xiaobo He ◽  
...  
Keyword(s):  
Small Molecules ◽  
High Throughput ◽  
High Throughput Screening ◽  
Firefly Luciferase ◽  
Screening Strategy ◽  
Effective Concentration ◽  
Renilla Luciferase ◽  
Bone Microenvironment ◽  
Receptor Activator

The ratio between osteoprotegerin (OPG) and the receptor activator of NF-κB ligand (RANKL) in the bone microenvironment indicates the level of osteoclastogenesis, and upregulation of this ratio would improve osteoporosis. In this study, we established a novel high-throughput screening (HTS) system using two stably transfected monoclonal cell lines that either express firefly luciferase under the OPG promoter control or concurrently express firefly and renilla luciferases under control of the OPG and RANKL promoters, respectively. With this system, we can conveniently and rapidly detect the effects of compounds on the expression of OPG and RANKL through changes in firefly and renilla luciferase activities. A total of 8160 compounds were screened using this system, yielding five compounds without previously reported activity. The compound with greatest potential is E05657 with high activity and low effective concentration in the HTS system. It increases the OPG/RANKL ratio and OPG secretion, decreases the NFATc1 expression, and reduces osteoclastogenesis in vitro. These results indicate that this novel HTS system can be used to identify small molecules with potential antiosteoporosis effects, and E05657 is a promising lead compound as a novel antiosteoporosis drug.

Development of a High-Throughput Human HepG2 Dual Luciferase Assay for Detection of Metabolically Activated Hepatotoxicants and Genotoxicants

2009 ◽  
Vol 28 (3) ◽  
pp. 162-176 ◽  
Author(s):  
Xuemei Liu ◽  
Jeffrey A. Kramer ◽  
Yi Hu ◽  
James M. Schmidt ◽  
Jianghong Jiang ◽  
...  
Keyword(s):  
High Throughput ◽  
Stress Responses ◽  
Liver Toxicity ◽  
Metabolic Activation ◽  
Negative Control ◽  
Renilla Luciferase ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Cmv Promoter ◽  
Dual Luciferase Assay

Hepatic toxicity remains a major concern for drug failure; therefore, a thorough examination of chemically induced liver toxicity is essential for a robust safety evaluation. Current hypotheses suggest that the metabolic activation of a drug to a reactive intermediate is an important process. In this article, we describe a new high-throughput GADD45β reporter assay developed for assessing potential liver toxicity. Most importantly, this assay utilizes a human cell line and incorporates metabolic activation and thus provides significant advantage over other comparable assays used to determine hepatotoxicity. Our assay has low compound requirement and relies upon 2 reporter genes cotransfected into the HepG2 cells. The gene encoding Renilla luciferase is fused to the CMV promoter and provides a control for cell numbers. The firefly luciferase gene is fused to the GADD45β promoter and used to report an increase in DNA damage. A dual luciferase assay is performed by measuring the firefly and Renilla luciferase activities in the same sample. Results are expressed as the ratio of the 2 luciferase activities; increases over the control are interpreted as evidence of stress responses. This mammalian dual luciferase reporter has been characterized with, and without, metabolic activation using positive and negative control agents. Our data demonstrate that this assay provides for an assessment of potential toxic metabolites, is adaptable to a high-throughput platform, and yields data that accurately and reproducibly detect hepatotoxicants.

scholarly journals A novel quantitative high-throughput screen identifies drugs that both activate SUMO conjugation via the inhibition of microRNAs 182 and 183 and facilitate neuroprotection in a model of oxygen and glucose deprivation

2015 ◽  
Vol 36 (2) ◽  
pp. 426-441 ◽  
Author(s):  
Joshua D Bernstock ◽  
Yang-ja Lee ◽  
Luca Peruzzotti-Jametti ◽  
Noel Southall ◽  
Kory R Johnson ◽  
...  
Keyword(s):  
Small Molecules ◽  
High Throughput ◽  
Cortical Neurons ◽  
High Throughput Screening ◽  
Neuroblastoma Cell ◽  
Glucose Deprivation ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Human Neuroblastoma Cell ◽  
Human Neuroblastoma

The conjugation/de-conjugation of Small Ubiquitin-like Modifier (SUMO) has been shown to be associated with a diverse set of physiologic/pathologic conditions. The clinical significance and ostensible therapeutic utility offered via the selective control of the global SUMOylation process has become readily apparent in ischemic pathophysiology. Herein, we describe the development of a novel quantitative high-throughput screening (qHTS) system designed to identify small molecules capable of increasing SUMOylation via the regulation/inhibition of members of the microRNA (miRNA)-182 family. This assay employs a SHSY5Y human neuroblastoma cell line stably transfected with a dual firefly-Renilla luciferase reporter system for identification of specific inhibitors of either miR-182 or miR-183. In this study, we have identified small molecules capable of inducing increased global conjugation of SUMO in both SHSY5Y cells and rat E18-derived primary cortical neurons. The protective effects of a number of the identified compounds were confirmed via an in vitro ischemic model (oxygen/glucose deprivation). Of note, this assay can be easily repurposed to allow high-throughput analyses of the potential drugability of other relevant miRNA(s) in ischemic pathobiology.

scholarly journals High-Throughput Luciferase Reporter Assay for Small-Molecule Inhibitors of MicroRNA Function

2012 ◽  
Vol 17 (6) ◽  
pp. 822-828 ◽  
Author(s):  
Colleen M. Connelly ◽  
Meryl Thomas ◽  
Alexander Deiters
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Hepatoma Cell ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Hepatoma Cell Line ◽  
Messenger Rnas ◽  
Reporter Assay ◽  
Human Hepatoma Cell

MicroRNAs (miRNAs) are endogenous, single-stranded, noncoding RNAs of 21 to 23 nucleotides that regulate gene expression, typically by binding the 3′ untranslated regions of target messenger RNAs. It is estimated that miRNAs are involved in the regulation of 30% of all genes and almost every genetic pathway. Recently, the misregulation of miRNAs has been linked to various human diseases including cancer and viral infections, identifying miRNAs as potential targets for drug discovery. Thus, small-molecule modifiers of miRNAs could serve as lead structures for the development of new therapeutic agents and be useful tools in the elucidation of detailed mechanisms of miRNA function. As a result, we have developed a high-throughput screen for potential small-molecule regulators of the liver-specific microRNA miR-122, which is involved in hepatocellular carcinoma development and hepatitis C virus infection. Our small-molecule screen employs a Huh7 human hepatoma cell line stably transfected with a Renilla luciferase sensor for endogenous miR-122. The assay was optimized and validated using an miR-122 antisense agent and a previously identified small-molecule miR-122 inhibitor. The described reporter assay will enable the high-throughput screening of small-molecule miR-122 inhibitors and can be readily extended to other miRNAs.

scholarly journals An AlphaScreen®-Based Assay for High-Throughput Screening for Specific Inhibitors of Nuclear Import

2011 ◽  
Vol 16 (2) ◽  
pp. 192-200 ◽  
Author(s):  
Kylie M. Wagstaff ◽  
Stephen M. Rawlinson ◽  
Anna C. Hearps ◽  
David A. Jans
Keyword(s):  
High Throughput ◽  
Nuclear Import ◽  
High Throughput Screening ◽  
Protein Import ◽  
Nuclear Transport ◽  
Dna Interaction ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Importin Α ◽  
Specific Inhibitors

Specific viral proteins enter the nucleus of infected cells to perform essential functions, as part of the viral life cycle. The integrase (IN) molecule of human immunodeficiency virus (HIV)–1 is of particular interest in this context due to its integral role in integrating the HIV genome into that of the infected host cell. Most IN-based antiviral compounds target the IN/DNA interaction, but since IN must first enter the nucleus before it can perform these critical functions, nuclear transport of IN is also an attractive target for therapeutic intervention. Here the authors describe a novel high-throughput screening assay for identifying inhibitors of nuclear import, particularly IN, based on amplified luminescent proximity homogeneous assay (AlphaScreen®) technology, which is high throughput, requires low amounts of material, and is efficient and cost-effective. The authors use the assay to screen for specific inhibitors of the interaction between IN and its nuclear transport receptor importin α/β, successfully identifying several inhibitors of the IN/importin α/β interaction. Importantly, they demonstrate that one of the identified compounds, mifepristone, is effective in preventing active nuclear transport of IN in transfected cells and hence may represent a useful anti-HIV therapeutic. The screen also identified broad-spectrum importin α/β inhibitors such as ivermectin, which may represent useful tools for nuclear transport research in the future. The authors validate the activity and specificity of mifepristone and ivermectin in inhibiting nuclear protein import in living cells, underlining the utility of the screening approach.

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