scholarly journals Affordable Luciferase Reporter Assay for Cell-Based High-Throughput Screening

2012 ◽  
Vol 18 (4) ◽  
pp. 453-461 ◽  
Author(s):  
Ellen Siebring-van Olst ◽  
Christie Vermeulen ◽  
Renee X. de Menezes ◽  
Michael Howell ◽  
Egbert F. Smit ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Firefly Luciferase ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Chemical Components ◽  
Simple Liquid ◽  
Luciferase Reporter Construct ◽  
Reagent Cost ◽  
Firefly Luciferase Gene

The firefly luciferase gene is commonly used in cell-based reporter assays. Convenient luciferase assay reagents for use in high-throughput screening (HTS) are commercially available. However, the high cost of these reagents is not within the means of some academic laboratories. Therefore, we set out to develop an affordable luciferase assay reagent applicable in an HTS format using simple liquid-handling steps. The reagent was homemade from individual chemical components and optimized for luminescence intensity and stability. We determined the minimal concentrations of the most expensive components, dithiothreitol (DTT) and D-luciferin, resulting in a total assay reagent cost of less than 1 cent per sample. Signal stability was maximized by omission of coenzyme A and reduction of DTT concentration. The assay was validated in a high-throughput setting using two cancer cell lines carrying a p53-dependent luciferase reporter construct and siRNAs modulating p53 transcriptional activity. Induction of p53 activity by silencing PPM1D or SYVN1 and reduction of p53 activity by silencing p53 remained constant over a 2-h measurement period, with good assay quality (Z′ factors mostly above 0.5). Hence, the luciferase assay described herein can be used for affordable reporter readout in cell-based HTS.

scholarly journals Novel Whole-Cell Antibiotic Biosensors for Compound Discovery

2007 ◽  
Vol 73 (20) ◽  
pp. 6436-6443 ◽  
Author(s):  
Andreas Urban ◽  
Stefan Eckermann ◽  
Beate Fast ◽  
Susanne Metzger ◽  
Matthias Gehling ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Protein Biosynthesis ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Environmental Biology ◽  
Drug Candidates ◽  
Novel Drug

ABSTRACT Cells containing reporters which are specifically induced via selected promoters are used in pharmaceutical drug discovery and in environmental biology. They are used in screening for novel drug candidates and in the detection of bioactive compounds in environmental samples. In this study, we generated and validated a set of five Bacillus subtilis promoters fused to the firefly luciferase reporter gene suitable for cell-based screening, enabling the as yet most-comprehensive high-throughput diagnosis of antibiotic interference in the major biosynthetic pathways of bacteria: the biosynthesis of DNA by the yorB promoter, of RNA by the yvgS promoter, of proteins by the yheI promoter, of the cell wall by the ypuA promoter, and of fatty acids by the fabHB promoter. The reporter cells mainly represent novel antibiotic biosensors compatible with high-throughput screening. We validated the strains by developing screens with a set of 14,000 pure natural products, representing a source of highly diverse chemical entities, many of them with antibiotic activity (6% with anti-Bacillus subtilis activity of ≤25 μg/ml]). Our screening approach is exemplified by the discovery of classical and novel DNA synthesis and translation inhibitors. For instance, we show that the mechanistically underexplored antibiotic ferrimycin A1 selectively inhibits protein biosynthesis.

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 Luciferase Measurements in High Throughput Screening

1996 ◽  
Vol 1 (2) ◽  
pp. 85-88 ◽  
Author(s):  
Alfred J. Kolb ◽  
Kenneth Neumann
Keyword(s):  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
Culture Media ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Cell Culture Media ◽  
Luminescence Signal ◽  
Temperature Controlled

Luminescence assays are becoming more popular in high throughput screening (HTS) laboratories with the luciferase reporter gene being the most common. As with other assays that are adapted to HTS, improvements have been made to the luciferase assay to make it better suited to the requirements of HTS. For the luciferase reporter gene, these improvements included stabilization of the enzyme, increasing the half-life of the luminescence signal to 5 h, and eliminating separation steps (centrifugation and aliquot transfer) after cell lysis. The improved assay, LucLite, is homogeneous and is measured directly in the cell culture media. In addition to reagent improvements, a temperature-controlled, multidetector microplate counter, TopCount, can quickly and accurately measure luminescence signals.

scholarly journals Green Fluorescent Protein Reporter Microplate Assay for High-Throughput Screening of Compounds againstMycobacterium tuberculosis

1998 ◽  
Vol 42 (2) ◽  
pp. 344-347 ◽  
Author(s):  
L. A. Collins ◽  
M. N. Torrero ◽  
S. G. Franzblau
Keyword(s):  
Green Fluorescent Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Low Cost ◽  
Firefly Luciferase ◽  
Drug Susceptibility ◽  
Luciferase Reporter ◽  
Microplate Assay ◽  
Green Fluorescent

ABSTRACT An optimal assay for high-throughput screening for new antituberculosis agents would combine the microplate format and low cost of firefly luciferase reporter assays and redox dyes with the ease of kinetic monitoring inherent in the BACTEC system. The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is a useful reporter molecule which requires neither substrates nor cofactors due to the intrinsically fluorescent nature of the protein. The gene encoding a red-shifted, higher-intensity GFP variant was introduced by electroporation into Mycobacterium tuberculosis H37Ra and M. tuberculosisH37Rv on expression vector pFPV2. A microplate-based fluorescence assay (GFP microplate assay [GFPMA]) was developed and evaluated by determining the MICs of existing antimycobacterial agents. The MICs of isoniazid, rifampin, ethambutol, streptomycin, amikacin, ofloxacin, ethionamide, thiacetazone, and capreomycin, but not cycloserine, determined by GFPMA were within 1 log2dilution of those determined with the BACTEC 460 system and were available in 7 days. Equivalent MICs of antituberculosis agents in the BACTEC 460 system for both the reporter and parent strains suggested that introduction of pFPV2 did not influence drug susceptibility, in general. GFPMA provides a unique tool with which the dynamic response of M. tuberculosis to the existing and potential antituberculosis agents can easily, rapidly, and inexpensively be monitored.

Large-scale High-throughput Screening Revealed 5'-(carbonylamino)-2,3'- bithiophene-4'-carboxylate as Novel Template for Antibacterial Agents

2020 ◽  
Vol 17 (5) ◽  
pp. 716-724
Author(s):  
Yan A. Ivanenkov ◽  
Renat S. Yamidanov ◽  
Ilya A. Osterman ◽  
Petr V. Sergiev ◽  
Vladimir A. Aladinskiy ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Antibacterial Agents ◽  
Sos Response ◽  
Luciferase Assay ◽  
Translational Machinery ◽  
E Coli ◽  
New Class

Background: The key issue in the development of novel antimicrobials is a rapid expansion of new bacterial strains resistant to current antibiotics. Indeed, World Health Organization has reported that bacteria commonly causing infections in hospitals and in the community, e.g. E. Coli, K. pneumoniae and S. aureus, have high resistance vs the last generations of cephalosporins, carbapenems and fluoroquinolones. During the past decades, only few successful efforts to develop and launch new antibacterial medications have been performed. This study aims to identify new class of antibacterial agents using novel high-throughput screening technique. Methods: We have designed library containing 125K compounds not similar in structure (Tanimoto coeff.< 0.7) to that published previously as antibiotics. The HTS platform based on double reporter system pDualrep2 was used to distinguish between molecules able to block translational machinery or induce SOS-response in a model E. coli system. MICs for most active chemicals in LB and M9 medium were determined using broth microdilution assay. Results: In an attempt to discover novel classes of antibacterials, we performed HTS of a large-scale small molecule library using our unique screening platform. This approach permitted us to quickly and robustly evaluate a lot of compounds as well as to determine the mechanism of action in the case of compounds being either translational machinery inhibitors or DNA-damaging agents/replication blockers. HTS has resulted in several new structural classes of molecules exhibiting an attractive antibacterial activity. Herein, we report as promising antibacterials. Two most active compounds from this series showed MIC value of 1.2 (5) and 1.8 μg/mL (6) and good selectivity index. Compound 6 caused RFP induction and low SOS response. In vitro luciferase assay has revealed that it is able to slightly inhibit protein biosynthesis. Compound 5 was tested on several archival strains and exhibited slight activity against gram-negative bacteria and outstanding activity against S. aureus. The key structural requirements for antibacterial potency were also explored. We found, that the unsubstituted carboxylic group is crucial for antibacterial activity as well as the presence of bulky hydrophobic substituents at phenyl fragment. Conclusion: The obtained results provide a solid background for further characterization of the 5'- (carbonylamino)-2,3'-bithiophene-4'-carboxylate derivatives discussed herein as new class of antibacterials and their optimization campaign.

scholarly journals Measurement of Effects of Antibiotics in Bioluminescent Staphylococcus aureus RN4220

2001 ◽  
Vol 45 (12) ◽  
pp. 3456-3461 ◽  
Author(s):  
Mervi Tenhami ◽  
Kaisa Hakkila ◽  
Matti Karp
Keyword(s):  
Staphylococcus Aureus ◽  
High Throughput Screening ◽  
Pathogenic Bacteria ◽  
Antimicrobial Agents ◽  
Light Emission ◽  
Detection System ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Luciferase Reporter Gene

ABSTRACT The spread of antibiotic resistance among pathogenic bacteria is a serious threat to humans and animals. Therefore, unnecessary use should be minimized, and new antimicrobial agents with novel mechanisms of action are needed. We have developed an efficient method for measuring the action of antibiotics which is applied to a gram-positive strain,Staphylococcus aureus RN4220. The method utilizes the firefly luciferase reporter gene coupled to the metal-induciblecadA promoter in a plasmid, pTOO24. Correctly timed induction by micromolar concentrations of antimonite rapidly triggers the luciferase gene transcription and translation. This sensitizes the detection system to the action of antibiotics, and especially for transcriptional and translational inhibitors. We show the results for 11 model antibiotics with the present approach and compare them to an analytical setup with a strain where luciferase expression is under the regulation of a constitutive promoter giving only a report of metabolic inhibition. The measurement of light emission from intact living cells is shown to correlate extremely well (r = 0.99) with the conventional overnight growth inhibition measurement. Four of the antibiotics were within a 20% concentration range and four were within a 60% concentration range of the drugs tested. This approach shortens the assay time needed, and it can be performed in 1 to 4 h, depending on the sensitivity needed. Furthermore, the assay can be automatized for high-throughput screening by the pharmaceutical industry.

scholarly journals Microplate Orbital Mixing Improves High-Throughput Cell-Based Reporter Assay Readouts

2006 ◽  
Vol 12 (1) ◽  
pp. 140-144 ◽  
Author(s):  
Michael K. Hancock ◽  
Myleen N. Medina ◽  
Brendan M. Smith ◽  
Anthony P. Orth
Keyword(s):  
High Throughput ◽  
Dynamic Range ◽  
Cell Lysis ◽  
Single Step ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Activity Measurements ◽  
Simple Detection ◽  
Reporter Assays ◽  
Orbital Mixing

Reporter assays are commonly used for high-throughput cell-based screening of compounds, cDNAs, and siRNAs due to robust signal, ease of miniaturization, and simple detection and analysis. Among the most widely used reporter genes is the bioluminescent enzyme luciferase, which, when exposed to its substrate luciferin upon cell lysis, yields linear signal over a dynamic range of several orders of magnitude. Commercially available luciferase assay formulations have been developed permitting homogeneous, single-step cell lysis and reporter activity measurements. Assay conditions employed with these formulations are typically designed to minimize well-to-well luminescence variability due to variability in dispensing, evaporation, and incomplete sample mixing. The authors demonstrate that incorporating a microplate orbital mixing step into 96- and 384-well microplate cell-based luciferase reporter assays can greatly improve reporter readouts. They have found that orbital mixing using commercially available mixers facilitates maximal luciferase signal generation from high cell density–containing samples while minimizing variability due to partial cell lysis, thereby improving assay precision. The authors fully expect that widespread availability of mixers with sufficiently small orbits and higher speed settings will permit gains in signal and precision in the 1536-well format as well.

scholarly journals Glucose enhances insulin promoter activity in MIN6 β-cells independently of changes in intracellular Ca2+ concentration and insulin secretion

1999 ◽  
Vol 342 (2) ◽  
pp. 275-280 ◽  
Author(s):  
Helen J. KENNEDY ◽  
Imran RAFIQ ◽  
Aristea E. POULI ◽  
Guy A. RUTTER
Keyword(s):  
Promoter Activity ◽  
Photon Counting ◽  
Firefly Luciferase ◽  
Receptor Activation ◽  
Luciferase Reporter ◽  
Β Cells ◽  
Viral Promoter ◽  
Luciferase Reporter Construct ◽  
Insulin Promoter ◽  
Firefly Luciferase Reporter

Recent studies have suggested that glucose may activate insulin gene transcription through increases in intracellular Ca2+ concentration, possibly acting via the release of stored insulin. We have investigated this question by dynamic photon-counting imaging of insulin- and c-fos-promoter-firefly luciferase reporter construct activity. Normalized to constitutive viral promoter activity, insulin promoter activity in MIN6 β-cells was increased 1.6-fold after incubation at 30 mM compared with 3 mM glucose, but was unaltered at either glucose concentration by the presence of insulin (100 nM) or the Ca2+ channel inhibitor, verapamil (100 μM). Increases in intracellular [Ca2+] achieved by plasma membrane depolarization with KCl failed to enhance either insulin or c-fos promoter activity in MIN6 cells, but increased c-fos promoter activity 5-fold in AtT20 cells. Together, these results demonstrate that glucose can exert a direct effect on insulin promoter activity in islet β-cells, via a signalling pathway which does not require increases in intracellular [Ca2+] nor insulin release and insulin receptor activation.

scholarly journals High-Throughput Screening for Linear Ubiquitin Chain Assembly Complex (LUBAC) Selective Inhibitors Using Homogenous Time-Resolved Fluorescence (HTRF)-Based Assay System

2018 ◽  
Vol 23 (10) ◽  
pp. 1018-1029 ◽  
Author(s):  
Ken Katsuya ◽  
Yuji Hori ◽  
Daisuke Oikawa ◽  
Tomohisa Yamamoto ◽  
Kayo Umetani ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Inflammatory Responses ◽  
Mass Spectrometry Analysis ◽  
Regulatory Subunit ◽  
Luciferase Reporter ◽  
Time Resolved Fluorescence ◽  
Ubiquitin Chain ◽  
Time Resolved ◽  
Gene Assay

The nuclear factor κB (NF-κB) pathway is critical for regulating immune and inflammatory responses, and uncontrolled NF-κB activation is closely associated with various inflammatory diseases and malignant tumors. The Met1-linked linear ubiquitin chain, which is generated by linear ubiquitin chain assembly complex (LUBAC), is important for regulating NF-κB activation. This process occurs through the linear ubiquitination of NF-κB essential modulator, a regulatory subunit of the canonical inhibitor of the NF-κB kinase complex. In this study, we have established a robust and efficient high-throughput screening (HTS) platform to explore LUBAC inhibitors, which may be used as tool compounds to elucidate the pathophysiological role of LUBAC. The HTS platform consisted of both cell-free and cell-based assays: (1) cell-free LUBAC-mediated linear ubiquitination assay using homogenous time-resolved fluorescence technology and (2) cell-based LUBAC assay using the NF-κB luciferase reporter gene assay. By using the HTS platform, we performed a high-throughput chemical library screen and identified several hit compounds with selectivity against a counterassay. Liquid chromatography–mass spectrometry analysis revealed that these compounds contain a chemically reactive lactone structure, which is transformed to give reactive α,β-unsaturated carbonyl compounds. Further investigation revealed that the reactive group of these compounds is essential for the inhibition of LUBAC activity.

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