scholarly journals Image-Based Screening for the Identification of Novel Proteasome Inhibitors

2007 ◽  
Vol 12 (2) ◽  
pp. 203-210 ◽  
Author(s):  
Linda Rickardson ◽  
Malin Wickström ◽  
Rolf Larsson ◽  
Henrik Lövborg
Keyword(s):  
Cell Line ◽  
Fluorescent Protein ◽  
Proteasome Inhibitors ◽  
Myeloma Cell ◽  
Cancer Drug ◽  
Screening Assay ◽  
Hek 293 ◽  
Compound Libraries ◽  
Human Embryo Kidney ◽  
Green Fluorescent

The proteasome is a new, interesting target in cancer drug therapy, and the proteasome inhibitor bortezomib has shown an effect in myeloma patients. It is of interest to efficiently discover and evaluate new proteasome inhibitors. The authors describe the development of an image-based screening assay for the identification of compounds with proteasome-inhibiting activity. The stably transfected human embryo kidney cell line HEK 293 ZsGreen Proteasome Sensor Cell Line expressing the ZsProSensor-1 fusion protein was used for screening and evaluation of proteasome inhibitors. Inhibition of the proteasome leads to accumulation of the green fluorescent protein ZsGreen, which is measured in the ArrayScan® High Content Screening system, in which cell morphology is studied simultaneously. When screening the LOPAC1280 substance library, several compounds with effect on the proteasome were found; among the hits were disulfiram and ammonium pyrrolidinedithiocarbamate (PDTC). Cytotoxic analysis of disulfiram and PDTC showed that the compounds induced cytotoxicity in the myeloma cell line RPMI 8226. The average Z' value for the assay was 0.66. The results indicate that the assay rapidly identifies new proteasome-inhibiting substances, and it will be further used as a tool for image-based screening of other chemically diverse compound libraries.

scholarly journals A new reporter cell line for studies with proteasome inhibitors in Trypanosoma brucei

2018 ◽  
Author(s):  
Danielle MN Moura ◽  
Osvaldo P de Melo Neto ◽  
Mark Carrington
Keyword(s):  
Cell Line ◽  
Trypanosoma Brucei ◽  
Fluorescent Protein ◽  
Proteasome Inhibitors ◽  
Reporter Cell ◽  
Ubiquitin Proteasome Pathway ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Green Fluorescent ◽  
Inhibitor Treatment

AbstractA Trypanosoma brucei cell line is described that produces a visual readout of proteasome activity. The cell line contains an integrated transgene encoding an ubiquitin-green fluorescent protein (GFP) fusion polypeptide responsive to the addition of proteasome inhibitors. A modified version of T. brucei ubiquitin unable to be recognized by deubiquitinases (UbG76V) was fused to eGFP and constitutively expressed. The fusion protein is unstable but addition of the proteasome inhibitor lactacystin stabilizes it and leads to visually detectable GFP. This cell line can be widely used to monitor the efficiency of inhibitor treatment through detection of GFP accumulation in studies involving proteasome-mediated proteolysis, screening of proteasome inhibitors or other events related to the ubiquitin-proteasome pathway.

scholarly journals A High-Content Screening Assay for Small Molecules That Stabilize Mutant Triose Phosphate Isomerase (TPI) as Treatments for TPI Deficiency

2021 ◽  
pp. 247255522110181
Author(s):  
Andreas Vogt ◽  
Samantha L. Eicher ◽  
Tracey D. Myers ◽  
Stacy L. Hrizo ◽  
Laura L. Vollmer ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Large Scale ◽  
Protein Quality ◽  
Fluorescent Protein ◽  
Triose Phosphate Isomerase ◽  
Pharmacological Intervention ◽  
Screening Assay ◽  
Triose Phosphate ◽  
The Common ◽  
Green Fluorescent

Triose phosphate isomerase deficiency (TPI Df) is an untreatable, childhood-onset glycolytic enzymopathy. Patients typically present with frequent infections, anemia, and muscle weakness that quickly progresses with severe neuromusclar dysfunction requiring aided mobility and often respiratory support. Life expectancy after diagnosis is typically ~5 years. There are several described pathogenic mutations that encode functional proteins; however, these proteins, which include the protein resulting from the “common” TPIE105D mutation, are unstable due to active degradation by protein quality control (PQC) pathways. Previous work has shown that elevating mutant TPI levels by genetic or pharmacological intervention can ameliorate symptoms of TPI Df in fruit flies. To identify compounds that increase levels of mutant TPI, we have developed a human embryonic kidney (HEK) stable knock-in model expressing the common TPI Df protein fused with green fluorescent protein (HEK TPIE105D-GFP). To directly address the need for lead TPI Df therapeutics, these cells were developed into an optical drug discovery platform that was implemented for high-throughput screening (HTS) and validated in 3-day variability tests, meeting HTS standards. We initially used this assay to screen the 446-member National Institutes of Health (NIH) Clinical Collection and validated two of the hits in dose–response, by limited structure–activity relationship studies with a small number of analogs, and in an orthogonal, non-optical assay in patient fibroblasts. The data form the basis for a large-scale phenotypic screening effort to discover compounds that stabilize TPI as treatments for this devastating childhood disease.

scholarly journals Development of an Intracellular Screen for New Compounds Able To Inhibit Mycobacterium tuberculosis Growth in Human Macrophages

2015 ◽  
Vol 60 (1) ◽  
pp. 640-645 ◽  
Author(s):  
Flavia Sorrentino ◽  
Ruben Gonzalez del Rio ◽  
Xingji Zheng ◽  
Jesus Presa Matilla ◽  
Pedro Torres Gomez ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Screening Assay ◽  
Human Macrophages ◽  
High Throughput Screening Assay ◽  
Primary Identification ◽  
Green Fluorescent ◽  
Development And Validation ◽  
New Compounds

ABSTRACTHere we describe the development and validation of an intracellular high-throughput screening assay for finding new antituberculosis compounds active in human macrophages. The assay consists of a luciferase-based primary identification assay, followed by a green fluorescent protein-based secondary profiling assay. Standard tuberculosis drugs and 158 previously recognized active antimycobacterial compounds were used to evaluate assay robustness. Data show that the assay developed is a short and valuable tool for the discovery of new antimycobacterial compounds.

Evaluation of resistance to aflatoxin contamination in kernels of maize genotypes using a GFP-expressing Aspergillus flavus strain

2013 ◽  
Vol 6 (2) ◽  
pp. 151-158 ◽  
Author(s):  
K. Rajasekaran ◽  
C.M. Sickler ◽  
R.L. Brown ◽  
J.W. Cary ◽  
D. Bhatnagar
Keyword(s):  
Fungal Infection ◽  
Aspergillus Flavus ◽  
Fluorescent Protein ◽  
Aflatoxin Contamination ◽  
Clear Correlation ◽  
Aleurone Layer ◽  
Screening Assay ◽  
Maize Germplasm ◽  
Antifungal Proteins ◽  
Green Fluorescent

Resistance or susceptibility of maize inbreds to infection by Aspergillus flavus was evaluated by the kernel screening assay. A green fluorescent protein-expressing strain of A. flavus was used to measure fungal spread and aflatoxin levels in real-time following fungal infection of kernels. Among the four inbreds tested, MI82 showed the most resistance and Ga209 the least. TZAR101 was also resistant to fungal infection, whereas Va35 was susceptible to fungal infection. However, Va35 produced lower aflatoxin levels compared to the susceptible line Ga209. Fluorescence microscopy indicated that the site of entry of the fungus into the kernel was consistently through the pedicel. Entry through the pericarp was never observed in undamaged kernels. In view of these results, incorporation or overexpression of antifungal proteins should be targeted to the pedicel and basal endosperm region in developing kernels. Once the fungus has entered through the pedicel, it spreads quickly through the open spaces between the pericarp and the aleurone layer, ultimately colonising the endosperm and scutellum and, finally, the embryo. A clear correlation was established between fungal fluorescence and aflatoxin levels. This method provides a quick, reliable means of evaluating resistance to A. flavus in undamaged kernels and provides breeders with a rapid method to evaluate maize germplasm.

High agonist-independent clearance of rabbit kinin B1 receptors in cultured cells

2003 ◽  
Vol 284 (5) ◽  
pp. H1647-H1654 ◽  
Author(s):  
Jean-Philippe Fortin ◽  
Johanne Bouthillier ◽  
François Marceau
Keyword(s):  
Fluorescent Protein ◽  
Cultured Cells ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Brefeldin A ◽  
Metabolic Inhibitors ◽  
Maximal Effect ◽  
Hek 293 ◽  
B1 Receptors ◽  
Green Fluorescent

We hypothesized that the inducible kinin B1 receptor (B1R) is rapidly cleared from cells when its synthesis subsides. The agonist-independent degradation of the rabbit B1Rs and related B2 receptors (B2Rs) was investigated. Endocytosis of the B1R-yellow fluorescent protein (YFP) conjugate was more intense than that of B2R-green fluorescent protein (GFP) based on fluorescence accumulation in HEK 293 cells treated with a lysosomal inhibitor. The cells expressing B1R-YFP contained more GFP/YFP-sized degradation product(s) than those expressing B2R-GFP (immunoblot, antibodies equally reacting with both fluorescent proteins). The binding site density of B1R-YFP decreased in the presence of protein synthesis or maturation inhibitors (anisomycin, brefeldin A), whereas that of B2R-GFP remained constant. Wild-type B1Rs were also cleared faster than B2Rs in rabbit smooth muscle cells treated with metabolic inhibitors. Contractility experiments based on brefeldin A-treated isolated rabbit blood vessels also functionally support that B1Rs are more rapidly eliminated than B2Rs (decreased maximal effect of agonist over 2 h). The highly regulated B1R is rapidly degraded, relative to the constitutive B2R.

scholarly journals Novel Real-Time Monitoring System for Human Cytomegalovirus- Infected Cells In Vitro That Uses a Green Fluorescent Protein-PML-Expressing Cell Line

2006 ◽  
Vol 50 (8) ◽  
pp. 2806-2813 ◽  
Author(s):  
T. Ueno ◽  
Y. Eizuru ◽  
H. Katano ◽  
T. Kurata ◽  
T. Sata ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Line ◽  
Real Time ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Fluorescent Protein ◽  
Clinical Isolates ◽  
Pml Bodies ◽  
Infected Cells ◽  
Green Fluorescent

ABSTRACT Promyelocytic leukemia (PML) bodies are discrete nuclear foci that are intimately associated with many DNA viruses. In human cytomegalovirus (HCMV) infection, the IE1 (for “immediate-early 1”) protein has a marked effect on PML bodies via de-SUMOylation of PML protein. Here, we report a novel real-time monitoring system for HCMV-infected cells using a newly established cell line (SE/15) that stably expresses green fluorescent protein (GFP)-PML protein. In SE/15 cells, HCMV infection causes specific and efficient dispersion of GFP-PML bodies in an IE1-dependent manner, allowing the infected cells to be monitored by fluorescence microscopy without immunostaining. Since a specific change in the detergent solubility of GFP-PML occurs upon infection, the infected cells can be quantified by GFP fluorescence measurement after extraction. With this assay, the inhibitory effects of heparin and neutralizing antibodies were determined in small-scale cultures, indicating its usefulness for screening inhibitory reagents for laboratory virus strains. Furthermore, we established a sensitive imaging assay by counting the number of nuclei containing dispersed GFP-PML, which is applicable for titration of slow-growing clinical isolates. In all strains tested, the virus titers estimated by the GFP-PML imaging assay were well correlated with the plaque-forming cell numbers determined in human embryonic lung cells. Coculture of SE/15 cells and HCMV-infected fibroblasts permitted a rapid and reliable method for estimating the 50% inhibitory concentration values of drugs for clinical isolates in susceptibility testing. Taken together, these results demonstrate the development of a rapid, sensitive, quantitative, and specific detection system for HCMV-infected cells involving a simple procedure that can be used for titration of low-titer clinical isolates.

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