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.

scholarly journals Development and pilot screen of novel high content assay for down regulators of expression of heterogenous nuclear ribonuclear protein H2

2020 ◽  
Author(s):  
Juan Diez ◽  
Sumitha Rajendrarao ◽  
Shadi A. Baajour ◽  
Praathibha Sripadhan ◽  
Timothy P. Spicer ◽  
...  
Keyword(s):  
Metastatic Melanoma ◽  
High Throughput Screening ◽  
Large Scale ◽  
Fluorescent Protein ◽  
High Content Screening ◽  
Screening Assay ◽  
Down Regulation ◽  
Metastatic Melanoma Cell ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

ABSTRACTDespite recent advances in melanoma drug discovery, the average overall survival of patients with late stage metastatic melanoma is approximately 3 years, suggesting a need for new approaches and melanoma therapeutic targets. Previously we identified heterogeneous nuclear ribonucleoprotein H2 as a potential target of anti-melanoma compound 2155-14 (Palrasu et al, Cell Physiol Biochem 2019;53:656-86). In the present study, we endeavored to develop an assay to enable a high throughput screening campaign to identify drug-like molecules acting via down regulation of heterogeneous nuclear ribonucleoprotein H that can be used for melanoma therapy and research.ResultsWe established a cell-based platform using metastatic melanoma cell line WM266-4 expressing hnRNPH2 conjugated with green fluorescent protein to enable assay development and screening. High Content Screening assay was developed and validated in 384 well plate format, followed by miniaturization to 1,536 well plate format. All plate-based QC parameters were acceptable: %CV = 6.7±0.3, S/B = 21±2.1, Z’ = 0.75±0.04. Pilot screen of FDA-approved drug library (n=1,400 compounds) demonstrated hit rate of 0.5%. Two compounds demonstrated pharmacological response and were authenticated by western blot analysis.ConclusionsWe developed a highly robust HTS-amenable high content screening assay capable of monitoring down regulation of hnRNPH2. This assay is thus capable of identifying authentic down regulators of hnRNPH1 and 2 in a large compound collection and, therefore, is amenable to a large-scale screening effort.

scholarly journals A Fluorescent-Based High-Throughput Screening Assay for Small Molecules That Inhibit the Interaction of MdmX with p53

2012 ◽  
Vol 18 (2) ◽  
pp. 191-198 ◽  
Author(s):  
Keiko Tsuganezawa ◽  
Yukari Nakagawa ◽  
Miki Kato ◽  
Shigenao Taruya ◽  
Fumio Takahashi ◽  
...  
Keyword(s):  
Small Molecules ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Diffusion Time ◽  
Correlation Spectroscopy ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Green Fluorescent ◽  
Fluorescence Quencher

A fluorescent-based high-throughput screening (HTS) assay for small molecules that inhibit the interaction of MdmX with p53 was developed and applied to identify new inhibitors. The assay evaluated the MdmX-p53 interaction by detecting the quenching of the fluorescence of green fluorescent protein (GFP) fused to the MdmX protein, after its interaction with a p53 peptide labeled with a fluorescence quencher. In this report, the developed HTS assay was applied to about 40 000 compounds, and 255 hit compounds that abrogated the GFP quenching were selected. Next, the obtained hits were reevaluated by other assays. First, their effects on the diffusion time of a fluorescently-labeled p53 peptide after incubation with the MdmX protein were tested by measuring the diffusion time using fluorescence correlation spectroscopy, and six stable hit compounds with IC50 values less than 5 µM were selected. Next, we further confirmed their inhibition of the MdmX-p53 interaction by surface plasmon resonance. To indicate the efficacy of the hit compound as a candidate anticancer drug, we showed that the hit compound triggered apoptosis after p53 and p21 accumulation in cultured MV4;11 leukemia cells. Thus, the new HTS assay is effective for obtaining novel MdmX-p53 interaction inhibitors that are valuable as candidate compounds for cancer treatment.

scholarly journals Development and Pilot Screen of Novel High Content Assay for Down Regulators of Expression of Heterogenous Nuclear Ribonuclear Protein H2

2021 ◽  
Vol 55 (3) ◽  
pp. 265-276
Keyword(s):  
Metastatic Melanoma ◽  
High Throughput Screening ◽  
Large Scale ◽  
Fluorescent Protein ◽  
High Content Screening ◽  
Screening Assay ◽  
Down Regulation ◽  
Metastatic Melanoma Cell ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

BACKGROUND/AIMS: Despite recent advances in melanoma drug discovery, the average overall survival of patients with late-stage metastatic melanoma is approximately 3 years, suggesting a need for new approaches and melanoma therapeutic targets. Previously we identified heterogeneous nuclear ribonucleoprotein H2 as a potential target of anti-melanoma compound 2155-14 (Palrasu et al., Cell Physiol Biochem 2019;53:656-686). In the present study, we endeavored to develop an assay to enable a high throughput screening campaign to identify drug-like molecules acting via down regulation of heterogeneous nuclear ribonucleoprotein H2 that can be used for melanoma therapy and research. METHODS: We established a cell-based platform using metastatic melanoma cell line WM266-4 expressing hnRNPH2 conjugated with green fluorescent protein to enable assay development and screening. High Content Screening assay was developed and validated in 384 well plate format, followed by miniaturization to 1,536 well plate format. RESULTS: All plate-based QC parameters were acceptable: %CV = 6.7±0.3, S/B = 21±2.1, Z' = 0.75±0.04. Pilot screen of FDA-approved drug library (n=1,400 compounds) demonstrated hit rate of 0.5%. Two compounds demonstrated pharmacological response and were authenticated by western blot analysis. CONCLUSION: We developed a highly robust HTS-amenable high content screening assay capable of monitoring down regulation of hnRNPH2. This assay is thus capable of identifying authentic down regulators of hnRNPH1 and 2 in a large compound collection and, therefore, is amenable to a large-scale screening effort.

The Fe-Core/Carbon-Shell Ultrafine Nanopowders as Platform for Biomolecules Grafting

2014 ◽  
Vol 1040 ◽  
pp. 194-198
Author(s):  
N.S. Surgutskaya ◽  
P.S. Postnikov ◽  
Alexandra G. Pershina ◽  
A.I. Galanov ◽  
Marina E. Trusova ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Carbon Shell ◽  
Shell Nanoparticles ◽  
Powder Surface ◽  
Metal Precursors ◽  
Covalent Grafting ◽  
Large Scale Synthesis ◽  
Green Fluorescent

The Fe-core/carbon-shell nanopowders are excellent platform for covalent grafting of biomolecules. The large-scale synthesis of Fe-core/carbon-shell nanoparticles via electropulse erosion of metal precursors in hydrocarbons was developed. The green fluorescent protein was covalently attached to the powder surface via diazonium functionalization and further carbodiimide activation.

scholarly journals Extracellular Vesicles-Encapsulated Yeast Prions and What They Can Tell Us about the Physical Nature of Propagons

2020 ◽  
Vol 22 (1) ◽  
pp. 90
Author(s):  
Mehdi Kabani
Keyword(s):  
Protein Quality ◽  
Fluorescent Protein ◽  
Physical Nature ◽  
Dependent Manner ◽  
Yeast Saccharomyces Cerevisiae ◽  
Yeast Prions ◽  
Daughter Cells ◽  
Cytoplasmic Proteins ◽  
Green Fluorescent ◽  
Functionally Diverse

The yeast Saccharomyces cerevisiae hosts an ensemble of protein-based heritable traits, most of which result from the conversion of structurally and functionally diverse cytoplasmic proteins into prion forms. Among these, [PSI+], [URE3] and [PIN+] are the most well-documented prions and arise from the assembly of Sup35p, Ure2p and Rnq1p, respectively, into insoluble fibrillar assemblies. Yeast prions propagate by molecular chaperone-mediated fragmentation of these aggregates, which generates small self-templating seeds, or propagons. The exact molecular nature of propagons and how they are faithfully transmitted from mother to daughter cells despite spatial protein quality control are not fully understood. In [PSI+] cells, Sup35p forms detergent-resistant assemblies detectable on agarose gels under semi-denaturant conditions and cytosolic fluorescent puncta when the protein is fused to green fluorescent protein (GFP); yet, these macroscopic manifestations of [PSI+] do not fully correlate with the infectivity measured during growth by the mean of protein infection assays. We also discovered that significant amounts of infectious Sup35p particles are exported via extracellular (EV) and periplasmic (PV) vesicles in a growth phase and glucose-dependent manner. In the present review, I discuss how these vesicles may be a source of actual propagons and a suitable vehicle for their transmission to the bud.

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.

scholarly journals ZF-AutoML: An Easy Machine-Learning-Based Method to Detect Anomalies in Fluorescent-Labelled Zebrafish

Inventions ◽  
2019 ◽  
Vol 4 (4) ◽  
pp. 72
Author(s):  
Ryota Sawaki ◽  
Daisuke Sato ◽  
Hiroko Nakayama ◽  
Yuki Nakagawa ◽  
Yasuhito Shimada
Keyword(s):  
Machine Learning ◽  
Image Analysis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Learning Program ◽  
High Throughput Analysis ◽  
Easy Method ◽  
High Fecundity

Background: Zebrafish are efficient animal models for conducting whole organism drug testing and toxicological evaluation of chemicals. They are frequently used for high-throughput screening owing to their high fecundity. Peripheral experimental equipment and analytical software are required for zebrafish screening, which need to be further developed. Machine learning has emerged as a powerful tool for large-scale image analysis and has been applied in zebrafish research as well. However, its use by individual researchers is restricted due to the cost and the procedure of machine learning for specific research purposes. Methods: We developed a simple and easy method for zebrafish image analysis, particularly fluorescent labelled ones, using the free machine learning program Google AutoML. We performed machine learning using vascular- and macrophage-Enhanced Green Fluorescent Protein (EGFP) fishes under normal and abnormal conditions (treated with anti-angiogenesis drugs or by wounding the caudal fin). Then, we tested the system using a new set of zebrafish images. Results: While machine learning can detect abnormalities in the fish in both strains with more than 95% accuracy, the learning procedure needs image pre-processing for the images of the macrophage-EGFP fishes. In addition, we developed a batch uploading software, ZF-ImageR, for Windows (.exe) and MacOS (.app) to enable high-throughput analysis using AutoML. Conclusions: We established a protocol to utilize conventional machine learning platforms for analyzing zebrafish phenotypes, which enables fluorescence-based, phenotype-driven zebrafish screening.

Sign in / Sign up

Export Citation Format

Share Document