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.

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 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.

Development of a High-Content Screening Assay Panel to Accelerate Mechanism of Action Studies for Oncology Research

2012 ◽  
Vol 17 (8) ◽  
pp. 1005-1017 ◽  
Author(s):  
Danli L. Towne ◽  
Emily E. Nicholl ◽  
Kenneth M. Comess ◽  
Scott C. Galasinski ◽  
Philip J. Hajduk ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Mechanism Of Action ◽  
Large Scale ◽  
Morphological Changes ◽  
Single Cells ◽  
High Content Screening ◽  
Individual Compound ◽  
Screening Assay ◽  
Oncology Research ◽  
Protein Functions

Efficient elucidation of the biological mechanism of action of novel compounds remains a major bottleneck in the drug discovery process. To address this need in the area of oncology, we report the development of a multiparametric high-content screening assay panel at the level of single cells to dramatically accelerate understanding the mechanism of action of cell growth–inhibiting compounds on a large scale. Our approach is based on measuring 10 established end points associated with mitochondrial apoptosis, cell cycle disruption, DNA damage, and cellular morphological changes in the same experiment, across three multiparametric assays. The data from all of the measurements taken together are expected to help increase our current understanding of target protein functions, constrain the list of possible targets for compounds identified using phenotypic screens, and identify off-target effects. We have also developed novel data visualization and phenotypic classification approaches for detailed interpretation of individual compound effects and navigation of large collections of multiparametric cellular responses. We expect this general approach to be valuable for drug discovery across multiple therapeutic areas.

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.

scholarly journals Automated High-Content Screening for Compounds That Disassemble the Perinucleolar Compartment

2009 ◽  
Vol 14 (9) ◽  
pp. 1045-1053 ◽  
Author(s):  
John T. Norton ◽  
Steven A. Titus ◽  
Dwayne Dexter ◽  
Christopher P. Austin ◽  
Wei Zheng ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cellular Proliferation ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Rna Binding Proteins ◽  
High Content Screening ◽  
Screening Assay ◽  
Phenotypic Marker ◽  
Perinucleolar Compartment

All solid malignancies share characteristic traits, including unlimited cellular proliferation, evasion of immune regulation, and the propensity to metastasize. The authors have previously described that a subnuclear structure, the perinucleolar compartment (PNC), is associated with the metastatic phenotype in solid tumor cancer cells. The percentage of cancer cells that contain PNCs (PNC prevalence) is indicative of the malignancy of a tumor both in vitro and in vivo, and thus PNC prevalence is a marker that reflects metastatic capability in a population of tumor cells. Although the function of the PNC remains to be determined, the PNC is highly enriched with small RNAs and RNA binding proteins. The initial chemical biology studies using a set of anticancer drugs that disassemble PNCs revealed a direct association of the structure with DNA. Therefore, PNC prevalence reduction as a phenotypic marker can be used to identify compounds that target cellular processes required for PNC maintenance and hence used to elucidate the nature of the PNC function. Here the authors report the development of an automated high-content screening assay that is capable of detecting PNC prevalence in prostate cancer cells (PC-3M) stably expressing a green fluorescent protein (GFP)—fusion protein that localizes to the PNC. The assay was optimized using known PNC-reducing drugs and non-PNC-reducing cytotoxic drugs. After optimization, the fidelity of the assay was probed with a collection of 8284 compounds and was shown to be robust and capable of detecting known and novel PNC-reducing compounds, making it the first reported high-content phenotypic screen for small changes in nuclear structure. ( Journal of Biomolecular Screening 2009:1045-1053)

scholarly journals Generation of Stably Transfected Mammalian Cell Lines as Fluorescent Screening Assay for NF- B Activation-Dependent Gene Expression

2003 ◽  
Vol 8 (5) ◽  
pp. 511-521 ◽  
Author(s):  
Christine E. Hellweg ◽  
Christa Baumstark-Khan ◽  
Gerda Horneck
Keyword(s):  
Gene Expression ◽  
High Throughput Screening ◽  
Time Course ◽  
Fluorescent Protein ◽  
Cellular Stress ◽  
Time Lag ◽  
Cellular Stress Response ◽  
Screening Assay ◽  
Mammalian Cell Lines ◽  
Tnf Α

Cellular stress protection responses lead to increased transcription of several genes via modulation of transcription factors. Activation of the Nuclear Factor κB (NF-κB) pathway as a possible antiapoptotic route represents one important cellular stress response. To identify conditions that are capable of modifying this pathway, a screening assay for detection of NF-κB-dependent gene activation using the reporter protein Enhanced Green Fluorescent Protein (EGFP) and its destabilized variant (d2EGFP) was developed. Human Embryonic Kidney (HEK/293) cells were stably transfected with a vector carrying EGFP or d2EGFP under control of a synthetic promoter containing 4 copies of the NF-κB response element. Treatment with tumor necrosis factor α (TNF-α) gave rise to substantial EGFP/d2EGFP expression in up to 90% of the cells and was therefore used to screen different stably transfected clones for induction of NF-κB-dependent gene expression. The time course of NF-κB activation leading to d2EGFP expression was measured in an oligonucleotide-based NF-κB-ELISA. NF-κB binding in-creased after 15-min incubation with TNF-α. In parallel, d2EGFP increased after 3 h and reached its maximum at 24 h. These results show (1) the time lag between NF-κB activation and d2EGFP transcription, translation, and protein folding and (2) the increased reporter gene expression after treatment with TNF-α to be caused by the activation of NF-κB. The detection of d2EGFP expression required FACS analysis or fluorescence microscopy, while EGFP could also be measured in the microplate reader, rendering the assay useful for high-throughput screening. ( Journal of Biomolecular Screening 2003:511-521)

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 A luciferase-based assay identifies niclosamide derivatives antagonizing Mcl-1 through post-translational down-regulation

2019 ◽  
Author(s):  
Qiang Liu ◽  
Jennifer M. Atkinson ◽  
Melat T. Gebru ◽  
Kristen Clements ◽  
George L. Moldovan ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Therapy Resistance ◽  
Mrna Levels ◽  
Screening Assay ◽  
Down Regulation ◽  
Major Mechanism ◽  
High Throughput Screening Assay

AbstractThe up-regulation of Mcl-1 expression is a major mechanism of cancer cell survival and therapy resistance. However, the underlying molecular mechanism remains incompletely understood, limiting the number of druggable approaches to selectively inhibit Mcl-1 function. In this study, we designed and employed a novel mechanistic high-throughput screening system to selectively uncover post-translational modulators of Mcl-1. We generated a cell-based high-throughput screening assay in which myeloid leukemia K562 cells constitutively express Mcl-1 or Bcl-xL fused with luciferase (Luc-Mcl-1 or Luc-Bcl-xL, respectively) under a viral promoter. 1,650 bioactive compounds were screened for their ability to selectively induce Mcl-1 down-regulation in a 2-hour assay. A family of niclosamide derivatives were eventually identified for their remarkable ability to decrease Mcl-1 protein stability, exemplified by N007. These salicylate derivatives did not alter Mcl-1 mRNA levels, but selectively induced proteasome-dependent Mcl-1 down-regulation independent of Noxa, Mule, or GSK3β. We also demonstrate that N007 potently induced cell death in leukemia cell lines, including those resistant to Bcl-2 inhibitors. Our work highlights the versatility of the mechanistic high-throughput screening approach as a valuable tool in identifying novel agents with the ability to down-regulate proteins crucial to human diseases.

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