A research proposal to study the life cycle of COV

SARS-CoV2 continues to affect the lives of the majority of the world, and although vaccines are beginning to become available, much of the world will still be unable to obtain them. Furthermore, some studies have suggested that there may have to be annual vaccines and as strains of the virus continue to increase, it is essential for us to move to the next stage of research and attempt to better understand the virus. By utilizing a chemical genetics approach where numerous ligands of distinct chemical libraries are screened through high-throughput screening, we may be able to form an ordered viral cycle of metabolic events that could help identify drug targets more efficiently and coordinate drug use to improve efficacy. A modified version of the virus (to decrease its ability of infection) along with the URA3 protein is then inserted into yeast cells (Saccharomyces cerevisiae) and screened. A simple assay involving the addition of 5’- fluoroorotic acid helps to determine ligand interference and after identifying the compounds, we can order their action into specific steps in the lifecycle and order the events of the life cycle.

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Development of a Novel High-Throughput Screen and Identification of Small-Molecule Inhibitors of the Gα–RGS17 Protein–Protein Interaction Using AlphaScreen

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057111410427 ◽

2011 ◽

Vol 16 (8) ◽

pp. 869-877 ◽

Cited By ~ 13

Author(s):

Duncan I. Mackie ◽

David L. Roman

Keyword(s):

Small Molecule ◽

High Throughput ◽

Protein Interaction ◽

High Throughput Screening ◽

Drug Targets ◽

Screening Method ◽

Fold Increase ◽

Rgs Proteins ◽

High Throughput Screen ◽

Protein Protein Interaction

In this study, the authors used AlphaScreen technology to develop a high-throughput screening method for interrogating small-molecule libraries for inhibitors of the Gαo–RGS17 interaction. RGS17 is implicated in the growth, proliferation, metastasis, and the migration of prostate and lung cancers. RGS17 is upregulated in lung and prostate tumors up to a 13-fold increase over patient-matched normal tissues. Studies show RGS17 knockdown inhibits colony formation and decreases tumorigenesis in nude mice. The screen in this study uses a measurement of the Gαo–RGS17 protein–protein interaction, with an excellent Z score exceeding 0.73, a signal-to-noise ratio >70, and a screening time of 1100 compounds per hour. The authors screened the NCI Diversity Set II and determined 35 initial hits, of which 16 were confirmed after screening against controls. The 16 compounds exhibited IC50 <10 µM in dose–response experiments. Four exhibited IC50 values <6 µM while inhibiting the Gαo–RGS17 interaction >50% when compared to a biotinylated glutathione-S-transferase control. This report describes the first high-throughput screen for RGS17 inhibitors, as well as a novel paradigm adaptable to many other RGS proteins, which are emerging as attractive drug targets for modulating G-protein-coupled receptor signaling.

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Cytotoxic Profiling of Annotated and Diverse Chemical Libraries Using Quantitative High-Throughput Screening

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555219873068 ◽

2019 ◽

Vol 25 (1) ◽

pp. 9-20 ◽

Cited By ~ 2

Author(s):

Olivia W. Lee ◽

Shelley Austin ◽

Madison Gamma ◽

Dorian M. Cheff ◽

Tobie D. Lee ◽

...

Keyword(s):

Cell Lines ◽

Cancer Cell ◽

High Throughput ◽

High Throughput Screening ◽

Drug Targets ◽

Large Scale ◽

Early Stage ◽

Cancer Cell Line ◽

Hek 293 ◽

Cytotoxic Compounds

Cell-based phenotypic screening is a commonly used approach to discover biological pathways, novel drug targets, chemical probes, and high-quality hit-to-lead molecules. Many hits identified from high-throughput screening campaigns are ruled out through a series of follow-up potency, selectivity/specificity, and cytotoxicity assays. Prioritization of molecules with little or no cytotoxicity for downstream evaluation can influence the future direction of projects, so cytotoxicity profiling of screening libraries at an early stage is essential for increasing the likelihood of candidate success. In this study, we assessed the cell-based cytotoxicity of nearly 10,000 compounds in the National Institutes of Health, National Center for Advancing Translational Sciences annotated libraries and more than 100,000 compounds in a diversity library against four normal cell lines (HEK 293, NIH 3T3, CRL-7250, and HaCat) and one cancer cell line (KB 3-1, a HeLa subline). This large-scale library profiling was analyzed for overall screening outcomes, hit rates, pan-activity, and selectivity. For the annotated library, we also examined the primary targets and mechanistic pathways regularly associated with cell death. To our knowledge, this is the first study to use high-throughput screening to profile a large screening collection (>100,000 compounds) for cytotoxicity in both normal and cancer cell lines. The results generated here constitute a valuable resource for the scientific community and provide insight into the extent of cytotoxic compounds in screening libraries, allowing for the identification and avoidance of compounds with cytotoxicity during high-throughput screening campaigns.

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Platforms for High-Throughput Screening and Force Measurements on Fungi and Oomycetes

Micromachines ◽

10.3390/mi12060639 ◽

2021 ◽

Vol 12 (6) ◽

pp. 639

Author(s):

Yiling Sun ◽

Ayelen Tayagui ◽

Sarah Sale ◽

Debolina Sarkar ◽

Volker Nock ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Drug Targets ◽

Control Strategies ◽

Pathogenic Fungi ◽

Antifungal Drugs ◽

Plant Diseases ◽

Force Measurements ◽

Technical Developments ◽

Alternative Drug

Pathogenic fungi and oomycetes give rise to a significant number of animal and plant diseases. While the spread of these pathogenic microorganisms is increasing globally, emerging resistance to antifungal drugs is making associated diseases more difficult to treat. High-throughput screening (HTS) and new developments in lab-on-a-chip (LOC) platforms promise to aid the discovery of urgently required new control strategies and anti-fungal/oomycete drugs. In this review, we summarize existing HTS and emergent LOC approaches in the context of infection strategies and invasive growth exhibited by these microorganisms. To aid this, we introduce key biological aspects and review existing HTS platforms based on both conventional and LOC techniques. We then provide an in-depth discussion of more specialized LOC platforms for force measurements on hyphae and to study electro- and chemotaxis in spores, approaches which have the potential to aid the discovery of alternative drug targets on future HTS platforms. Finally, we conclude with a brief discussion of the technical developments required to improve the uptake of these platforms into the general laboratory environment.

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Development of NS3/4A Protease-Based Reporter Assay Suitable for Efficiently Assessing Hepatitis C Virus Infection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00601-09 ◽

2009 ◽

Vol 53 (11) ◽

pp. 4825-4834 ◽

Cited By ~ 20

Author(s):

Kao-Lu Pan ◽

Jin-Ching Lee ◽

Hsing-Wen Sung ◽

Teng-Yuang Chang ◽

John T.-A. Hsu

Keyword(s):

Cell Culture ◽

Life Cycle ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Infection ◽

High Throughput ◽

High Throughput Screening ◽

Reporter System ◽

Viral Protease ◽

Peptide Cleavage

ABSTRACT A cell culture system for the production of hepatitis C virus (HCV) whole virions has greatly accelerated studies of the virus life cycle and the discovery of anti-HCV agents. However, the quantification of the HCV titers in a whole-virus infection/replication system currently relies mostly on reverse transcription-PCR or immunofluorescence assay, which would be cumbersome for high-throughput drug screening. To overcome this problem, this study has generated a novel cell line, Huh7.5-EG(Δ4B5A)SEAP, that carries a dual reporter, EG(Δ4B5A)SEAP. The EG(Δ4B5A)SEAP reporter is a viral protease-cleavable fusion protein in which the enhanced green fluorescence protein is linked to secreted alkaline phosphatase (SEAP) in frame via Δ4B5A, a short peptide cleavage substrate for NS3/4A viral protease. This study demonstrates that virus replication/infection in the Huh7.5-EG(Δ4B5A)SEAP cells can be quantitatively indicated by measuring the SEAP activity in cell culture medium. The levels of SEAP released from HCV-infected Huh7.5-EG(Δ4B5A)SEAP cells correlated closely with the amounts of HCV in the inocula. The Huh7.5-EG(Δ4B5A)SEAP cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target multiple stages of the HCV life cycle. The Z′-factor of this assay ranged from 0.64 to 0.74 in 96-well plates, indicating that this reporter system is suitable for high-throughput screening of prospective anti-HCV agents.

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Approaches to Miniaturized High-Throughput Screening of Chemical Libraries

Integrated Microfabricated Biodevices ◽

10.1201/9780203908006.ch15 ◽

2002 ◽

Author(s):

Damien Dunnington ◽

Zhuyin Li ◽

Alan Binnie ◽

Henning Vollert

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Chemical Libraries

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Application of Life Cycle Assessment and Machine Learning for High-Throughput Screening of Green Chemical Substitutes

10.26434/chemrxiv.12210860.v1 ◽

2020 ◽

Author(s):

Xinzhe Zhu ◽

Chi-Hung Ho ◽

Xiaonan Wang

Keyword(s):

Machine Learning ◽

Life Cycle Assessment ◽

Life Cycle ◽

Production Process ◽

Environmental Impacts ◽

High Throughput ◽

High Throughput Screening ◽

Molecular Descriptors ◽

Trifluoroacetic Anhydride ◽

Chemical Materials

<p><a></a><a>The production process of many active pharmaceutical ingredients such as sitagliptin could cause severe environmental problems due to the use of toxic chemical materials and production infrastructure, energy consumption and wastes treatment. The environmental impacts of sitagliptin production process were estimated with life cycle assessment (LCA) method, which suggested that the use of chemical materials provided the major environmental impacts. Both methods of Eco-indicator 99 and ReCiPe endpoints confirmed that chemical feedstock accounted 83% and 70% of life-cycle impact, respectively. Among all the chemical materials used in the sitagliptin production process, </a><a>trifluoroacetic anhydride </a>was identified as the largest influential factor in most impact categories according to the results of ReCiPe midpoints method. Therefore, high-throughput screening was performed to seek for green chemical substitutes to replace the target chemical (i.e. trifluoroacetic anhydride) by the following three steps. Firstly, thirty most similar chemicals were obtained from two million candidate alternatives in PubChem database based on their molecular descriptors. Thereafter, deep learning neural network models were developed to predict life-cycle impact according to the chemicals in Ecoinvent v3.5 database with known LCA values and corresponding molecular descriptors. Finally, 1,2-ethanediyl ester was proved to be one of the potential greener substitutes after the LCA data of these similar chemicals were predicted using the well-trained machine learning models. The case study demonstrated the applicability of the novel framework to screen green chemical substitutes and optimize the pharmaceutical manufacturing process.</p>

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