scholarly journals Optimised techniques for high-throughput screening of differentiated SH-SY5Y cells and application for neurite outgrowth assays

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anusha Dravid ◽  
Brad Raos ◽  
Darren Svirskis ◽  
Simon J. O’Carroll
Keyword(s):  
Neurite Outgrowth ◽  
High Throughput ◽  
High Throughput Screening ◽  
Initial Density ◽  
Neuronal Model ◽  
Neuronal Markers ◽  
Cellular Processes ◽  
Undifferentiated State ◽  
Mature Neurons ◽  
High Throughput Assay

AbstractNeuronal models are a crucial tool in neuroscientific research, helping to elucidate the molecular and cellular processes involved in disorders of the nervous system. Adapting these models to a high-throughput format enables simultaneous screening of multiple agents within a single assay. SH-SY5Y cells have been widely used as a neuronal model, yet commonly in an undifferentiated state that is not representative of mature neurons. Differentiation of the SH-SY5Y cells is a necessary step to obtain cells that express mature neuronal markers. Despite this understanding, the absence of a standardised protocol has limited the use of differentiated SH-SY5Y cells in high-throughput assay formats. Here, we describe techniques to differentiate and re-plate SH-SY5Y cells within a 96-well plate for high-throughput screening. SH-SY5Y cells seeded at an initial density of 2,500 cells/well in a 96-well plate provide sufficient space for neurites to extend, without impacting cell viability. Room temperature pre-incubation for 1 h improved the plating homogeneity within the well and the ability to analyse neurites. We then demonstrated the efficacy of our techniques by optimising it further for neurite outgrowth analysis. The presented methods achieve homogenously distributed differentiated SH-SY5Y cells, useful for researchers using these cells in high-throughput screening assays.

scholarly journals Differentiated SH-SY5Y Cells for High-Throughput Screening in a 96-Well Plate Format: Protocol Optimisation and Application for Neurite Outgrowth Assays

2021 ◽  
Author(s):  
Anusha Dravid ◽  
Brad Raos ◽  
Darren Svirskis ◽  
Simon J O’Carroll
Keyword(s):  
Neurite Outgrowth ◽  
High Throughput ◽  
High Throughput Screening ◽  
Initial Density ◽  
Neuronal Model ◽  
Neuronal Markers ◽  
Cellular Processes ◽  
Undifferentiated State ◽  
Mature Neurons ◽  
High Throughput Assay

Abstract Neuronal models are a crucial tool in neuroscientific research, helping to elucidate the molecular and cellular processes involved in disorders of the nervous system. Adapting these models to a high-throughput format enables simultaneous screening of multiple agents within a single assay. SH-SY5Y cells have been widely used as a neuronal model, yet commonly in an undifferentiated state that is not representative of mature neurons. Differentiation of the SH-SY5Y cells is a necessary step to obtain cells that express mature neuronal markers. Despite this understanding, the absence of a standardised protocol has limited the use of differentiated SH-SY5Y cells in high-throughput assay formats. Here, we describe a protocol to differentiate and re-plate SH-SY5Y cells within a 96-well plate for high-throughput screening. SH-SY5Y cells seeded at an initial density of 2,500 cells/well in a 96-well plate provide sufficient space for neurites to extend, without impacting cell viability. Room temperature pre-incubation for 1 hour improved the plating homogeneity within the well and the ability to analyse neurites. We then demonstrated the efficacy of this protocol by optimising it further for neurite outgrowth analysis. The presented protocol achieves homogenously distributed differentiated SH-SY5Y cells, useful for researchers using these cells in high-throughput screening assays.

scholarly journals Development of a High-Throughput Screening Assay to Identify Inhibitors of the SARS-CoV-2 Guanine-N7-Methyltransferase Using RapidFire Mass Spectrometry

2021 ◽  
pp. 247255522110006
Author(s):  
Lesley-Anne Pearson ◽  
Charlotte J. Green ◽  
De Lin ◽  
Alain-Pierre Petit ◽  
David W. Gray ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Mutational Analysis ◽  
Nonstructural Protein ◽  
Translation Efficiency ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Starting Point ◽  
Approved Drugs ◽  
High Throughput Assay

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a significant threat to human health. Despite its similarity to related coronaviruses, there are currently no specific treatments for COVID-19 infection, and therefore there is an urgent need to develop therapies for this and future coronavirus outbreaks. Formation of the cap at the 5′ end of viral RNA has been shown to help coronaviruses evade host defenses. Nonstructural protein 14 (nsp14) is responsible for N7-methylation of the cap guanosine in coronaviruses. This enzyme is highly conserved among coronaviruses and is a bifunctional protein with both N7-methyltransferase and 3′-5′ exonuclease activities that distinguish nsp14 from its human equivalent. Mutational analysis of SARS-CoV nsp14 highlighted its role in viral replication and translation efficiency of the viral genome. In this paper, we describe the characterization and development of a high-throughput assay for nsp14 utilizing RapidFire technology. The assay has been used to screen a library of 1771 Food and Drug Administration (FDA)-approved drugs. From this, we have validated nitazoxanide as a selective inhibitor of the methyltransferase activity of nsp14. Although modestly active, this compound could serve as a starting point for further optimization.

scholarly journals Identification and Characterization of a New Series of Ghrelin O-Acyl Transferase Inhibitors

2017 ◽  
Vol 23 (2) ◽  
pp. 154-163 ◽  
Author(s):  
Mariko Yoneyama-Hirozane ◽  
Kohei Deguchi ◽  
Takeshi Hirakawa ◽  
Tsuyoshi Ishii ◽  
Tomoyuki Odani ◽  
...  
Keyword(s):  
High Throughput ◽  
Inhibitory Activity ◽  
High Throughput Screening ◽  
Enzyme Linked Immunosorbent Assay ◽  
Acyl Transferase ◽  
Lead Compound ◽  
Time Resolved ◽  
Acyl Ghrelin ◽  
Antiobesity Drugs ◽  
High Throughput Assay

Ghrelin O-acyl transferase (GOAT; MBOAT4) catalyzes O-acylation at serine-3 of des-acyl ghrelin. Acyl ghrelin is secreted by stomach X/A-like cells and plays a role in appetite and metabolism. Therefore, GOAT has been expected to be a novel antiobesity target because it is responsible for acyl ghrelin production. Here, we report homogeneous time-resolved fluorescence (HTRF) and enzyme-linked immunosorbent assay (ELISA) methods utilizing human GOAT-expressing microsomes as a novel high-throughput assay system for the discovery of hit compounds and optimization of lead compounds. Hit compounds exemplified by compound A (2-[(2,4-dichlorobenzyl)sulfanyl]-1,3-benzoxazole-5-carboxylic acid) were identified by high-throughput screening using the HTRF assay and confirmed to have GOAT inhibitory activity using the ELISA. Based on the hit compound information, the novel lead compound (compound B, (4-chloro-6-{[2-methyl-6-(trifluoromethyl)pyridin-3-yl]methoxy}-1-benzothiophen-3-yl)acetic acid) was synthesized and exhibited potent GOAT inhibition with oral bioavailability. Both the hit compound and lead compound showed octanoyl-CoA competitive inhibitory activity. Moreover, these two compounds decreased acyl ghrelin production in the stomach of mice after their oral administration. These novel findings demonstrate that GOAT is a druggable target, and its inhibitors are promising antiobesity drugs.

Application of the BD ACTOne™ Technology for the High-Throughput Screening of Gs-Coupled Receptor Antagonists

2007 ◽  
Vol 12 (8) ◽  
pp. 1068-1073 ◽  
Author(s):  
András Visegrády ◽  
András Boros ◽  
Zsolt Némethy ◽  
Béla Kiss ◽  
György M. Keserű
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Calcium Influx ◽  
Live Cells ◽  
Cyclic Nucleotide Gated Channels ◽  
Novel Technology ◽  
Adenosine Cyclic Monophosphate ◽  
Compound Screening ◽  
High Throughput Assay ◽  
Act One

A novel technology for monitoring the changes of 3,′5′-adenosine cyclic monophosphate (cAMP) in live cells suitable for drug screening relies on the use of cyclic nucleotide-gated channels as biosensors coexpressed with the appropriate target receptor. The technique (termed BD ACT One™) offers measurement of cAMP-dependent calcium influx or membrane depolarization with conventional fluorescent methods both in kinetic and in endpoint modes, optimal for high-throughput and subsequent compound screening. The utility of the technique is reported here based on assay development and high-throughput screening for small-molecule antagonists of the peptide parathyroid hormone 2 receptor (PTH2R). The dual-signaling properties of the receptor were retained in the recombinant system, and the observed pharmacological profile corresponded to data from radiolabeled cAMP determination. The membrane-potential-based high-throughput assay produced reproducible actives and led to the identification of several chemical scaffolds with potential utility as PTH2R ligands. ( Journal of Biomolecular Screening 2007:1068-1073)

scholarly journals A Homogeneous, High-Throughput Fluorescence Anisotropy-Based DNA Supercoiling Assay

2010 ◽  
Vol 15 (9) ◽  
pp. 1088-1098 ◽  
Author(s):  
Adam Shapiro ◽  
Haris Jahic ◽  
Swati Prasad ◽  
David Ehmann ◽  
Jason Thresher ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescence Anisotropy ◽  
High Throughput Screening ◽  
Topoisomerase I ◽  
Dna Supercoiling ◽  
New Drugs ◽  
E Coli ◽  
Cellular Processes ◽  
The Difference ◽  
Cellular Replication

The degree of supercoiling of DNA is vital for cellular processes, such as replication and transcription. DNA topology is controlled by the action of DNA topoisomerase enzymes. Topoisomerases, because of their importance in cellular replication, are the targets of several anticancer and antibacterial drugs. In the search for new drugs targeting topoisomerases, a biochemical assay compatible with automated high-throughput screening (HTS) would be valuable. Gel electrophoresis is the standard method for measuring changes in the extent of supercoiling of plasmid DNA when acted upon by topoisomerases, but this is a low-throughput and laborious method. A medium-throughput method was described previously that quantitatively distinguishes relaxed and supercoiled plasmids by the difference in their abilities to form triplex structures with an immobilized oligonucleotide. In this article, the authors describe a homogeneous supercoiling assay based on triplex formation in which the oligonucleotide strand is labeled with a fluorescent dye and the readout is fluorescence anisotropy. The new assay requires no immobilization, filtration, or plate washing steps and is therefore well suited to HTS for inhibitors of topoisomerases. The utility of this assay is demonstrated with relaxation of supercoiled plasmid by Escherichia coli topoisomerase I, supercoiling of relaxed plasmid by E. coli DNA gyrase, and inhibition of gyrase by fluoroquinolones and nalidixic acid.

scholarly journals High-Throughput Screening of Psychotropic Compounds: Impacts on Swimming Behaviours in Artemia franciscana

Toxics ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 64
Author(s):  
Shanelle A. Kohler ◽  
Matthew O. Parker ◽  
Alex T. Ford
Keyword(s):  
High Throughput ◽  
Swimming Speed ◽  
High Throughput Screening ◽  
Artemia Franciscana ◽  
Animal Behaviour ◽  
Crustacean Species ◽  
Significant Difference ◽  
Increased Sensitivity ◽  
Light Side ◽  
High Throughput Assay

Animal behaviour is becoming increasingly popular as an endpoint in ecotoxicology due to its increased sensitivity and speed compared to traditional endpoints. However, the widespread use of animal behaviours in environmental risk assessment is currently hindered by a lack of optimisation and standardisation of behavioural assays for model species. In this study, assays to assess swimming speed were developed for a model crustacean species, the brine shrimp Artemia franciscana. Preliminary works were performed to determine optimal arena size for this species, and weather lux used in the experiments had an impact on the animals phototactic response. Swimming speed was significantly lower in the smallest arena, whilst no difference was observed between the two larger arenas, suggesting that the small arena was limiting swimming ability. No significant difference was observed in attraction to light between high and low light intensities. Arena size had a significant impact on phototaxis behaviours. Large arenas resulted in animals spending more time in the light side of the arena compared to medium and small, irrespective of light intensity. The swimming speed assay was then used to expose specimens to a range of psychotropic compounds with varying modes of action. Results indicate that swimming speed provides a valid measure of the impacts of behaviour modulating compounds on A. franciscana. The psychotropic compounds tested varied in their impacts on animal behaviour. Fluoxetine resulted in increased swimming speed as has been found in other crustacean species, whilst oxazepam, venlafaxine and amitriptyline had no significant impacts on the behaviours measured. The results from this study suggest a simple, fast, high throughput assay for A. franciscana and gains insight on the impacts of a range of psychotropic compounds on the swimming behaviours of a model crustacean species used in ecotoxicology studies.

scholarly journals Development of a High-Throughput Cell-Based Assay for Superoxide Production in HL-60 Cells

2010 ◽  
Vol 15 (4) ◽  
pp. 388-397 ◽  
Author(s):  
Patricia M. Seitz ◽  
Rona Cooper ◽  
Gregory J. Gatto ◽  
Fernando Ramon ◽  
Thomas D. Sweitzer ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Superoxide Production ◽  
Test Set ◽  
Cellular Processes ◽  
Detection Assay ◽  
Z Value ◽  
Compound Concentration ◽  
Compound Test ◽  
Early Drug

Superoxide affects many normal and pathogenic cellular processes, and the detection of superoxide produced by cells is therefore of interest for potential therapeutic applications. To develop a high-throughput cell-based assay for the detection of extracellular superoxide production that could be run in a 384-well or 1536-well format, 2 luminescent reagents, Lucigenin and Diogenes, and one fluorescent reagent, Oxyburst Green BSA, were tested. HL-60 cells, which had been differentiated to a neutrophil-like phenotype with DMSO and frozen in large batches, were used in assays. All 3 superoxide detection reagents performed well statistically in terms of IC50 reproducibility and met a desired Z′ value requirement of >0.4. When tested against a 1408-compound test set at 5 or 10 µM compound concentration, a higher hit rate was obtained with the 2 luminescent reagents compared with that obtained with the fluorescent Oxyburst Green BSA reagent. The Oxyburst Green BSA assay was ultimately chosen for compound profiling and high-throughput screening activities. This 1536 superoxide detection assay using cryopreserved differentiated HL-60 cells represents a shifting paradigm toward the utilization of more therapeutically relevant cells in early drug development activities.

scholarly journals Antagonism screen for inhibitors of bacterial cell wall biogenesis uncovers an inhibitor of undecaprenyl diphosphate synthase

2015 ◽  
Vol 112 (35) ◽  
pp. 11048-11053 ◽  
Author(s):  
Maya A. Farha ◽  
Tomasz L. Czarny ◽  
Cullen L. Myers ◽  
Liam J. Worrall ◽  
Shawn French ◽  
...  
Keyword(s):  
Cell Wall ◽  
High Throughput ◽  
High Throughput Screening ◽  
Teichoic Acid ◽  
Antibacterial Drug ◽  
Wall Teichoic Acid ◽  
Chemical Screening ◽  
Cellular Processes ◽  
Cell Wall Biogenesis ◽  
Antagonistic Interactions

Drug combinations are valuable tools for studying biological systems. Although much attention has been given to synergistic interactions in revealing connections between cellular processes, antagonistic interactions can also have tremendous value in elucidating genetic networks and mechanisms of drug action. Here, we exploit the power of antagonism in a high-throughput screen for molecules that suppress the activity of targocil, an inhibitor of the wall teichoic acid (WTA) flippase in Staphylococcus aureus. Well-characterized antagonism within the WTA biosynthetic pathway indicated that early steps would be sensitive to this screen; however, broader interactions with cell wall biogenesis components suggested that it might capture additional targets. A chemical screening effort using this approach identified clomiphene, a widely used fertility drug, as one such compound. Mechanistic characterization revealed the target was the undecaprenyl diphosphate synthase, an enzyme that catalyzes the synthesis of a polyisoprenoid essential for both peptidoglycan and WTA synthesis. The work sheds light on mechanisms contributing to the observed suppressive interactions of clomiphene and in turn reveals aspects of the biology that underlie cell wall synthesis in S. aureus. Further, this effort highlights the utility of antagonistic interactions both in high-throughput screening and in compound mode of action studies. Importantly, clomiphene represents a lead for antibacterial drug discovery.

scholarly journals Upscaling of hiPS Cell–Derived Neurons for High-Throughput Screening

2016 ◽  
Vol 22 (3) ◽  
pp. 274-286
Author(s):  
Stefanie Traub ◽  
Heiko Stahl ◽  
Holger Rosenbrock ◽  
Eric Simon ◽  
Ralf Heilker
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Detection Sensitivity ◽  
Neuronal Markers ◽  
Downstream Target ◽  
Electrophysiological Recordings ◽  
Downstream Target Gene ◽  
The Central Nervous System ◽  
Induced Pluripotent

The advent of human-induced pluripotent stem (hiPS) cell–derived neurons promised to provide better model cells for drug discovery in the context of the central nervous system. This work demonstrates both the upscaling of cellular expansion and the acceleration of neuronal differentiation to accommodate the immense material needs of a high-throughput screening (HTS) approach. Using GRowth factor–driven expansion and INhibition of NotCH (GRINCH) during maturation, the derived cells are here referred to as GRINCH neurons. GRINCH cells displayed neuronal markers, and their functional activity could be demonstrated by electrophysiological recordings. In an application of GRINCH neurons, the brain-derived neurotrophic factor (BDNF)–mediated activation of tropomyosin receptor kinase (TrkB) was investigated as a promising drug target to treat synaptic dysfunctions. To assess the phosphorylation of endogenous TrkB in the GRINCH cells, the highly sensitive amplified luminescent proximity homogeneous assay LISA (AlphaLISA) format was established as a primary screen. A high-throughput reverse transcription (RT)–PCR format was employed as a secondary assay to analyze TrkB-mediated downstream target gene expression. In summary, an optimized differentiation protocol, highly efficient cell upscaling, and advanced assay miniaturization, combined with increased detection sensitivity, pave the way for a new generation of predictive cell-based drug discovery.

scholarly journals Multidimensional Profiling of CSF1R Screening Hits and Inhibitors

2011 ◽  
Vol 16 (9) ◽  
pp. 1007-1017 ◽  
Author(s):  
Joost C. M. Uitdehaag ◽  
Cecile M. Sünnen ◽  
Antoon M. van Doornmalen ◽  
Nikki de Rouw ◽  
Arthur Oubrie ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Active Form ◽  
Dissociation Rate ◽  
The Past ◽  
Compound Libraries ◽  
Competitive Kinetics ◽  
Homogeneous Assay ◽  
High Throughput Assay ◽  
Stimulating Factor

Over the past years, improvements in high-throughput screening (HTS) technology and compound libraries have resulted in a dramatic increase in the amounts of good-quality screening hits, and there is a growing need for follow-on hit profiling assays with medium throughput to further triage hits. Here the authors present such assays for the colony-stimulating factor 1 receptor (CSF1R, Fms), including tests for cellular activity and a homogeneous assay to measure affinity for inactive CSF1R. They also present a high-throughput assay to measure target residence time, which is based on competitive binding kinetics. To better fit koff rates, they present a modified mathematical model for competitive kinetics. In all assays, they profiled eight reference inhibitors (imatinib, sorafenib, sunitinib, tandutinib, dasatinib, GW2580, Ki20227, and J&J’s pyrido[2,3-d]pyrimidin-5-one). Using the known biochemical selectivities of these inhibitors, which can be quantified using metrics such as the selectivity entropy, the authors have determined which assay readout best predicts hit selectivity. Their profiling shows surprisingly that imatinib has a preference for the active form of CSF1R and that Ki20227 has an unusually slow target dissociation rate. This confirms that follow-on hit profiling is essential to ensure that the best hits are selected for lead optimization.

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