scholarly journals A live-cell screen for altered Erk dynamics reveals principles of proliferative control

2019 ◽  
Author(s):  
Alexander G. Goglia ◽  
Maxwell Z. Wilson ◽  
Jillian Silbert ◽  
Lena P. Basta ◽  
Siddhartha G. Jena ◽  
...  
Keyword(s):  
High Throughput ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Pulse Frequency ◽  
Live Cell ◽  
Live Cells ◽  
Drug Induced ◽  
Primary Mouse ◽  
Cell Assays ◽  
High Throughput Screens

Complex, time-varying responses have been observed widely in cell signaling, but how specific dynamics are generated or regulated is largely unknown. One major obstacle has been that high-throughput screens for identifying pathway components are typically incompatible with the live-cell assays used to monitor dynamics. Here, we address this challenge by performing a drug screen for altered Erk signaling dynamics in primary mouse keratinocytes. We screened a library of 429 kinase inhibitors, monitoring Erk activity over 5 h in more than 80,000 single live cells. The screen revealed both known and uncharacterized modulators of Erk dynamics, including inhibitors of non-EGFR receptor tyrosine kinases (RTKs) that increased Erk pulse frequency and overall activity. Using drug treatment and direct optogenetic control, we demonstrate that drug-induced changes to Erk dynamics alter the conditions under which cells proliferate. Our work opens the door to high-throughput screens using live-cell biosensors and reveals that cell proliferation integrates information from Erk dynamics as well as additional permissive cues.

scholarly journals Development of a Robust High-Throughput Screening Platform for Inhibitors of the Striatal-Enriched Tyrosine Phosphatase (STEP)

2021 ◽  
Vol 22 (9) ◽  
pp. 4417
Author(s):  
Lester J Lambert ◽  
Stefan Grotegut ◽  
Maria Celeridad ◽  
Palak Gosalia ◽  
Laurent JS De Backer ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
Tyrosine Kinases ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Synaptic Function ◽  
Label Free ◽  
Protein Tyrosine

Many human diseases are the result of abnormal expression or activation of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Not surprisingly, more than 30 tyrosine kinase inhibitors (TKIs) are currently in clinical use and provide unique treatment options for many patients. PTPs on the other hand have long been regarded as “undruggable” and only recently have gained increased attention in drug discovery. Striatal-enriched tyrosine phosphatase (STEP) is a neuron-specific PTP that is overactive in Alzheimer’s disease (AD) and other neurodegenerative and neuropsychiatric disorders, including Parkinson’s disease, schizophrenia, and fragile X syndrome. An emergent model suggests that the increase in STEP activity interferes with synaptic function and contributes to the characteristic cognitive and behavioral deficits present in these diseases. Prior efforts to generate STEP inhibitors with properties that warrant clinical development have largely failed. To identify novel STEP inhibitor scaffolds, we developed a biophysical, label-free high-throughput screening (HTS) platform based on the protein thermal shift (PTS) technology. In contrast to conventional HTS using STEP enzymatic assays, we found the PTS platform highly robust and capable of identifying true hits with confirmed STEP inhibitory activity and selectivity. This new platform promises to greatly advance STEP drug discovery and should be applicable to other PTP targets.

scholarly journals Droplet-microarray on superhydrophobic–superhydrophilic patterns for high-throughput live cell screenings

RSC Advances ◽  
2016 ◽  
Vol 6 (44) ◽  
pp. 38263-38276 ◽  
Author(s):  
Anna A. Popova ◽  
Konstantin Demir ◽  
Titus Genisius Hartanto ◽  
Eric Schmitt ◽  
Pavel A. Levkin
Keyword(s):  
High Throughput ◽  
Microarray Platform ◽  
Live Cell ◽  
Live Cells

Droplet-microarray platform based on superhydrophobic–superhydrophilic patterning allows for miniaturized high throughput drug and transfection screenings of live cells in separated nanoliter droplets.

scholarly journals A novel live cell assay to measure diacylglycerol lipase α activity

2016 ◽  
Vol 36 (3) ◽  
Author(s):  
Praveen K. Singh ◽  
Rachel Markwick ◽  
Fiona V. Howell ◽  
Gareth Williams ◽  
Patrick Doherty
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
Human Cell Line ◽  
Live Cell ◽  
Diacylglycerol Lipase ◽  
Cell Assay ◽  
Retrograde Signalling ◽  
Cell Assays ◽  
The Central Nervous System ◽  
Rate Limiting

Diacylglycerol lipase α (DAGLα) hydrolyses DAG to generate the principal endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLα dependent cannabinoid (CB) signalling has been implicated in numerous processes including axonal growth and guidance, adult neurogenesis and retrograde signalling at the synapse. Recent studies have implicated DAGLα as an emerging drug target for several conditions including pain and obesity. Activity assays are critical to the drug discovery process; however, measurement of diacylglycerol lipase (DAGL) activity using its native substrate generally involves low-throughput MS techniques. Some relatively high-throughput membrane based assays utilizing surrogate substrates have been reported, but these do not take into account the rate-limiting effects often associated with the ability of a drug to cross the cell membrane. In the present study, we report the development of a live cell assay to measure DAGLα activity. Two previously reported DAGLα surrogate substrates, p-nitrophenyl butyrate (PNPB) and 6,8-difluoro-4-methylumbelliferyl octanoate (DiFMUO), were evaluated for their ability to detect DAGLα activity in live cell assays using a human cell line stably expressing the human DAGLα transgene. Following optimization, the small molecule chromogenic substrate PNPB proved to be superior by providing lower background activity along with a larger signal window between transfected and parental cells when compared with the fluorogenic substrate DiFMUO. The assay was further validated using established DAGL inhibitors. In summary, the live cell DAGLα assay reported here offers an economical and convenient format to screen for novel inhibitors as part of drug discovery programmes and compliments previously reported high-throughput membrane based DAGL assays.

scholarly journals Development of a Homogeneous Time-Resolved Fluorescence Assay for High Throughput Screening to Identify Lck Inhibitors: Comparison with Scintillation Proximity Assay and Streptavidin-Coated Plate Assay

2000 ◽  
Vol 5 (6) ◽  
pp. 463-470 ◽  
Author(s):  
Natsue Ohml ◽  
Jonathan M. Wingfield ◽  
Hidenori Yazawa ◽  
Osamu Inagaki
Keyword(s):  
High Throughput ◽  
Tyrosine Kinases ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Time Resolved Fluorescence ◽  
Screening Assay ◽  
Time Resolved ◽  
Atp Binding Site ◽  
High Throughput Screening Assay ◽  
Coated Plate

This study details the development of a homogeneous time-resolved fluorescence (HTRF) high throughput screening assay to identify inhibitors of Lck. HTRF was compared with scintillation proximity and streptavidin-coated plate assays. Because of the differences in the sensitivity of detection of phosphotyrosine among the three assays, different amounts of enzyme were used. However, the concentrations of the other assay components were standardized. When using similar assay conditions, the calculated IC50 values of inhibitory compounds were independent of assay format. Furthermore, filtration experiments revealed that phosphorylation of a biotinyl poly-Glu,Ala, Tyr peptide substrate was less than autophosphorylation of the Lck enzyme; this was due to the low Km value for biotinyl poly-Glu,Ala,Tyr. In the HTRF assay, small amounts of enzyme and high concentrations of ATP could be used, thereby minimizing the effects of autophosphorylation. Higher ATP concentration would also minimize the effect of ATP competitors. Using this technology, it may be possible to find novel kinase inhibitors that do not act at the ATP binding site of protein tyrosine kinases.

scholarly journals Development of a High-Throughput Screening Method for LIM Kinase 1 Using a Luciferase-Based Assay of ATP Consumption

2011 ◽  
Vol 17 (4) ◽  
pp. 460-468 ◽  
Author(s):  
Mokdad Mezna ◽  
Ai Ching Wong ◽  
Margaret Ainger ◽  
Rebecca W. Scott ◽  
Tim Hammonds ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Kinase Inhibitors ◽  
Screening Method ◽  
Human Diseases ◽  
Luciferase Assay ◽  
Lim Kinase ◽  
Binding Pockets ◽  
High Throughput Screens

Kinases are attractive drug targets because of the central roles they play in signal transduction pathways and human diseases. Their well-formed adenosine triphosphate (ATP)–binding pockets make ideal targets for small-molecule inhibitors. For drug discovery purposes, many peptide-based kinase assays have been developed that measure substrate phosphorylation using fluorescence-based readouts. However, for some kinases these assays may not be appropriate. In the case of the LIM kinases (LIMK), an inability to phosphorylate peptide substrates resulted in previous high-throughput screens (HTS) using radioactive labeling of recombinant cofilin protein as the readout. We describe the development of an HTS-compatible assay that measures relative ATP levels using luciferase-generated luminescence as a function of LIMK activity. The assay was inexpensive to perform, and proof-of-principle screening of kinase inhibitors demonstrated that compound potency against LIMK could be determined; ultimately, the assay was used for successful prosecution of automated HTS. Following HTS, the secondary assay format was changed to obtain more accurate measures of potency and mechanism of action using more complex (and expensive) assays. The luciferase assay nonetheless provides an inexpensive and reliable primary assay for HTS that allowed for the identification of LIMK inhibitors to initiate discovery programs for the eventual treatment of human diseases.

scholarly journals High-Throughput Molecular Imaging for the Identification of FADD Kinase Inhibitors

2010 ◽  
Vol 15 (9) ◽  
pp. 1063-1070 ◽  
Author(s):  
Amjad P. Khan ◽  
Katrina A. Schinske ◽  
Shyam Nyati ◽  
Mahaveer S. Bhojani ◽  
Brian D. Ross ◽  
...  
Keyword(s):  
High Throughput ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Live Cells ◽  
Death Domain ◽  
A Cell ◽  
Induced Apoptosis ◽  
Cycle Regulation ◽  
Potential Use

Fas-associated protein with death domain (FADD) was originally reported as a proapoptotic adaptor molecule that mediates receptor-induced apoptosis. Recent studies have revealed a potential role of FADD in NF-κB activation, embryogenesis, and cell cycle regulation and proliferation. Overexpression of FADD and its phosphorylation have been associated with the transformed phenotype in many cancers and is therefore a potential target for therapeutic intervention. In an effort to delineate signaling events that lead to FADD phosphorylation and to identify novel compounds that impinge on this pathway, the authors developed a cell-based reporter for FADD kinase activity. The reporter assay, optimized for a high-throughput screen (HTS), measures bioluminescence in response to modulation of FADD kinase activity in live cells. In addition, the potential use of the reporter cell line in the rapid evaluation of pharmacologic properties of HTS hits in mouse models has been demonstrated.

Yeast-based high-throughput screens for discovery of kinase inhibitors for neglected diseases

2020 ◽  
Author(s):  
T.A. Tavella ◽  
G.C. Cassiano ◽  
F.T.M. Costa ◽  
P. Sunnerhagen ◽  
E. Bilsland
Keyword(s):  
High Throughput ◽  
Kinase Inhibitors ◽  
Neglected Diseases ◽  
High Throughput Screens

α-Methylene-β-Lactone Probe for Measuring Live-Cell Reactions of Small Molecules

2020 ◽  
Author(s):  
Lei Wang ◽  
Louis Riel ◽  
Bekim Bajrami ◽  
Bin Deng ◽  
Amy Howell ◽  
...  
Keyword(s):  
Mass Spectra ◽  
Live Cell ◽  
Live Cells ◽  
The Novel ◽  
Proteomics Analysis ◽  
Chemical Proteomics ◽  
Tandem Mass Spectra ◽  
Modified Peptides ◽  
Covalent Probes ◽  
Ht 29

The novel use of the α-methylene-β-lactone (MeLac) moiety as a warhead of multiple electrophilic sites is reported. In this study, we demonstrate that a MeLac-alkyne is a competent covalent probe and reacts with diverse proteins in live cells. Proteomics analysis of affinity-enriched samples identifies probe-reacted proteins, resolves their modified peptides/residues, and thus characterizes probe-protein reactions. Unique methods are developed to evaluate confidence in the identification of the reacted proteins and modified peptides. Tandem mass spectra of the peptides reveal that MeLac reacts with nucleophilic cysteine, serine, lysine, threonine, and tyrosine residues, through either Michael addition or acyl addition. A peptide-centric proteomics platform, using MeLac-alkyne as the measurement probe, successfully analyzes the Orlistat selectivity in live HT-29 cells. MeLac is a versatile warhead demonstrating enormous potential to expedite the development of covalent probes and inhibitors in interrogating protein (re)activity. MeLac-empowered platforms in chemical proteomics are widely adaptable for measuring the live-cell action of reactive molecules.

scholarly journals A high-throughput screening assay based on automated microscopy for monitoring antibiotic susceptibility of Mycobacterium tuberculosis phenotypes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sadaf Kalsum ◽  
Blanka Andersson ◽  
Jyotirmoy Das ◽  
Thomas Schön ◽  
Maria Lerm
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Imaging System ◽  
Aggregate Size ◽  
Live Cell ◽  
Screening Assay ◽  
High Throughput Screening Assay

Abstract Background Efficient high-throughput drug screening assays are necessary to enable the discovery of new anti-mycobacterial drugs. The purpose of our work was to develop and validate an assay based on live-cell imaging which can monitor the growth of two distinct phenotypes of Mycobacterium tuberculosis and to test their susceptibility to commonly used TB drugs. Results Both planktonic and cording phenotypes were successfully monitored as fluorescent objects using the live-cell imaging system IncuCyte S3, allowing collection of data describing distinct characteristics of aggregate size and growth. The quantification of changes in total area of aggregates was used to define IC50 and MIC values of selected TB drugs which revealed that the cording phenotype grew more rapidly and displayed a higher susceptibility to rifampicin. In checkerboard approach, testing pair-wise combinations of sub-inhibitory concentrations of drugs, rifampicin, linezolid and pretomanid demonstrated superior growth inhibition of cording phenotype. Conclusions Our results emphasize the efficiency of using automated live-cell imaging and its potential in high-throughput whole-cell screening to evaluate existing and search for novel antimycobacterial drugs.

scholarly journals Role of the ABL tyrosine kinases in the epithelial–mesenchymal transition and the metastatic cascade

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Jillian Hattaway Luttman ◽  
Ashley Colemon ◽  
Benjamin Mayro ◽  
Ann Marie Pendergast
Keyword(s):  
Solid Tumors ◽  
Tyrosine Kinases ◽  
Epithelial To Mesenchymal Transition ◽  
Kinase Inhibitors ◽  
Epithelial Mesenchymal Transition ◽  
Therapeutic Effects ◽  
Mesenchymal Transition ◽  
Metastatic Cascade ◽  
Abl Kinases ◽  
Treatment Paradigm

AbstractThe ABL kinases, ABL1 and ABL2, promote tumor progression and metastasis in various solid tumors. Recent reports have shown that ABL kinases have increased expression and/or activity in solid tumors and that ABL inactivation impairs metastasis. The therapeutic effects of ABL inactivation are due in part to ABL-dependent regulation of diverse cellular processes related to the epithelial to mesenchymal transition and subsequent steps in the metastatic cascade. ABL kinases target multiple signaling pathways required for promoting one or more steps in the metastatic cascade. These findings highlight the potential utility of specific ABL kinase inhibitors as a novel treatment paradigm for patients with advanced metastatic disease.

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