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

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 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 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.

High-Throughput Screening to Identify Small Molecules That Selectively Inhibit APOL1 Protein Level in Podocytes

2021 ◽  
pp. 247255522110262
Author(s):  
Jonathan Choy ◽  
Yanqing Kan ◽  
Steve Cifelli ◽  
Josephine Johnson ◽  
Michelle Chen ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Strategy ◽  
Time Resolved ◽  
Protein Levels ◽  
Increased Risk ◽  
Compound Screening ◽  
High Throughput Screens ◽  
Screening Library

High-throughput phenotypic screening is a key driver for the identification of novel chemical matter in drug discovery for challenging targets, especially for those with an unclear mechanism of pathology. For toxic or gain-of-function proteins, small-molecule suppressors are a targeting/therapeutic strategy that has been successfully applied. As with other high-throughput screens, the screening strategy and proper assays are critical for successfully identifying selective suppressors of the target of interest. We executed a small-molecule suppressor screen to identify compounds that specifically reduce apolipoprotein L1 (APOL1) protein levels, a genetically validated target associated with increased risk of chronic kidney disease. To enable this study, we developed homogeneous time-resolved fluorescence (HTRF) assays to measure intracellular APOL1 and apolipoprotein L2 (APOL2) protein levels and miniaturized them to 1536-well format. The APOL1 HTRF assay served as the primary assay, and the APOL2 and a commercially available p53 HTRF assay were applied as counterscreens. Cell viability was also measured with CellTiter-Glo to assess the cytotoxicity of compounds. From a 310,000-compound screening library, we identified 1490 confirmed primary hits with 12 different profiles. One hundred fifty-three hits selectively reduced APOL1 in 786-O, a renal cell adenocarcinoma cell line. Thirty-one of these selective suppressors also reduced APOL1 levels in conditionally immortalized human podocytes. The activity and specificity of seven resynthesized compounds were validated in both 786-O and podocytes.

An overview of analytical methods for monitoring bacterial transglycosylation

2014 ◽  
Vol 6 (19) ◽  
pp. 7590-7596 ◽  
Author(s):  
Bart Blanchaert ◽  
Erwin Adams ◽  
Ann Van Schepdael
Keyword(s):  
High Throughput ◽  
Analytical Methods ◽  
High Throughput Screens

This review highlights the fluorescence and radioactively labeled assays and high-throughput screens for the search for antibiotics targeting bacterial transglycosylation.

Discovery of indenopyrazoles as EGFR and VEGFR-2 tyrosine kinase inhibitors by in silico high-throughput screening

2008 ◽  
Vol 18 (1) ◽  
pp. 285-288 ◽  
Author(s):  
Taikou Usui ◽  
Hyun Seung Ban ◽  
Junpei Kawada ◽  
Takatsugu Hirokawa ◽  
Hiroyuki Nakamura
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
High Throughput ◽  
In Silico ◽  
High Throughput Screening ◽  
Kinase Inhibitors

Technology for high-throughput screens: the present and future using zebrafish

2004 ◽  
Vol 15 (6) ◽  
pp. 564-571 ◽  
Author(s):  
Donald R Love ◽  
Franz B Pichler ◽  
Andrew Dodd ◽  
Brent R Copp ◽  
David R Greenwood
Keyword(s):  
High Throughput ◽  
High Throughput Screens
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