scholarly journals Discovery of Novel Gq-Biased LPA1 Negative Allosteric Modulators

2017 ◽  
Vol 22 (7) ◽  
pp. 859-866
Author(s):  
Yuji Shimizu ◽  
Masaharu Nakayama
Keyword(s):  
G Protein ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Allosteric Modulators ◽  
Label Free ◽  
Cellular Processes ◽  
Recombinant Cells ◽  
And Migration ◽  
Cellular Phenotypes

Lysophosphatidic acid (LPA) activates the G-protein-coupled receptor LPA1, which regulates various cellular processes, including cell proliferation and migration. Although LPA1 transduces cellular responses via Gq, Gi, and G12/13, associations between these signaling molecules and cellular phenotypes remain poorly characterized due to the lack of signal-specific pharmacological tools. Here, we characterized novel signal-biased modulators using multiple assays, including label-free impedance assays. LPA caused dramatic changes in cellular impedance in LPA1-expressing recombinant cells, which were susceptible to G-protein and protein kinase inhibitors. Subsequently, Gq-biased LPA1 negative allosteric modulators (NAMs) were identified using high-throughput screening, and a nonbiased antagonist differently affected the LPA-induced cellular impedance. These NAMs provide pharmacological tools for further investigations of the biology of LPA1.

scholarly journals A Nonradioactive Fluorimetric SPE-Based Ceramide Kinase Assay Using NBD-C6-Ceramide

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Helena Van Overloop ◽  
Gerd Van der Hoeven ◽  
Paul P. Van Veldhoven
Keyword(s):  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Solid Phase ◽  
Protein Kinase Inhibitors ◽  
Kinase Assay ◽  
Cellular Processes ◽  
Ceramide Kinase ◽  
C6 Ceramide ◽  
Ceramide 1

Ceramide kinase (CERK) has been implicated in important cellular processes such as inflammation and apoptosis. Its activity is usually measured using radiolabeled ceramide or [γ-32P]-ATP, followed by extraction, thin-layer chromatography, and detection of the formed labeled ceramide-1-phosphate. To eliminate the use of radioactivity, we developed similarly but independently from the approach by Don and Rosen (2008), a fluorescence-based ceramide kinase assay, using N-[7-(4-nitrobenz-2-oxa-1,3-diazole)]-6-aminohexanoyl-sphingenine (NBD-C6-ceramide) as substrate. ItsKmvalue (4 μM) was comparable to that of N-hexanoyl-sphingenine (C6-ceramide). The produced fluorescent NBD-C6-ceramide-1-phosphate was captured by means of solid-phase extraction on an aminopropyl phase, resulting in a fast and sensitive CERK measurement. By performing this assay in a 96-well format, it is also suitable for high-throughput screening (HTS) to search for CERK modulators. A limited screen revealed that some protein kinase inhibitors (e.g., U-0126;IC504 μM) and ceramide analogues (e.g., fenretinide, AMG-9810;IC501.1 μM) affect CERKin vitro.

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.

A robust and quantitative assay platform for multiplexed, high throughput screening of protein kinase inhibitors

2016 ◽  
Vol 52 (81) ◽  
pp. 12112-12115 ◽  
Author(s):  
Jieon Lee ◽  
Il-Soo Park ◽  
Ginam Park ◽  
Kyukwang Cho ◽  
Hee-Sung Park ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Protein Kinase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Protein Kinase Activity ◽  
Activity Assay ◽  
Kinase Activity Assay

We present a new platform for multiplexed protein kinase activity assay using TiO2decorated graphene oxide (GO), which is applicable to high throughput inhibitor screening.

G-protein-coupled receptor S1P1 acts within endothelial cells to regulate vascular maturation

Blood ◽  
2003 ◽  
Vol 102 (10) ◽  
pp. 3665-3667 ◽  
Author(s):  
Maria L. Allende ◽  
Tadashi Yamashita ◽  
Richard L. Proia
Keyword(s):  
Endothelial Cells ◽  
G Protein ◽  
Receptor Expression ◽  
S1p1 Receptor ◽  
Cellular Processes ◽  
Sphingosine 1 Phosphate ◽  
And Migration ◽  
The One ◽  
G Protein Coupled ◽  
Vascular Maturation

AbstractSphingosine-1-phosphate (S1P) stimulates signaling pathways via G-protein-coupled receptors and triggers diverse cellular processes, including growth, survival, and migration. In S1P1 receptor-deficient embryos, blood vessels were incompletely covered by vascular smooth muscle cells (VSMCs), indicating the S1P1 receptor regulates vascular maturation. Because S1P1 receptor expression is not restricted to a particular cell type, it was not known whether the S1P1 receptor controlled VSMC coverage of vessels in a cell-autonomous fashion by functioning directly in VSMCs or indirectly through its activity in endothelial cells (ECs). By using the Cre/loxP system, we disrupted the S1P1 gene solely in ECs. The phenotype of the conditional mutant embryos mimicked the one obtained in the embryos globally deficient in S1P1. Thus, vessel coverage by VSMCs is directed by the activity of the S1P1 receptor in ECs. (Blood. 2003;102:3665-3667)

scholarly journals mTOR inhibitor everolimus reduces invasiveness of melanoma cells

Human Cell ◽  
2019 ◽  
Vol 33 (1) ◽  
pp. 88-97 ◽  
Author(s):  
Dorota Ciołczyk-Wierzbicka ◽  
Dorota Gil ◽  
Marta Zarzycka ◽  
Piotr Laidler
Keyword(s):  
Mtor Inhibitor ◽  
Kinase Inhibitors ◽  
Mammalian Target Of Rapamycin ◽  
Mek Inhibitor ◽  
Melanoma Cells ◽  
Protein Kinase Inhibitors ◽  
Akt Inhibitor ◽  
Cancer Hallmarks ◽  
Cellular Processes ◽  
Cell Invasiveness

Abstract The mammalian target of rapamycin (mTOR) plays a key role in several cellular processes: proliferation, survival, invasion, and angiogenesis, and therefore, controls cell behavior both in health and in disease. Dysregulation of the mTOR signaling is involved in some of the cancer hallmarks, and thus the mTOR pathway is an important target for the development of a new anticancer therapy. The object of this study is recognition of the possible role of mTOR kinase inhibitors—everolimus single and in combination with selected downstream protein kinases inhibitors: LY294002 (PI3 K), U0126 (ERK1/2), GDC-0879 (B-RAF), AS-703026 (MEK), MK-2206 (AKT), PLX-4032 (B-RRAF) in cell invasion in malignant melanoma. Treatment of melanoma cells with everolimus led to a significant decrease in the level of both phosphorylated: mTOR (Ser2448) and mTOR (Ser2481) as well as their downstream effectors. The use of protein kinase inhibitors produced a significant decrease in metalloproteinases (MMPs) activity, as well as diminished invasion, especially when used in combination. The best results in the inhibition of both MMPs and cell invasiveness were obtained for the combination of an mTOR inhibitor— everolimus with a B-RAF inhibitor—PLX-4032. Slightly less profound reduction of invasiveness was obtained for the combinations of an mTOR inhibitor—everolimus with ERK1/2 inhibitor—U126 or MEK inhibitor—AS-703026 and in the case of MMPs activity decrease for PI3 K inhibitor—LY294002 and AKT inhibitor—MK-2206. The simultaneous use of everolimus or another new generation rapalog with selected inhibitors of crucial signaling kinases seems to be a promising concept in cancer treatment.

scholarly journals High-Throughput Screening by Mass Spectrometry: Comparison with the Scintillation Proximity Assay with a Focused-File Screen of AKT1/PKBα

2007 ◽  
Vol 12 (4) ◽  
pp. 473-480 ◽  
Author(s):  
Andrea K. Quercia ◽  
William A. Lamarr ◽  
Jayhyuk Myung ◽  
Can C. Özbal ◽  
James A. Landro ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Label Free ◽  
False Negatives ◽  
Scintillation Proximity Assay ◽  
Therapy Target ◽  
Hit Identification ◽  
Selection Of

Mass spectrometry is an emerging format for label-free high-throughput screening. The main limitation of mass spectrometry is throughput, due to the requirement to purify samples prior to ionization. Here the authors compare an automated high-throughput mass spectrometry (HTMS) system (RapidFire™) with the scintillation proximity assay (SPA). The cancer therapy target AKT1/PKBα was screened against a focused library of kinase inhibitors and IC50 values determined for all compounds that exhibit > 50% inhibition. A selection of additional compounds that exhibited ≤ 50% inhibition in the primary screen was chosen as controls to confirm inactives. The selection of compounds is expected to identify common actives, common inactives, false positives, and false negatives. Agreement is found between HTMS and SPA in terms of primary hit identification and hit confirmation. ( Journal of Biomolecular Screening 2007:473-480)

scholarly journals Development of a Fluorescence Polarization Bead-Based Coupled Assay to Target Different Activity/Conformation States of a Protein Kinase

2004 ◽  
Vol 9 (4) ◽  
pp. 309-321 ◽  
Author(s):  
Zhuomei Lu ◽  
Zhizhang Yin ◽  
Linda James ◽  
Rosalinda Syto ◽  
Jill M. Stafford ◽  
...  
Keyword(s):  
Protein Kinase ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Catalytic Domain ◽  
Protein Kinase Inhibitors ◽  
Biologically Active ◽  
Specific Substrate ◽  
Inactive Form ◽  
Substrate Peptide ◽  
Coupled Assay

Most of the protein kinase inhibitors being developed are directed toward the adenosine triphosphate (ATP) binding site that is highly conserved in many kinases. A major issue with these inhibitors is the specificity for a given kinase. Structure determination of several kinases has shown that protein kinases adopt distinct conformations in their inactive state, in contrast to their strikingly similar conformations in their active states. Hence, alternative assay formats that can identify compounds targeting the inactive form of a protein kinase are desirable. The authors describe the development and optimization of an Immobilized Metal Assay for Phosphochemicals (IMAP™)-based couple™d assay using PDK1 and inactive Akt-2 enzymes. PDK1 phosphorylates Akt-2 at Thr 309 in the catalytic domain, leading to enzymatic activation. Activation of Akt by PDK1 is measured by quantitating the phosphorylation of Akt-specific substrate peptide using the IMAP assay format. This IMAP-coupled assay has been formatted in a 384-well microplate format with a Z′ of 0.73 suitable for high-throughput screening. This assay was evaluated by screening the biologically active sample set LOPAC™ and validated with the protein kinase C inhibitor staurosporine. The IC50 value generated was comparable to the value obtained by the radioactive 33P-γ-ATP flashplate transfer assay. This coupled assay has the potential to identify compounds that target the inactive form of Akt and prevent its activation by PDK1, in addition to finding inhibitors of PDK1 and activated Akt enzymes.

Fluorescent biosensors for high throughput screening of protein kinase inhibitors

2013 ◽  
Vol 9 (2) ◽  
pp. 253-265 ◽  
Author(s):  
Camille Prével ◽  
Morgan Pellerano ◽  
Thi Nhu Ngoc Van ◽  
May C. Morris
Keyword(s):  
Protein Kinase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Fluorescent Biosensors
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