Homogeneous and Nonradioactive High-Throughput Screening Platform for the Characterization of Kinase Inhibitors in Cell Lysates

Guenat, Sylvie; Rouleau, Nathalie; Bielmann, Christelle; Bédard, Julie; Maurer, Fabienne; Allaman-Pillet, Nathalie; Nicod, Pascal; Bielefeld-Sévigny, Martina; Beckmann, Jacques S.; Bonny, Christophe; Bossé, Roger; Roduit, Raphaël

doi:10.1177/1087057106294697

Cited by 24 publications

(18 citation statements)

References 26 publications

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“…The expression of non-phosphorylated kinases was not changed after UV stress for all three tested kinases. To confirm western blot observations, we measured the activity of JNKs by a non-radioactive AlphaScreen assay [16]. In this assay, the luminescence of the acceptor beads was proportional to JNKs activity, as shown in panel B of where JNK activity was largely induced by UV.…”

Section: Jnk1/2 and P38 Are Activated By Uv Treatmentmentioning

confidence: 95%

“…After 2 h of incubation, AlphaScreen signal was measured on Envision Ò system. The measurement of JNK activity was performed as described previously [16]; briefly kinase reagents (10 nM B-GST-cJun, 30 nM anti P-cJun antibody and 50 lM of ATP) were first diluted in kinase buffer (20 mM Tris-HCl pH 7.6, 10 mM MgCl 2 , 1 mM DTT, 100 lM Na 3 VO 4 , 0.01% Tween-20) and added to 1 lg of proteins for 30 min at 23°C. Detection was performed by an addition of 10 ll of beads mix (Protein A acceptor 20 lg/ml and Streptavidin donor 20 lg/ml), diluted in detection buffer (20 mM Tris-HCl pH 7.4, 20 mM NaCl, 80 mM EDTA, 0.3% BSA), followed by a 1 h incubation at 23°C in the dark.…”

Section: Rpe Cells Isolation and Cell Culturementioning

confidence: 99%

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MAP kinase pathways in UV-induced apoptosis of retinal pigment epithelium ARPE19 cells

Roduit

Schorderet

2008

Apoptosis

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The retinal pigment epithelium (RPE) is constantly exposed to external injuries which lead to degeneration, dysfunction or loss of RPE cells. The balance between RPE cells death and proliferation may be responsible for several diseases of the underlying retina, including age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Signaling pathways able to control cells proliferation or death usually involve the MAPK (mitogen-activated protein kinases) pathways, which modulate the activity of transcription factors by phosphorylation. UV exposure induces DNA breakdown and causes cellular damage through the production of reactive oxygen species (ROS) leading to programmed cell death. In this study, human retinal pigment epithelial cells ARPE19 were exposed to 100 J/m(2) of UV-C and MAPK pathways were studied. We first showed the expression of the three major MAPK pathways. Then we showed that activator protein-1 (AP-1) was activated through phosphorylation of cJun and cFos, induced by JNK and p38, respectively. Specific inhibitors of both kinases decreased their respective activities and phosphorylation of their nuclear targets (cJun and cFos) and reduced UV-induced cell death. The use of specific kinases inhibitors may provide excellent tools to prevent RPE apoptosis specifically in RPE diseases involving ROS and other stress-related compounds such as in AMD.

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Section: Jnk1/2 and P38 Are Activated By Uv Treatmentmentioning

confidence: 95%

Section: Rpe Cells Isolation and Cell Culturementioning

confidence: 99%

MAP kinase pathways in UV-induced apoptosis of retinal pigment epithelium ARPE19 cells

Roduit

Schorderet

2008

Apoptosis

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“…At that surface, one component produces a product that serves to activate a second component, thereby producing a detectable signal. In many aspects of drug discovery, principally high throughput screening (HTS), AlphaScreen has been widely deployed in aspects of cell signaling research, drug discovery and biomarker quantification [1-5]. This is wide adoption results from both the simplicity of assay protocols, but also from the high sensitivity of the assay.…”

Section: Introductionmentioning

confidence: 99%

The Use of AlphaScreen Technology in HTS: Current Status

Eglen¹,

Reisine²,

Roby³

et al. 2008

Curr Chem Genomics

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221

184

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AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) is versatile assay technology developed to measuring analytes using a homogenous protocol. This technology is an example of a bead-based proximity assay and was developed from a diagnostic assay technology known as LOCI (Luminescent Oxygen Channeling Assay). Here, singlet oxygen molecules, generated by high energy irradiation of Donor beads, travel over a constrained distance (approx. 200 nm) to Acceptor beads. This results in excitation of a cascading series of chemical reactions, ultimately causing generation of a chemiluminescent signal.In the past decade, a wide variety of applications has been reported, ranging from detection of analytes involved in cell signaling, including protein:protein, protein:peptide, protein:small molecule or peptide:peptide interactions. Numerous homogeneous HTS-optimized assays have been reported using the approach, including generation of second messengers (such as accumulation of cyclic AMP, cyclic GMP, inositol [1, 4, 5] trisphosphate or phosphorylated ERK) from liganded GPCRs or tyrosine kinase receptors, post-translational modification of proteins (such as proteolytic cleavage, phosphorylation, ubiquination and sumoylation) as well as protein-protein and protein-nucleic acid interactions.Recently, the basic AlphaScreen technology was extended in that the chemistry of the Acceptor bead was modified such that emitted light is more intense and spectrally defined, thereby markedly reducing interference from biological fluid matrices (such as trace hemolysis in serum and plasma). In this format, referred to as AlphaLISA, it provides an alternative technology to classical ELISA assays and is suitable for high throughput automated fluid dispensing and detection systems.Collectively, AlphaScreen and AlphaLISA technologies provide a facile assay platform with which one can quantitate complex cellular processes using simple no-wash microtiter plate based assays. They provide the means by which large compound libraries can be screened in a high throughput fashion at a diverse range of therapeutically important targets, often not readily undertaken using other homogeneous assay technologies. This review assesses the current status of the technology in drug discovery, in general, and high throughput screening (HTS), in particular.

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“…The most important advantage of AlphaScreen over TR-FRET is that the AlphaScreen can distinguish between an agonist and an antagonist by the selective usage of coactivator or corepressor peptides. The AlphaScreen system is generally applicable over a wide variety of biomolecular targets, which can supplant solid-support binding assays in many applications, such as receptor-ligand interactions [191], lipid signaling [192], protein kinase monitoring [193], and other types of signaling [194]. …”

Section: Strategies For the Discovery Of Novel Ligands For Nuclearmentioning

confidence: 99%

Targeting Nuclear Receptors with Marine Natural Products

Yang

2014

Marine Drugs

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Nuclear receptors (NRs) are important pharmaceutical targets because they are key regulators of many metabolic and inflammatory diseases, including diabetes, dyslipidemia, cirrhosis, and fibrosis. As ligands play a pivotal role in modulating nuclear receptor activity, the discovery of novel ligands for nuclear receptors represents an interesting and promising therapeutic approach. The search for novel NR agonists and antagonists with enhanced selectivities prompted the exploration of the extraordinary chemical diversity associated with natural products. Recent studies involving nuclear receptors have disclosed a number of natural products as nuclear receptor ligands, serving to re-emphasize the translational possibilities of natural products in drug discovery. In this review, the natural ligands of nuclear receptors will be described with an emphasis on their mechanisms of action and their therapeutic potentials, as well as on strategies to determine potential marine natural products as nuclear receptor modulators.

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Homogeneous and Nonradioactive High-Throughput Screening Platform for the Characterization of Kinase Inhibitors in Cell Lysates

Cited by 24 publications

References 26 publications

MAP kinase pathways in UV-induced apoptosis of retinal pigment epithelium ARPE19 cells

MAP kinase pathways in UV-induced apoptosis of retinal pigment epithelium ARPE19 cells

The Use of AlphaScreen Technology in HTS: Current Status

Targeting Nuclear Receptors with Marine Natural Products

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