Miniaturization and Validation of a High-Throughput Serine Kinase Assay Using the Alpha Screen Platform

Leoprechting, Achim Von; Kumpf, R.; Menzel, Susanne; Reulle, Dominique; Griebel, Ralf; Valler, Martin J.; Büttner, Frank

doi:10.1177/1087057104268805

Cited by 38 publications

(29 citation statements)

References 11 publications

(14 reference statements)

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“…The application of AlphaScreen technology for highthroughput enzymatic assays has been described previously (Von Leoprechting et al, 2004;Warner et al, 2004;Burns et al, 2006). We adapted this assay format for the identification of inhibitors of PPM1D phosphatase activity.…”

Section: High-throughput Screen To Identify Chemical Inhibitors Of Ppm1dmentioning

confidence: 99%

A chemical inhibitor of PPM1D that selectively kills cells overexpressing PPM1D

et al. 2007

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The PPM1D gene is aberrantly amplified in a range of common cancers and encodes a protein phosphatase that is a potential therapeutic target. However, the issue of whether inhibition of PPM1D in human tumour cells that overexpress this protein compromises their viability has not yet been fully addressed. We show here, using an RNA interference (RNAi) approach, that inhibition of PPM1D can indeed reduce the viability of human tumour cells and that this effect is selective; tumour cell lines that overexpress PPM1D are sensitive to PPM1D inhibition whereas cell lines with normal levels are not. Loss of viability associated with PPM1D RNAi in human tumour cells occurs via the activation of the kinase P38. To identify chemical inhibitors of PPM1D, a high-throughput screening of a library of small molecules was performed. This strategy successfully identified a compound that selectively reduces viability of human tumour cell lines that overexpress PPM1D. As expected of a specific inhibitor, the toxicity to PPM1D overexpressing cell lines after inhibitor treatment is P38 dependent. These results further validate PPM1D as a therapeutic target and identify a proof-of-principle small molecule inhibitor.

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Section: High-throughput Screen To Identify Chemical Inhibitors Of Ppm1dmentioning

confidence: 99%

A chemical inhibitor of PPM1D that selectively kills cells overexpressing PPM1D

et al. 2007

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“…In total, 361 templates were selected for biochemical screening. The focused set of 361 compounds was evaluated in a 384-well format Amplified Luminescent Proximity Homogeneous Assay Screen (AlphaScreen) 33 for inhibition of CHK1 kinase activity, measured by the reduction in phosphorylation of a CHK1 substrate peptide. The assay was adapted for screening at high ligand concentration (250 μM), with each test plate run under three different conditions.…”

Section: Resultsmentioning

confidence: 99%

Identification of Inhibitors of Checkpoint Kinase 1 through Template Screening

Matthews¹,

Klair²,

Burns³

et al. 2009

J. Med. Chem.

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Checkpoint kinase 1 (CHK1) is an oncology target of significant current interest. Inhibition of CHK1 abrogates DNA damage-induced cell cycle checkpoints and sensitizes p53 deficient cancer cells to genotoxic therapies. Using template screening, a fragment-based approach to small molecule hit generation, we have identified multiple CHK1 inhibitor scaffolds suitable for further optimization. The sequential combination of in silico low molecular weight template selection, a high concentration biochemical assay and hit validation through protein-ligand X-ray crystallography provided 13 template hits from an initial in silico screening library of ca. 15000 compounds. The use of appropriate counter-screening to rule out nonspecific aggregation by test compounds was essential for optimum performance of the high concentration bioassay. One low molecular weight, weakly active purine template hit was progressed by iterative structure-based design to give submicromolar pyrazolopyridines with good ligand efficiency and appropriate CHK1-mediated cellular activity in HT29 colon cancer cells.

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“…Assays commonly used in HTS against protein kinases follow substrate or ATP turnover as measured by a variety of approaches, including luminescence 18 , fluorescent polymer superquenching 19 , homogenous time resolved fluorescence (HTRF) 20 , scintillation proximity assay (SPA) 21 , AlphaScreen 22 , electrophoretic shift 23 and fluorescence polarization (FP). 24 The identification of non-ATP competitive ligands that modulate kinase function through stabilizing active or inactive conformational states (e.g.…”

Section: Limitations In High-throughput Screening Of Protein Kinasesmentioning

confidence: 99%

“…To prove probe specificity and demonstrate that this assay is not reporting artifacts such as protein or peptide aggregation, a truncated version of IP20, PKI (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24), was shown to compete with FAM-IP20 for binding to C-subunit in a dose dependent manner, with an IC 50 of 2.7 µM (Figure 2c). …”

Section: Interaction Of Fam-ip20 With the Catalytic Subunitmentioning

confidence: 99%

Assay Principle for Modulators of Protein−Protein Interactions and Its Application to Non-ATP-Competitive Ligands Targeting Protein Kinase A

et al. 2006

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Targeting sites that modulate protein-protein interactions represents an ongoing challenge for drug discovery. We have devised an assay principle, named Ligand-Regulated Competition (LiReC), in an effort to find non-ATP competitive small molecule regulators for type Iα cAMP-dependent Protein Kinase A (PKA-Iα), a protein complex that is implicated in disease. Our assay based on the LiReC principle utilizes a competitive fluorescent peptide probe to assess the integrity of the PKA-Iα complex upon introduction of an allosteric ligand. The developed fluorescence polarization method screens for small molecules that specifically protect (antagonists) or conversely activate (agonists) this protein complex. In high throughput format, various cyclic nucleotide-derived agonists and antagonists are successfully detected with high precision. Furthermore, assay performance (Z'-factors above 0.7) far exceeds the minimum requirement for small molecule screening. To identify compounds that operate through novel modes of action, our method shields the ATP binding site and purposely excludes ATP-competitive ligands. These proof-of-principle experiments highlight the potential of the LiReC technique and suggest its application to other protein complexes, thereby providing a novel approach to identify and characterize modulators (small molecules, proteins, peptides, or nucleic acids) of protein-protein systems.

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Miniaturization and Validation of a High-Throughput Serine Kinase Assay Using the Alpha Screen Platform

Cited by 38 publications

References 11 publications

A chemical inhibitor of PPM1D that selectively kills cells overexpressing PPM1D

A chemical inhibitor of PPM1D that selectively kills cells overexpressing PPM1D

Identification of Inhibitors of Checkpoint Kinase 1 through Template Screening

Assay Principle for Modulators of Protein−Protein Interactions and Its Application to Non-ATP-Competitive Ligands Targeting Protein Kinase A

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