Immobilized Metal Ion Affinity-Based Fluorescence Polarization (IMAP): Advances in Kinase Screening

Sportsman, J. Richard; Gaudet, Elizabeth A.; Boge, Annegret

doi:10.1089/154065804323056549

Cited by 50 publications

(40 citation statements)

References 17 publications

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“…Non-RI protein kinase assays based on fluorescence polarization, such as the Far-Red PolarScreen TM fluorescence polarization kinase assay kit (Invitrogen) and KinEASE (Upstate Cell Signaling Solutions, Charlottesville, VA, USA), have been developed. A highly sensitive fluorescence polarization assay combined with bead technology has also been reported (IMAP-FP assay) (Molecular Devices, Sunnyvale, CA, USA) (13). Detecting the changes in ATP levels associated with the kinase reaction is another way to monitor kinase activity.…”

Section: Various Techniques To Detect Activities Of Protein Kinasesmentioning

confidence: 99%

Recent Advances in Technologies for Analyzing Protein Kinases

et al. 2007

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Abstract. Most cellular events are regulated by protein phosphorylation mediated by protein kinases, whose malfunction is involved in the etiology of various disorders. The elucidation of the biochemical properties of the protein phosphorylation reaction will lead not only to a better understanding of the signal transduction mechanism, but also to developing new therapeutic agents. In this review, we briefly summarize the technologies to detect or characterize protein kinases with special emphasis on recently developed and / or commercially available techniques.

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Section: Various Techniques To Detect Activities Of Protein Kinasesmentioning

confidence: 99%

Recent Advances in Technologies for Analyzing Protein Kinases

et al. 2007

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“…24 The identification of non-ATP competitive ligands that modulate kinase function through stabilizing active or inactive conformational states (e.g. allosteric modulators) would provide a novel mechanism-of-action for kinase drugs.…”

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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“…11 So far all the developed methods based on fluorescence polarization for phosphatase assays need substrates labeled with proper fluorophores and huge molecules that bind specifically to phosphates such as antibodies, 12,13 polyarginine, 14 or IMAP TM , 15 and such requirements make the assays less straightforward. Development of methods to detect the phosphatase activity with less number of experimental steps would make assays more cost-effective and less laborious and eventually improve high-throughput screening of phosphatase inhibitors as well as measuring activity of phosphatases.…”

Section: Introductionmentioning

confidence: 99%

Label-Free and Real-Time Monitoring of Phosphatase Reactions Using a Phosphate-Specific and Fluorescent Probe

2008

Bulletin of the Korean Chemical Society

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A phosphate-specific and fluorescent probe was prepared for label-free phosphatase assays based on fluorescence polarization. By using the probe, dephosphorylation reactions of DNA and protein substrates by calf intestinal alkaline phosphatase (CIP) could effectively be monitored in real-time. Since this assay method does not require additional materials such as labeled substrates and phosphospecific antibodies to obtain fluorescence polarization signals, it is simple, cost-effective, and expected to be useful not only for measuring activity of phosphatases but also for high-throughput screening of phosphatase inhibitors.

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Immobilized Metal Ion Affinity-Based Fluorescence Polarization (IMAP): Advances in Kinase Screening

Cited by 50 publications

References 17 publications

Recent Advances in Technologies for Analyzing Protein Kinases

Recent Advances in Technologies for Analyzing Protein Kinases

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

Label-Free and Real-Time Monitoring of Phosphatase Reactions Using a Phosphate-Specific and Fluorescent Probe

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