Biomedical subjects

Frauke Rininsland

Publications and source records attributed to Frauke Rininsland.

6 recordsLinked to original sources

PubMed2006

Biotinylated peptides for rapid identification of substrates and inhibitors of kinases and phosphatases with fluorescence superquenching.

Aberrant regulation of kinase and phosphatase activities is implicated in various diseases, including cancer, diabetes, and inflammation. Thus, high-throughput screening (HTS) has become a focused strategy for the identification of kinase and phosphatase inhibitors. With a growing number of these enzymes becoming available for HTS, rapid identification of substrates has become pertinent. Several substrate panel screening assays exist that allow the researcher to test dye-labeled peptides for kinase or phosphatase activity. Here we introduce a method that uses readily available biotinylated peptides instead of dye-labeled substrates, which are costly and limited in availability. After enzymatic phosphorylation, biotinylated peptides are coupled to streptavidin-quencher conjugates, which then associate with a fluorescent polymer via phosphate-metal ion interaction between the reacted biotinylated peptide complex and the polymer. As a result, quencher and polymer are brought into a proximity that allows electron transfer from the polymer to the dye. The Dylight(647) (Pierce, Rockford, IL) dye was identified as an efficient electron transfer molecule that allows assays to be monitored using two emission wavelengths simultaneously, 490 nm from the polymer and 685 nm from the transferred emission of the dye. Assays are homogeneous and show comparable sensitivities to assays performed with direct-labeled dyes. When applied to a limited screen using previously characterized peptides, substrates for two kinases and one phosphatase were correctly identified. Further, ratiometric analysis of polymer quenching and transferred emission accurately detected inhibitors in a compound screen against protein kinase A, protein kinase Calpha, and protein tyrosine phosphatase 1B with limited interferences from colored compounds and with Z factors of >0.7.

Biotinylation↗

PubMed2005

High-throughput kinase assays with protein substrates using fluorescent polymer superquenching.

BACKGROUND: High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. RESULTS: Using a modified QTL Lightspeed assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP), Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1). Phosphorylation of the proteins was detected by Protein Kinase Calpha (PKCalpha) and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4). Enzyme inhibition yielded IC50 values that were comparable to those obtained using peptide substrates. Statistical parameters that are used in the high-throughput community to determine assay robustness (Z'-value) demonstrate the suitability of this format for high-throughput screening applications for detection of inhibitors of enzyme activity. CONCLUSION: The QTL Lightspeed protein detection system provides a simple mix and measure "turn on" assay for the detection of kinase activity using natural protein substrates. The platform is robust and allows for identification of inhibitors of kinase activity.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Metal ion-mediated polymer superquenching for highly sensitive detection of kinase and phosphatase activities.

An assay technology for high-throughput screening of kinase and phosphatase activities is introduced. The format is based upon superquenching of fluorescent-conjugated polymers by dye-labeled kinase/phosphatase peptide substrates. The sensor platform is composed of highly fluorescent-conjugated polyelectrolytes colocated with the phosphate coordinating metal ion gallium on microspheres. Phosphorylated peptide substrates containing a quencher bind specifically to the metal ions by means of phosphate groups, resulting in quench of polymer fluorescence. The modulation of fluorescence signal is proportional to kinase or phosphatase activity and is monitored as a turn-off or turn-on signal, respectively. The assay is homogeneous and simple and can be run either as an endpoint measurement or in a kinetic mode. The assay meets the sensitivity required for high-throughput screening of kinase or phosphatase inhibitors and is a valuable tool for drug discovery. A modified version of the assay allows for the detection of protein phosphorylation.

Amino Acid Sequence↗

PubMed2004

Fluorescent-conjugated polymer superquenching facilitates highly sensitive detection of proteases.

Sensor formats have been developed for detecting the activity of proteolytic enzymes based on fluorescent conjugated polymer superquenching. These sensors employ a reactive peptide sequence within a tether linking a quencher to a biotin. The peptide binds to sensors containing colocated biotin-binding protein and fluorescent polymer by means of biotin-biotin binding protein interactions, resulting in a strong quenching of polymer fluorescence. Enzyme-mediated cleavage of the peptide results in a reversal of the fluorescence quenching. These assays for protease activity are simple, sensitive, fast, and have the specificity required for screening chemical libraries for novel protease inhibitors in a high-throughput screening assay environment. These assays have been demonstrated for enterokinase, caspase-3/7, and beta-secretase.

Aspartic Acid Endopeptidases↗

PubMed2004

Applications of fluorescent polymer superquenching to high throughput screening assays for protein kinases.

Protein kinases are involved in the regulation of cellular metabolism, growth, differentiation, and proliferation. Aberrations in their function can lead to diseases such as cancer and inflammation. Protein kinases are therefore possible targets for drug therapies. To address the need for high throughput screening of potential inhibitors, QTL has developed a homogeneous and robust kinase assay for use in multiwell plate format. The QTL Lightspeed fluorescence superquenching-based kinase assays do not require specialized equipment, nor do they involve the use of radioactive hazardous materials or antibodies. QTL Lightspeed kinase assays directly measure the enzymatic activity of the target and do not involve secondary (detector) enzyme. In this article, we compare QTL Lightspeed protein kinase assays using Protein Kinase A, Protein Kinase Balpha/Akt1, and ribosomal S6 kinase-2 as examples with other commercially available kinase kits. Our data show that QTL Lightspeed kinase assays offer significant advantages over the current commercial kits in terms of both sensitivity and performance. The QTL Lightspeed kinase assay also offers a kinetic assay mode where the substrate phosphorylation can be monitored in real-time.

Dose-Response Relationship, Drug↗

PubMed2001

Biosensors from conjugated polyelectrolyte complexes.

A charge neutral complex (CNC) was formed in aqueous solution by combining an orange light emitting anionic conjugated polyelectrolyte and a saturated cationic polyelectrolyte at a 1:1 ratio (per repeat unit). Photoluminescence (PL) from the CNC can be quenched by both the negatively charged dinitrophenol (DNP) derivative, (DNP-BS(-)), and positively charged methyl viologen (MV(2+)). Use of the CNC minimizes nonspecific interactions (which modify the PL) between conjugated polyelectrolytes and biopolymers. Quenching of the PL from the CNC by the DNP derivative and specific unquenching on addition of anti-DNP antibody (anti-DNP IgG) were observed. Thus, biosensing of the anti-DNP IgG was demonstrated.

Animals↗