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

Abstract

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 dyelabeled kinase͞phosphatase 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. P hosphorylation and dephosphorylation of proteins by kinase and phosphatase enzymes mediate the regulation of cellular metabolism, growth, differentiation, and proliferation (1-3). Aberrations in kinase and phosphatase activities can lead to inflammation and diseases such as cancer (4, 5). More than 500 kinases and phosphatases are thought to be involved in the regulation of cellular activity and are possible targets for drug therapy (6). Of the kinases, Ϸ90% phosphorylate serine residues, 10% threonine, and 0.1% tyrosine residues (7). Although it has become possible to develop anti-phospho-tyrosine antibodies (8), those against phospho-serine and threonine residues are of low affinity and are often specific to only one kinase (9). Currently, non-antibody-based high-throughput screening (HTS) assays are based on methods such as time-resolved fluorescence (TRF) (10), fluorescence polarization (FP) (11-13), or fluorescence resonance energy transfer (FRET) (14). These assays require specialized equipment and͞or suffer from low fluorescence intensity change as a function of enzyme activity and generally cannot be used to detect phosphorylation of natural, chemically unmodified protein substrates. The use of native substrates is attractive because inhibitor screens may yield novel inhibitors that affect the enzyme docking site, which can be at a site distant from the active site.We sought to enhance sensitivity in the measurement of enzymatic activity by amplifying the fluorescence signal using superquenching (15)(16)(17)(18)(19)(20)(21)(22)(23)(24). This phenomenon has been described in several reports and is based on the finding that photoluminescence of conjugated polymers and related polymeric ensembles can be quenched by means of energy and͞or electron transfer to small molecule quenchers (15)(16)(17)22). In previous studies, it was found that one quencher molecule can quench the photoluminescence of up to several hundred polymer repeat units (25)(26)(27).Our sensor platform compri...

Identification of a Small Molecule Inhibitor of the Human DNA Repair Enzyme Polynucleotide Kinase/Phosphatase

“…The fluorescence-based phosphatase assay that we adapted was originally developed to monitor protein phosphatase activity (35). The assay involves a fluorescent sensor molecule coupled with trivalent metal ions that bind to phosphate groups.…”

“…Most fluorescence-based high throughput screening assays for phosphatase activity have been directed toward protein phosphatases and rely on immunodetection using antibodies to the phosphorylated peptide substrate. The superquenching assay, originally devised by Rininsland and colleagues (35), presented an alternative approach that depended on the presence of a phosphate group for chemical recognition. It required some optimization involving protonation of the substrate to enhance the influence of the terminal phosphomonoester group over the internucleotide phosphodiester groups of the DNA substrate.…”

Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrate

“…We subsequently sought to produce pores bearing a sensor peptide attached by a single terminus, as these would better resemble the synthetic, linear peptides often used in other methods for measuring kinase substrate interactions (38,47,48). This had previously been achieved by forming an αHL pore with one subunit chemically coupled through a cysteine residue at the trans mouth to a peptide sensor element by using an S-pyridyl-functionalized tetra (ethylene glycol) linker (14).…”

“…† Interaction of full-length heat-stable protein kinase inhibitor (PKI) with PKA catalytic subunit (48). ‡ Interaction of GST-PKI fusion with PKA catalytic subunit in the presence of 1 mM ATP and 10 mM MgCl 2 (47).…”

Recent Advances in Technologies for Analyzing Protein Kinases

“…The IQ kinase assay (Pierce, Rockford, IL, USA) (16) and QTL Lightspeed TM assay (QTL Biosystems, Santa Fe, NM, USA) (17) were designed on the basis of such methodology. Recently, the TruLight TM kinase assay (Merck Biosciences, San Diego, CA, USA) was developed.…”