Biocidal activity of a light-absorbing fluorescent conjugated polyelectrolyte.

Herein we describe studies that indicate a cationic conjugated polyelectrolyte shows biocidal activity against gram-negative bacteria (Escherichia coli, E. coli, BL21, with plasmids for Azurin and ampicillin resistance) and gram-positive bacterial spores (Bacillus anthracis, Sterne, B. anthracis, Sterne). These studies were carried out with aqueous suspensions of the conjugated polyelectrolyte, with the polyelectrolyte in supported formats and with samples in which the conjugated polyelectrolyte was coated on the bacteria. The results are interesting in that the biocidal activity is light-induced and appears effective due to the ability of the conjugated polyelectrolyte to form a surface coating on both types of bacteria. The effects observed here should be general and suggest that a range of conjugated polyelectrolytes in different formulations may provide a useful new class of biocides for both dark and light-activated applications.

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.

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.

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.

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.

Superquenching in cyanine pendant poly(L-lysine) dyes: dependence on molecular weight, solvent, and aggregation.

A series of poly(L-lysines) ranging in number of repeat units (N(PRU)) from 6 to 900 has been synthesized and the photophysics of the series and monomer cyanine dye have been studied in solution. In water or aqueous dimethyl sulfoxide, the oligomers and polymers exhibit high sensitivity to fluorescence quenching by oppositely charged electron acceptors; in this study, 9,10-anthraquinone-2,6-disulfonate was used as a quencher for the cationic fluorescent polyelectrolytes. Quenching constants (K(SV)) measured in 50:50 (v/v) dimethyl sulfoxide-water increase monotonically with increase in N(PRU) ranging from 630 M(-1) for monomer to 1.2 x 10(9) M(-1) for dilute solutions of the polymer having N(PRU) approximately 900. The polymers having N(PRU) > 100 exhibit predominantly J-aggregate absorption and fluorescence and enhanced susceptibility to quenching. For the polymers exhibiting strong J-aggregation, the effective exciton domain quenched by a single quencher reaches approximately 100 PRU. The results of this study permit a semiquantitative analysis of superquenching of fluorescent polyelectrolytes in solution and the factors that control it.