January 1, 2006 (Vol. 26, No. 1)

Harnessing the Power of Superquenching with TruLight Assays

Protein kinases are enzymes that transfer a phosphoryl group to serine, threonine, or tyrosine side chains of a target protein, thereby changing its activity. Dysregulation of protein kinases has been implicated in many types of cancer and also inflammation.

Protein kinases are a major focus of drug discovery research due to the successful development of kinase inhibitor drugs, such as Gleevec and Iressa, for different types of cancer. Of the 518 protein kinases in the human genome, 37 are targeted by small molecule inhibitors in clinical trials1, and an increasingly larger number are being studied by many pharmaceutical and biotechnology companies.

High-throughput screening (HTS) of kinases with large compound libraries to find inhibitors is an important part of the drug discovery process.

HTS kinase assays must meet several requirements. They must be homogeneous, in other words they must not require washing steps. Additionally, they must provide high quality data, since HTS conditions are relatively unforgiving, yielding only one data point for each compound that is screened.

To ensure data quality, the Z value was introduced as a universal measure of assay quality and robustness2. The Z value is based on the difference between the signal of the positive control and that of the negative control and also factors in the standard deviations of each signal. An assay with a Z value of 1.0 is deemed to be perfect, while assays with Z values above 0.5 are needed for HTS.

HTS kinase assays must also be designed to maximally detect inhibition. To achieve maximal inhibition, enzymologists suggest screening at enzyme velocities that are as close to initial velocities as possible, and this can be achieved at 10% substrate conversion3.

At higher substrate conversion, apparent IC50 values can be much higher than expected, meaning that weak inhibitors may be missed. Therefore, an ideal assay would generate Z values greater than 0.5 at 10% substrate conversion.

In addition, the assays should be able to tolerate high ATP concentrations to ensure maximal velocity for a wide range of protein kinases. Assays meeting these criteria would also allow detection of kinases that have a low turnover. In other words, those that phosphorylate only ~10% of substrate under optimal conditions.

Fig 1: TruLight Kinase Assays use a proprietary technology utilizing microspheres that are coated with fluorescent polymers and a metal ion coordination complex.

Standard Methods

A variety of kinase assay methods is currently available4. HTS kinase assays vary greatly in the methodologies and detection methods used. While assays utilizing radioactive detection have long been the standard, the disposal costs and safety concerns make newer fluorescence-based assays more attractive.

Fluorescence-based kinase assays utilize different detection methods for protein or peptide phosphorylation. Assays based on Fluorescence Polarization (FP) take advantage of the fact that smaller particles rotate faster in solution than larger particles. Binding of an antibody or particle to a fluorescently labeled phosphorylated peptide can be measured utilizing FP measurements.

Assays utilizing fluorescence resonance energy transfer (FRET) and fluorescence quenching rely on the fact that fluorescent energy is transferred from a fluorophore when it is brought in close proximity with a molecule that can receive the energy.

The development of phospho-specific antibodies, which recognize phosphorylated peptides or proteins, has been important in the advancement of many fluorescence-based technologies. Fluorescent assays based on phospho-specific antibodies can be sensitive but often rely on the generation of a new phospho-specific antibody for each kinase target to be studied.

In addition, these techniques are often limited to detecting tyrosine phosphorylation, because antibodies detecting serine or threonine phosphorylation do not exhibit the needed specificity. Fluorescent kinase assays obviating the use of antibodies are more versatile but often lack the sensitivity needed to identify weak inhibitors under HTS conditions.

Fig. 2: Superquenching is used to detect kinase activity as a decrease in fluorescence. Key to chart: x=Trulight

Superquenching Technology

EMD Biosciences (www.emdbiosciences. com) TruLight Kinase Assays, which do not require antibodies or radioactivity, achieve high-sensitivity by utilizing fluorescence superquenching, a proprietary technology related to fluorescence quenching5.

Fluorescence superquenching occurs for a variety of light-emitting polyelectrolytes6. Small-molecule quenchers can quench the photoluminescence of up to several hundred polymer repeat units. This results in an “amplification” of the quenching potential and the ability to detect fewer quenchers.

TruLight Kinase Assays utilize quenchers that are conjugated to kinase substrate peptides. Fluorescent polymers capable of being superquenched are co-localized with metal ions on microsphere sensors, providing a means for phosphorylated peptides to bind (Figure 1)7. Kinase activity is detected as a decrease in fluorescence when the quencher-labeled phosphorylated peptides bind (Figure 2). All of the assay components have been optimized to meet HTS data requirements.

As a result of superquenching, phosphorylated peptides are detected at lower levels, making TruLight Kinase Assays more sensitive than conventional FRET-based assays (Figure 3). The assays are optimized to exhibit maximum signal change at less than 25% enzyme conversion and exhibit Z values of 0.7 at 10% conversion (Table). This increased sensitivity leads to better detection of weak inhibitors, since IC50 values are lower than those observed at higher enzyme conversion. Additional benefits include cost savings due to lower enzyme requirements and the ability to screen kinases with low turnover.

The assays exhibit ATP tolerances of 100 mM or greater and are compatible with most solvents. Also, they have signal stabilities compatible with HTS. A further advantage for HTS is that ratiometric monitoring at higher wavelengths (~600 nm) can be used with TruLight Kinase Assays to reduce interference from colored or fluorescent compounds.

TruLight Kinase Assays have been developed for Akt1, Aurora A, ERK1/2, p38a, p70S6K, PKA, PKCa, PKCbI/II, PKCe, RSK-2, and Src. The lower limit of detection for these assays varies from 1.2800 picomolar.

Additionally, a Universal Kinase/ Phosphatase Assay is available, whereby the user supplies the quencher-labeled peptide substrate to study any kinase for which a substrate is known. The user determines the substrate, quencher, and reaction conditions needed for the target enzyme.

One adaptation of the TruLight Kinase Assay platform uses protein rather than peptide substrates8. In this mode, the phosphorylated protein substrate binds to the microsphere, and unoccupied sites are “sensed” by subsequent addition of quencher-labeled phospho-peptide. An increase in the fluorescent signal is observed as the kinase reaction proceeds, because the phosphorylated protein substrate blocks the tracer peptide quencher from binding the sensor.

In addition to increasing the versatility of the assays, the ability to utilize protein substrates allows the user to identify novel inhibitors that act via a mechanism that cannot be reproduced using peptide substrates.

Fig. 3: Superquenching leads to sensitive detection of kinase activity.

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