November 1, 2009 (Vol. 29, No. 19)

Measuring Various Post-Translational Modifications with One Detection Reagent

There is growing enthusiasm in the pharmaceutical industry for whole-system functional assays that reveal the action of potential drugs on integrated systems such as intact cells in order to find allosteric effects and off-target effects.

In target-based drug discovery, high-throughput screening (HTS) of compounds against a single defined molecular target is performed in order to identify drug candidates that can perturb the action of a cellular component that is presumed to mediate a specific phenotype. Common targets include catalytically active proteins such as kinases, phosphatases, phosphodiesterases, and proteases.

Despite the huge amount of resources put into target-based drug discovery, a disappointingly small number of drugs have been developed using this approach. This is due in part to the fact that many diseases are not caused by aberrant activities of only one target cellular component but are multifactorial. They typically involve various complex pathway interactions implicated in signal transduction and are dynamically regulated in living cells by effects such as feedback inhibition.

To complicate matters, many inducer molecules that create various phenotypes share cellular-signaling pathways with a number of endogenous regulatory molecules. As a consequence, target-based drug discovery can result in a drug with limited impact or in a drug that creates harmful side effects that are revealed only at later stages of clinical or preclinical testing.

To increase the efficiency of drug development, it is therefore advantageous to concurrently measure a network of signaling events that connect a target with a phenotype within the context of the cell. Gyrasol Technologies has addressed this challenge by developing a detection platform that measures various post-translational modifications implicated in signaling pathways simultaneously, in a single or multiplexed fashion using only one detection reagent.

These assays are homogeneous, sensitive, and adaptable to HTS and for measuring activities of endogenous enzymes in cellular lysates. In addition, since the sensor can be chemically synthesized in bulk quantities, this platform is more cost-effective than other available screening platforms.

Assay Platform

The Gyrasol platform is based on metal ion-mediated association of a sensor to a fluorophore-labeled substrate (Figure 1). The sensor consists of a metal ion coordinated to a phosphonate-based chelator, which retains its ability to bind to phosphates present on substrates.

Upon association of the sensor to the substrate the fluorescence of the fluorophore is quenched. Because the metal-ion mediated association to phosphoryl groups is generic, the sensor can detect activities of various enzymes involved in signaling (kinases, phosphatases, phosphodiesterases, and proteases) by using various substrates such as peptides, cyclic nucleotides, DNA, or lipids within defined assay components or in lysates of cells (Figure 2). Dose responses are linear, allowing for semiquantitative data analysis using maximum or minimum controls for conversion. Alternatively, the extent of substrate conversion can be quantified using synthetic calibrator molecules.

In contrast to other commonly used metal ions that are components of enzyme activity detection platforms, the particular metal ion used in the Gyrasol Sensor can coordinate to phosphates at physiological pH. As a result, kinetic monitoring can be performed for some enzymes, simplifying mode-of-action inhibitor analysis.

Activity assays have an advantage over antibody-based phosphoprotein detection platforms since measurement of a protein modification does not necessarily correlate with modulation of catalytic activity. Additionally, phosphoprotein detection assays may miss inhibitors that modulate the activity of a protein by altering its conformation and/or intramolecular associations (allosteric effects).

Figure 1. Enzyme dose-response curves obtained with intramolecular and intermolecular quench assays

Intramolecular Quenching

The mechanism of fluorescence quench by the sensor is electron transfer, which involves the physical exchange of an electron between the donor and acceptor molecule. The efficiency of photo-induced electron transfer is highest with ~20 Å distance between donor and acceptor fluorophores, which corresponds to approximately 13 amino acids.

In order to overcome this limitation, a strategy was used to form a bridge between the site of a posttranslational modification and the fluorophore (Figure 1, left). This was accomplished by employing a Chromeo 642 fluorophore (Active Motif) containing a phosphonate group that acts as a chelator for the metal ion. Upon chelation, the metal ion forms a ternary complex consisting of [fluor phosphonate (P1)]-[metal ion (M)]-[substrate phosphate (P2)]. Formation of this intramolecular ternary complex is thought to reduce the distance between donor and acceptor molecules to an extent that results in marked increase in sensitivity.

Assays using Chromeo 642 phosphonated fluorophores resulted in 35-fold higher sensitivity of detection of protein kinase A (PKA) enzyme activity than could be achieved using a nonphosphonated TAMRA peptide of the same sequence (LRRASLG) (Figure 1). In cellular lysates, endogenous PKA activity was detectable in as little as 260 ng total rat brain lysate using Chromeo 642-labeled substrates, whereas no activity was detected using the TAMRA-labeled peptide.

Figure 2. Various types of post-translational modifications can be detected with one sensor.

Activity Assays

The electron transfer sensor allows the researcher to measure activities of kinases, phosphatases, proteases, and phosphodiesterases with one assay platform, enabling the establishment of signaling-pathway signatures within lysates of cells that have undergone perturbations.

As an example, the cyclic AMP dependant activation of PKA was measured with a phosphodiesterase assay and a kinase activity assay in one experiment and quantified with a single detection reagent (Figure 3, left). cAMP is an important cellular second messenger that is regulated by GPCRs. The levels of cAMP are balanced by phosphodiesterases, which hydrolyze the 3´ cyclic bond.

cAMP mediates various signaling events by binding to the regulatory domain of PKA, one of its downstream targets. Upon binding, the regulatory domain disassociates from the catalytic domain and the enzyme becomes active. With increasing concentrations of the generic cAMP phosphodiesterase inhibitor IBMX, endogenous phosphodiesterase mediated cleavage of a fluorescein-labeled cAMP was decreased in lysates of rat brain (Figure 3, left).

The resulting increase in cAMP levels correlated with increase in PKA activity that was monitored using a Chromeo 642-labeled substrate (LRRASLG). Connecting GPCR signaling through measurement of phosphodiesterase-mediated cAMP hydrolysis with downstream protein kinase activity using one assay system illustrates the viability of the sensor as a tool to generate drug-induced signaling signatures.

Figure 3. Sensor performance in cellular lysates


In contrast to FRET, electron transfer does not require spectral overlap between the donor and acceptor molecules. Thus, the fluorescence of fluorophores of any spectral properties can be quenched with electron transfer sensors, providing a multiplexed readout of several substrates labeled with various fluorophores in a reaction mixture.

As an example, phosphodiesterase-mediated cleavage of fluorescein-labeled cAMP was simultaneously monitored with hydrolysis of a TAMRA-labeled cGMP in one sample of 2 µg whole brain lysate (Figure 3).

The sensor promises to be a cost-effective new tool for studying chemical compounds that offer potential disease intervention strategies, bringing drug discovery closer to a more relevant systems biology approach and a better understanding of a drug’s action on catalytic events within interconnected cellular pathways. 

Wendy Weatherford is the director of R&D at Gyrasol Technologies, Alexander Karasyov, Ph.D., is senior scientist at Active Motif Chromeon, James Schwaber, Ph.D., is director and professor at Thomas Jefferson University, and Frauke Rininsland, Ph.D. ([email protected]), is CSO at Gyrasol Technologies. Web:

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