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.
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).