A Continuous Kinetic Assay to Quantitate Specific Protein Kinase Activity in Unfractionated Cell Lysates

Abstract

The human kinome contains 538 protein kinases implicated in > 400 diseases. Protein kinases form a network of signaling pathways and are modified by multiple post-translational modifications, including phosphorylation, which can positively or negatively regulate the kinase enzymatic activity, the protein complexes that form, and the location in the cell. Given this rich target complexity, >30% of all drug development is focused on kinases. Of the 103 approved small-molecule drugs, only 15% target the human kinome and are primarily just one type (ATP-competitive) that can readily result in off-target inhibition and resistance during cancer treatment. Most kinase inhibitor development uses in vitro assays with low ATP concentrations and a truncated and epitope-tagged recombinant kinase.

As a result, drug efficacy in patients can be limited due to poor inhibitor performance under physiological conditions (e.g., mM levels of ATP and full-length kinases being complexed with other proteins). Contrarily, in vitro assays using unfractionated cell lysates provide a closer approximation of the native kinase environment, including the complexity of interactions with other cellular components. Selectively quantifying the enzymatic activity of all protein kinases in crude cell or tissue homogenates would enable an improved understanding of kinase signaling biology, drug development, and personalized medicine. However, despite the extensive literature on kinase activity assays, there are no direct and continuous activity assays compatible with robust drug discovery with unfractionated lysates due to insufficient selectivity of peptide substrates for a given kinase. To that end, we utilized a continuous assay format coupled with high-throughput Sox-sensor-peptide library synthesis using physiological sequences to identify selective sensor peptide substrates for multiple high-profile kinases implicated in cancer.

Here, we describe a highly selective sensor peptide for ERK1/2 from the mitogen-activated protein kinases (MAPKs) family, the enzymes that are critical to progrowth/oncogenic signaling. We demonstrated selectivity initially with the recombinant enzymes for ERK1/2 and a panel of other related MAPKs or CMGC kinases. Next, we extended the selectivity screen to 392 kinases using a large panel kinome profiling service. These screens demonstrated high selectivity for ERK1/2 isoforms. We then tested our best sensor peptide substrate for ERK1/2 using unfractionated lysates from the HEK293T cell line with ERK2 overexpression. We demonstrated high activity that could be increased 50-fold by adding recombinant MEK1 or 2, the upstream kinase that activates ERK1 and 2, and blocked by adding an ERK2-selective inhibitor. These results demonstrate a method to systematically generate a selective sensor peptide for ERK1/2 to continuously monitor this kinase activity in crude cells or tissue lysates. This poster will fully describe the methods and results pertaining to ERK-selective sensor peptide development and validation and the ability to extend to other kinases of interest.

Key Takeaways

• AQT0491 sensor peptide is a simple linear sequence generated using standard solid phase peptide synthesis, rather than complex chimeric peptide structure.
• AQT0491 is commercially scalable and is selective towards ERK1/2.
• Experiments using ERK2 overexpression lysates show that ERK2 selectively phosphorylates AQT0491 and the enzyme activity can be inhibited using an ERK2-selective inhibitor.
• Together, these studies show that the ERK2 sensor peptide AQT0491, which also detects ERK1 activity, can be advanced to further optimization efforts using endogenous cell lysates and tissue homogenates to provide a more physiologically relevant, easy-to-use assay.
• More importantly, the continuous kinetic readout eliminates random noise and enables rapid generation of high-value information on kinase activity in cell lysates.