Abstract 1676: A continuous kinetic assay to quantitate specific protein kinase activity in unfractionated cell lysates

Abstract

Abstract Protein kinases form a network of signaling pathways and are modified by multiple post-translational modifications, including phosphorylation, which regulate the kinase enzymatic activity, protein complexes formation, and its cellular location. Given this rich target complexity, >30% of all drug development is focused on kinases. However, these target only 15% of the human kinome and are primarily just one type (ATP-competitive) that can readily result in off-target inhibition and the development of resistance during cancer treatment. Most kinase inhibitor development is carried out in vitro assays with an epitope-tagged recombinant kinase. As a result, efficacy in patients can be limited due to poor inhibitor performance under physiological conditions (e.g., mM ATP and kinases being complexed with other proteins). In contrast, in vitro assays using unfractionated cell lysates provide a close approximation of the native kinase environment, including the complexity of interactions with other cellular components. Being able to selectively quantify 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 large amount of literature on kinase assays, only a fraction is compatible with unfractionated lysates since most peptide substrates are not selective 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. As an example, we identified a highly selective sensor peptide for ERK1 and 2 mitogen-activated protein kinases (MAPKs) involved in pro-growth/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. The screen also 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 and demonstrated high activity that could be increased 50-fold by the addition of recombinant MEK1 or 2, the upstream kinase that activates ERK1 and 2, and completely blocked with the addition of 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 cell 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. Citation Format: Venkatesh Nemmara, Susan Cornell-Kennon, Zhibing Lu, Gabriela Pikul, Zacchary Belisle, Matthew Hakar, Jefferson Chin, Erik Schaefer, Earl May. A continuous kinetic assay to quantitate specific protein kinase activity in unfractionated cell lysates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1676.