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Abstract
Palmitoylation is a widespread, reversible lipid modification that has been implicated in regulating a variety of cellular processes. Approximately one thousand proteins are annotated as being palmitoylated, and for some of these, including several oncogenes of the Ras and Src families, palmitoylation is indispensable for protein function. Despite this wealth of disease-relevant targets, there are currently few effective pharmacological tools to interfere with protein palmitoylation. One reason for this lack of development is the dearth of assays to efficiently screen for small molecular inhibitors of palmitoylation. To address this shortcoming, we have developed a robust, high-throughput compatible, click chemistry-based approach to identify small molecules that interfere with the palmitoylation of Ras, a high value therapeutic target that is mutated in up to a third of human cancers. This assay design shows excellent performance in 384-well format and is sensitive to known, non-specific palmitoylation inhibitors. Further, we demonstrate an ideal counter-screening strategy, which relies on a target peptide from an unrelated protein, the Src-family kinase Fyn. The screening approach described here provides an integrated platform to identify specific modulators of palmitoylated proteins, demonstrated here for Ras and Fyn, but potentially applicable to pharmaceutical targets involved in a variety of human diseases.
Highlights
Despite its ubiquity and biomedical relevance, there are few chemical tools available for the perturbation of S-palmitoylation, and none have been pursued for clinical translation
As a therapeutically-relevant target, we focused on the oncogene Ras, a small GTPase that acts as a key switch in a number of cell signaling pathways that regulate cell growth, survival, proliferation, and differentiation[17,18]
Only one of the 23 known palmitoyl transferase (PAT) has been associated with Ras palmitoylation - the zDHHC9-GCP16 complex[3,26,27,28]
Summary
Despite its ubiquity and biomedical relevance, there are few chemical tools available for the perturbation of S-palmitoylation, and none have been pursued for clinical translation. Despite decades of research, Ras has proven intransigent to pharmacological intervention, temporarily earning the unfortunate moniker of ‘undruggable target’ due to its high affinity for GTP and the lack of clear allosteric binding pockets[21] Ras interacts both with upstream regulators and downstream effectors at the plasma membrane, making membrane anchoring indispensable for Ras-mediated signaling[20,22], and suggesting that inhibition of this anchoring could be a viable therapeutic strategy[23]. The peptides retain the covalently linked primary lipid modifications (C-terminal farnesyl and N-terminal myristoyl groups, of fully processed N-Ras and Fyn, respectively) to ensure specificity We demonstrate that these peptides are enzymatically palmitoylated in vitro and that known palmitoylation inhibitors can be effectively detected in 384-well format. We demonstrate a robust counter-screening strategy that constitutes a comprehensive platform for discovery and development of palmitoylation-targeted pharmaceuticals
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