Click-Chemistry Based High Throughput Screening Platform for Modulators of Ras Palmitoylation.

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.

Ras and its signals diffuse through the cell on randomly moving nanoparticles.

Spatiotemporal modulation of Ras signaling from different intracellular compartments requires mechanisms allowing Ras and its signals to navigate across cells. Here, we describe one mechanism by which clusters of palmitoylated H-Ras and N-Ras isoforms but not nonpalmitoylated K-Ras diffuse through the cytoplasm, independently of ATP, on fast, randomly moving, small cytosolic nanoparticles ("rasosomes"). Rasosomes forced to diffuse out of live cells and trapped by Ras antibody beads appear as round structures of 80- to 100-nm diameter. Association of H-Ras with rasosomes requires Ras palmitoylation and the hypervariable sequence (hvr) upstream of the palmitoylated cysteines. H-Ras hvr mutants that fail to interact with rasosomes are biologically inactive. Epidermal growth factor stimulation rapidly increases active H-Ras-GTP and phosphorylated extracellular signal-regulated kinase (ERK) on rasosomes. Similarly, rasosomes carrying H-Ras(G12V) but not H-Ras are loaded with active ERK. Thus, the rasosome represents a hitherto unknown particle that enables Ras signal information to spread rapidly across cells.