title: Click-Chemistry Based High Throughput Screening Platform for Modulators of Ras Palmitoylation.
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.
authors.affiliation: Department of Integrated Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Ganesan
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
authors.forename: Peyton
authors.initials: P
authors.lastname: Shieh
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305, USA.|Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA.
authors.forename: Carolyn R
authors.initials: CR
authors.lastname: Bertozzi
authors.affiliation: Department of Integrated Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
authors.forename: Ilya
authors.initials: I
authors.lastname: Levental
mesh_terms: D057930:Click Chemistry; D004353:Drug Evaluation, Preclinical; D057166:High-Throughput Screening Assays / Q000379:methods*; D054878:Lipoylation; D051076:Proto-Oncogene Proteins c-fyn / Q000494:pharmacology; D018631:ras Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000493:pharmacokinetics
chemical_list: D051076:Proto-Oncogene Proteins c-fyn; D018631:ras Proteins