The identification and characterization of changes in the level of farnesylated proteins in response to the treatment of farnesyltransferase inhibitors (FTIs), a newly introduced family of antitumor agents currently undergoing clinical evaluation, represents a major scientific challenge. Extant proteomics methods are limited to quantifying a few thousands of the most abundant proteins and, therefore, are unsuitable for the targeted profiling of less abundant farnesylated expression. The major goal of this application is to develop and validate a powerful technology, Tagging via Azido Substrate (TAS), for the efficient isolation of farnesylated proteins. This technology will then be applied to the proteomics analysis of farnesylated proteins in physiologically relevant models. The TAS technology involves the introduction of a synthetic azide-modified farnesyl substrate, either farnesyl azide diphosphate (FPP-azide) or farnesyl azide alcohol (F-azide-OH), which replaces the natural substrate during cellular protein farnesylation. The resulting farnesyl-azide (F-azide)-modified proteins will be affinity-purified through an azide-specific conjugation reaction (Staudinger reaction) using a phosphine capture reagent linked to photo-cleavable beads, which can then be released by UV light-induced photo-cleavage. Since affinity purification relies on covalent bonding resulting from a specific conjugation reaction between an azide and phosphine capture reagent, other proteins without the F-azide modification can be effectively removed by thorough washing. Thus, the TAS technology will allow farnesylated proteins to be isolated with high yield, high specificity, and low contamination. The initial focus of the R21 portion of this proposal is on the development of TAS technology and its application to the isolation of farnesylated proteins. These studies will be extended subsequently to geranylgeranylated proteins. The R33 proposal will aim at applications of the TAS technology to the identification of novel FTI targets. These studies will provide fundamental information for the understanding of molecular mechanisms of FTI functions and are likely to identify novel targets for antitumor drug design.
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