Synthesis and applications of caged thiols for studying protein prenylation.

University of Minnesota Ph.D. dissertation. February 2012. Major: Chemistry. Advisor: Dr. Mark D. Distefano. 1 computer file (PDF); xxi, 106 pages.Ras proteins are a subfamily of small GTP-binding proteins that are involved in various critical cellular processes including cell growth, survival and nuclear transport. It has been reported that roughly 30% of human cancers are derived from mutations of Ras, and prenylation is a key step that activates their oncogenicity. Commercial inhibitors of prenylation have been successful at arresting Ras activation and can be categorized into two families: farnesyltransferase inhibitors (FTIs) and geranylgeranyltransferase inhibitors (GGTIs). The focus of this thesis is to explore the use of photoremovable protecting groups (caging groups) to better understand the process of prenylation by caging the critical thiol residues of FTIs, GGTIs and peptides. The caging group bromohydroxy coumarin (bhc) was covalently bound to the thiol of the FTI L-744,832 in order to inactivate the inhibitor. This caged FTI was evaluated with respect to its one- and two-photon uncaging kinetics and ability to release FTI upon photolysis. Analysis shows that bhc photolysis occurs more rapidly compared to the most frequently used family of nitrobenzyl-based cages, and that FTI is produced with good yields upon one- and two-photon excitation. Bhc-FTI was then tested on different cell lines in order to show that upon irradiation FTI is released that inhibits Ras farnesylation (observed via Western blot analysis), Ras membrane localization (detected by confocal microscopy), and downstream signaling (fibroblast morphology). This same approach was utilized to cage FTI with bromohydroxy quinoline (BHQ). The covalent inactivation of FTI with BHQ was employed to cage the active site thiol (BHQ-FTI) and active site amine (BHQ-FTI urethane). Kinetic evaluation suggests that BHQ-FTI uncages faster than bhc-FTI but it produces little FTI upon photolysis due to the formation of unreactive photoproducts. Despite its poor yield, one photon cell experiments with BHQ-FTI resulted in the inhibition of Ras farnesylation, Ras membrane localization and downstream signaling. Quantitation and biological experiments with BHQ-FTI urethane are ongoing. Peptides that are substrates of protein farnesyltransferase (PFTase) were caged with bhc and BHQ at their crucial thiol that is targeted for farnesylation. Upon one-photon photolysis peptides caged with BHQ show poor yields of free peptide while bhc-caged ones result in good peptide production. One of these caged peptides was subjected to an in vitro farnesylation assay to show that no farnesylation occurs, but upon one- and two-photon irradiation farnesylated peptide can be detected. Application of this caged peptide to study the mechanism of farnesylation via X-ray crystallography is under way. Certain Ras proteins are alternatively geranylgeranylated and retain full function when farnesylation has been inhibited; as a result, GGTI-286 was caged with bhc to study this phenomenon. The synthesis of this GGTI and the inactivation of its thiol via covalent bonding with bhc is described here. The kinetic analysis of bhc-GGTI as well as its quantitation and biological testing are a work still in progress