HTS-Based Identification of Novel Rce1p Inhibitors(RMI)

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DESCRIPTION (provided by applicant): Our long-term goal is to define the enzymatic properties of the Ras Converting Enzyme (Rce1p), a relatively uncharacterized protease that is required for CaaX protein-biosythensis. CaaX proteins are lipid-modified molecules that often function as signaling molecules in important cellular pathways. Ras and RhoB are key examples. Because of the role that CaaX proteins have in cellular transformation (e. g. activated forms of Ras are associated with 30% of all cancers), strategies that regulate the biosynthesis of these proteins are being explored as novel anti-cancer therapies. Rce1P is a new target in these strategies. Rce1p is an atypical protease, having multiple membrane spans and lacking a canonical protease motif. To date, the mechanism of Rce1p remains undefined, in part due to a lack of inhibitors that can be used as probes to investigate its catalytic properties. We have developed a coherent set of biochemical and genetic, chemical, and molecular assays that we are using for defining the active site and enzymatic properties of Rce1p and for identifying and characterizing novel Rcelp inhibitors. 1 of our assays is used to monitor the Rce1p-dependent cleavage of a quenched fluorescent peptide substrate. We have used this assay to evaluate the effect of novel inhibitors and mutations on the kinetic parameters of Rcelp. This assay is readily adaptable for HTS, and 1 of the aims of this proposal is fully assess the utility of this assay for HTS by optimizing the signal-to-noise ratio, determining reproducibility, and conducting a pilot screen for Rce1p inhibitors. As a second aim, we are proposing to integrate our other Rce1p assays into secondary screens that can be used to confirm HTS hits and to further assess the target specificity of candidate inhibitors. The ultimate goal of this project is the identification of novel Rce1p inhibitors that can serve as tools for providing insight into the mechanism of Rce1p; some of these compounds may also be of potential therapeutic value.