Mutations in Ras isoforms such as K-Ras, N-Ras, and H-Ras contribute to roughly 
85, 15, and 1% of human cancers, respectively. Proper membrane targeting of 
these Ras isoforms, a prerequisite for Ras activity, requires farnesylation or 
geranylgeranylation at the C-terminal CAAX box. We devised an in vivo screening 
strategy based on monitoring Ras activation and phenotypic physiological outputs 
for assaying synthetic Ras function inhibitors (RFI). Ras activity was 
visualized by the translocation of RBD Raf1 -GFP to activated Ras at the plasma 
membrane. By using this strategy, we screened one synthetic farnesyl substrate 
analog (AGOH) along with nine putative inhibitors and found that only m-CN-AGOH 
inhibited Ras activation. Phenotypic analysis of starving cells could be used to 
monitor polarization, motility, and the inability of these treated cells to 
aggregate properly during fruiting body formation. Incorporation of AGOH and 
m-CN-AGOH to cellular proteins was detected by western blot. These screening 
assays can be incorporated into a high throughput screening format using 
Dictyostelium discoideum and automated microscopy to determine effective RFIs. 
These RFI candidates can then be further tested in mammalian systems.