BISUBSTRATE INHIBITORS OF FARNESYLTRANSFERASE - A NOVEL CLASS OF SPECIFIC INHIBITORS OF RAS TRANSFORMED-CELLS

We describe the biological properties of a new class of potent farnesy ltransferase (FT) inhibitors designed as bisubstrate analog inhibitors . These inhibitors incorporate the structural motifs of both farnesyl pyrophosphate and the CAAX tetrapeptide, the two substrates of the rea ction catalyzed by FT. Both the phosphinate inhibitor, EMS-185878, and the phosphonate inhibitor, BMS-184467, exhibited higher in vitro FT s electivity than some of the previously reported CVFM peptidomimentics and benzodiazepine analogs. Xenopus oocyte maturation induced by micro injected oncogenic Ras proteins was blocked by coinjected EMS-184467 a nd EMS-185878. However, both inhibitors showed poor cell activity pres umably because of the doubly charged nature of the compounds. Thus, ma sking the charge on the carboxylate ion markedly improved the cell per meability of EMS-185878, leading to EMS-186511, the methyl carboxyl es ter prodrug. EMS-186511 inhibited FT activity in whole cells as determ ined by inhibition of p21 Ras protein processing, inhibition of farnes ylation of proteins including Ras and the accumulation of unfarnesylat ed Ras proteins in the cytosolic fraction. While the cellular effects of these bisubstrate analog inhibitors had no significant effect on gr owth of untransformed NIH3T3 cells, they produced pronounced inhibitio n of Ras transformed cell growth. Both the anchorage dependent and ind ependent growth of ras transformed cells were severely curtailed by mi cromolar concentrations of EMS-186511, We also found that both H-ras a nd K-pas transformed cells are affected by this bisubstrate inhibitor. However, K-ras transformed cells appear to be less sensitive. The inh ibition of FT activity in cells and the ensuing inhibition of ras tran sformed cell growth is further manifested in distinct morphological ch anges in cells. Cells flattened, became less refractile and grew in co ntact inhibited monolayer. Moreover, the highly diffused character of the actin cytoskeleton in the ras transformed cells was dramatically r everted to an organized network of stress cables crisscrossing the ent ire cells upon treatment with EMS-186511. All of these effects of EMS- 186511 are limited to ras transformed cells that utilize farnesylated Ras, but are not seen in transformed cells that use geranylgeranyl Ras or myristoyl Ras. Significantly, these FT inhibitors did not produce any signs of gross cytotoxicity in untransformed, ras transformed cell s or other oncogene transformed cells.