Ras famesyl-protein transferase

Singh SB, Ball RG, Bills GF, Cascales C, Gibbs JB, Goetz MA, Hoogsteen K, Jenkins RG, Liesch JM, Lingham RB, Silverman KC, Zink DL (1996) Chemistry and Biology of Cylindrols Novel Inhibitors of Ras Famesyl-Protein Transferase from Cylindrocarpon lucidum. J Org Chem 61 7727... 

To function, Ras must be attached to the plasma membrane. Translocation from the cytoplasm to membrane requires a series of posttranslational modifications that begin with farnesylation of the cysteine residue, the fourth amino acid residue from the C terminus of the protein, by famesyl protein transferase (FPTase) (64). Attachment of the hydrophobic 15-carbon lipid farnesyl group allows Ras molecule insertion into the plasma membrane and is crucial for Ras signaling activity and transformation properties. As farnesylation is required for oncogenic Ras function, FPTase inhibitors (FTIs) are obvious candidate antineoplastic agents. Several drugs that inhibit Ras farnesylation are at various stages of clinical development (65). 

Displacing the Essential Metal Ion in Biomolecules. It is estimated that approximately one third of all enzymes require metal as a cofactor or as a structural component. Those that involve metals as a structural component do so either for catalytic capability, for redox potential, or to confer steric arrangements necessary to protein function. Metals can cause toxicity via substitution reactions in which the native, essential metal is displaced/replaced by another metal. In some cases, the enzyme can still function after such a displacement reaction. More often, however, enzyme function is diminished or completely abolished. For example, Cd can substitute for Zn in the protein famesyl protein transferase, an important enzyme in adding famesyl groups to proteins such as Ras. In this case, Cd diminishes the activity of the protein by 50%. Pb can substitute for Zn in 8-aminolevulinic acid dehydratase (ALAD), and it causes inhibition in vivo and in vitro. ALAD contains eight subunits, each of which requires Zn. Another classic example of metal ions substituting for other metal ions is Pb substitution for Ca in bones. 

Gibbs, J.B., et al. (1993). Selective inhibition of famesyl-protein transferase blocks Ras processing in vivo. J Biol Chem 268 7617-7620. 

The chaetomellic acids A and B (43 and 44), isolated from Chaetomella acutiseta found in seeds of peas, are potent and highly specific famesyl-mimic inhibitors of ras farnesyl-protein transferase. Farnesylation by this enzyme is the first and obligatory step for the post-translational modification of ras. The modified ras protein appears to be involved in tumorigenesis (approximately 25% of tumours) and there is evidence that inhibitors of the transferase have the potential to be antitumorigenic agents. These metabolites can be considered to be assembled by condensation of a palmitoyl and oleic acid chain C2 with pyruvate or oxaloacetate (Scheme 2) [38, 39]. Two syntheses have been reported for chaetomallic acid A [39, 40], one of which is patterned on the probable biogenesis [39]. 

Scheme 1. Post-translational modification by famesylation. The cysteine as part of the consensus sequence CAAX (C, cysteine A, aliphatic amino acid residue X, serine or methionine) near the C-terminus in the Ras protein is famesylated by famesylpyrophosphate (FPP) catalyzed by famesyl protein transferase (FPTase). The famesylated protein can then attach itself to the plasma membrane. If mutated Ras proteins are famesylated and attached to the cell membrane this will lead to transformation.

Liu, M., Bryant, M.S., Chen, J., Lee, S., Yaremko, B., et al. (1998) Antitumor activity of SCH 66336, an orally bioavailable tricyclic inhibitor of famesyl protein transferase, in human tumor xenograft models and wap-ras transgenic mice. Cancer Res. 58 4947-4956. 

Both the mixed stems and stem bark, and the stems CHCls-soluble extracts of L. wallichii Kurz, were found to display significant inhibitory activity in a famesyl protein transferase (FPTase) assay system. It has been suggested that inhibitors of this enzyme may be considered as potential anticancer agents for tumors in which products of the ras oncogene contribute to transformation. The bioassay directed fractionation of the two active extracts [213] led to the isolation of the known lupane lactones, ochraceolide A (128), ochraceolide B (129), and the new compound dihydroochraceolide A (135), among other known triterpenes. The structure of 135 was confirmed by reduction of 128, Fig. (37) and the stereochemistry to the epoxide group of 129, not determined when this compound was first isolated from K. ochracea [211], was established by preparation of both epoxide isomers, 129 and the new semisynthetic derivative, 20-epi-ochraceolide B (136) from 128. 

Famesylation of the Ras protein occurs at the C-terminal CAAX sequence (A aliphatic amino acid, X Ser or Thr). The famesyl residue is attached, with the help of a farnesyl protein transferase, via a thioether bond to the Cys residue of the CAAX sequence. Next, the last three amino acids are cleaved off by proteases and the carboxyl group of the C-terminal cysteine residue undergoes a methylesterification (Fig. 9.6). In addition, the Ras proteins have a palmitinic acid anchor at different Cys residues in the vicinity of the C terminus. The membrane localization of the Ki-Ras protein is also supported by a polybasic sequence close to the C terminus (see 3.7 and Fig. 3.12). 

An example of the signal-transduction protein-targeted inhibitor design which illustrates both peptide scaffold- and nonpeptide template-based approaches is that for the Ras famesyl transferase inhibitor discovery. Such compounds show potential as new therapeutic agents for Ras-related carcinogenesis [81]. Substrate sequences for famesyl transferase have the consensus Cy s-A A, - A A2-Met motif (AA refers to Val or lie). Both substrate-based... 

NO (nM concentxation) has been reported to activate Ras post-translational modification via S-nitrosylation of critical cysllS residue which stimulates guanine nucleotide exchange (Gallo et al. 1998). We have reported that NO (60 nM) treatment of MDA-MB-231 cells led to Ras-mediated increase in PI-3 kinase/Akt and Raf/MEK/ERK signal transduction pathways which was independent of cGMP. It was shown that pre-treatment of the cells by famesyl transferase inhibitors (FTIs), which block the post-translational modification of Ras protein, decreased the basal levels of pERKl/2, pAKT, and cyclin D1 to induce cytostasis (Pervin et al. 2007). 

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