Said M. Sebti

Ras CAAX peptidomimetic FTI-277 selectively blocks oncogenic Ras signaling by inducing cytoplasmic accumulation of inactive Ras-Raf complexes

Ras-induced malignant transformation requires Ras farnesylation, a lipid posttranslational modification catalyzed by farnesyltransferase (FTase). Inhibitors of this enzyme have been shown to block Ras-dependent transformation, but the mechanism by which this occurs remains largely unknown. We have designed FTI-276, a peptide mimetic of the COOH-terminal Cys-Val-Ile-Met of K-Ras4B that inhibited potently FTase in vitro (IC = 500 pM) and was highly selective for FTase over geranylgeranyltransferase I (GGTase I) (IC = 50 nM). FTI-277, the methyl ester derivative of FTI-276, was extremely potent (IC = 100 nM) at inhibiting H-Ras, but not the geranylgeranylated Rap1A processing in whole cells. Treatment of H-Ras oncogene-transformed NIH 3T3 cells with FTI-277 blocked recruitment to the plasma membrane and subsequent activation of the serine/threonine kinase c-Raf-1 in cells transformed by farnesylated Ras (H-RasF), but not geranylgeranylated, Ras (H-RasGG). FTI-277 induced accumulation of cytoplasmic non-farnesylated H-Ras that was able to bind Raf and form cytoplasmic Ras/Raf complexes in which Raf kinase was not activated. Furthermore, FTI-277 blocked constitutive activation of mitogen-activated protein kinase (MAPK) in H-RasF, but not H-RasGG, or Raf-transformed cells. FTI-277 also inhibited oncogenic K-Ras4B processing and constitutive activation of MAPK, but the concentrations required were 100-fold higher than those needed for H-Ras inhibition. The results demonstrate that FTI-277 blocks Ras oncogenic signaling by accumulating inactive Ras/Raf complexes in the cytoplasm, hence preventing constitutive activation of the MAPK cascade.

Inhibitors of prenyl transferases

Because farnesylation of Ras is required for its cancer-causing activity, several classes of farnesyl transferase inhibitors have recently been developed as potential anticancer drugs. During the last 12 months, important advances have been made in this field. In this review, we focus on three topics: targets of farnesyl transferase inhibitors other than Ras, alternative preny-lation of K-Ras by the closely related prenyl transferase, geranyl geranyl transferase I, and the effects of geranyl geranyl transferase I inhibitors on cell cycle, apoptosis, and human tumor growth.

Inhibition of Ras prenylation: A novel approach to cancer chemotherapy

The demonstration that Ras requires prenylation for its cancer-causing activity led several groups of investigators to an intense search for farnesyltransferase and geranylgeranyltransferase inhibitors as potential anticancer drugs. Rational design of small organic molecules that mimic the carboxyl terminal tetrapeptide prenylation site on Ras resulted in pharmacological agents capable of inhibiting Ras processing and selectively antagonizing oncogenic signaling, and suppressing human tumor growth in mouse models without side effects. These agents presently are undergoing advanced preclinical studies. This review describes the efforts of several groups to design, synthesize and evaluate the biological activities of several classes of prenyltransferase inhibitors. Several important issues, such as mechanism of action of prenyltransferase inhibitors and potential mechanisms of resistance to inhibition of K-Ras farnesylation, are also discussed.

Inhibiting geranylgeranylation blocks growth and promotes apoptosis in pulmonary vascular smooth muscle cells

The activity of small GTP-binding proteins is regulated by a critical step in posttranslational processing, namely, the addition of isoprenoid lipids farnesyl and geranylgeranyl, mediated by the enzymes farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I), respectively. We have developed compounds that inhibit these enzymes specifically and in this study sought to determine their effects on smooth muscle cells (SMC) from the pulmonary microvasculature. We found that the GGTase I inhibitor GGTI-298 suppressed protein geranylgeranylation and blocked serum-dependent growth as measured by thymidine uptake and cell counts. In the absence of serum, however, GGTI-298 induced apoptosis in these cells as measured by both DNA staining and flow cytometry. The FTase inhibitor FTI-277 selectively inhibited protein farnesylation but had a minor effect on growth and no effect on apoptosis. To further investigate the role of geranylgeranylated proteins in apoptosis, we added the cholesterol synthesis inhibitor lovastatin, which inhibits the biosynthesis of farnesyl and geranylgeranyl pyrophosphates. This also induced apoptosis, but when geranylgeraniol was added to replenish cellular pools of geranylgeranyl pyrophosphate, apoptosis was reduced to baseline. In contrast, farnesol achieved only partial rescue of the cells. These results imply that geranylgeranylated proteins are required for growth and protect SMC against apoptosis. GGTase I inhibitors may be useful in preventing hyperplastic remodeling and may have the potential to induce the apoptotic regression of established vascular lesions.

Bleomycin: A pharmacologic tool in the study of the pathogenesis of interstitial pulmonary fibrosis

Bleomycin is a unique DNA-interactive antitumor agent that has become a popular tool in studies of the pathogenesis of interstitial pulmonary fibrosis. The biochemical and morphological changes seen in the lungs of many species after bleomycin simulate those seen in humans. The availability of these animal models of interstitial pulmonary fibrosis also provides the opportunity to investigate novel pharmacological approaches to preventing this disease.

Farnesyltransferase as a target for anticancer drug design

The currently understood function for Ras in signal transduction is in mediating the transmission of signals from external growth factors to the cell nucleus. Mutated forms of this GTP-binding protein are found in 30% of human cancers with particularly high prevalence in colon and pancreatic carcinomas. These mutations destroy the GTPase activity of Ras and cause the protein to be locked in its active, GTP bound form. As a result, the signaling pathways are activated, leading to uncontrolled tumor growth. Ras function in signaling requires its association with the plasma membrane. This is achieved by posttranslational farnesylation of a cysteine residue present as part of the CA1A2X carboxyl terminal tetrapeptide of all Ras proteins. The enzyme that recognizes and farnesylates the CA1A2X sequence, Ras farnesyltransferase (FTase), has become an important target for the design of inhibitors that might be interesting as antitumor agents. Several approaches have been taken in the search for in vivo active inhibitors of farnesyltransferase. These include the identification of natural products such as the chaetomellic and zaragozic acids that mimic farnesylpyrophosphate, bisubstrate transition state analogs combining elements of the farnesyl and tetrapeptide substrates and peptidomimetics that reproduce features of the carboxyl terminal tetrapeptide CA1A2X sequence. This last group of compounds has been most successful in showing highly potent inhibition of FTase and selective blocking of Ras processing in a range of Ras transformed tumor cell lines at concentrations as low as 10 nM. Certain peptidomimetics will also block tumor growth in various mouse models, with apparently few toxic side effects. These results suggest that farnesyltransferase inhibitors hold considerable promise as anticancer drugs in the clinic.

Peptidomimetic inhibitors of p21ras farnesyltransferase: Hydrophobic functionalization leads to disruption of p21ras membrane association in whole cells

Abstract In this paper we describe the synthesis of several hydrophobic inhibitors of p21ras farnesyltransferase. These peptidomimetic structures, containing cysteine and methionine residues separated by an aromatic spacer, are functionalized as their methyl esters and N,S-bis-benzyloxycarbonyl (Cbz) derivatives and are shown to penetrate NIH 3T3 cells to disrupt p21 ras plasma membrane association.

Metabolic inactivation of bleomycin analogs by bleomycin hydrolase

La bleomycine est inactivee in vivo et in vitro par une enzyme appelee bleomycine hydrolase, et transformee en deaminobleomycine a activite biologique reduite. Les caracteristiques de cette enzyme (localisation tissulaire, poids moleculaire…) et ses parametres cinetiques, sont presentes

CYSTEINE PROTEINASE INHIBITORS AND BLEOMYCIN-SENSITIVE AND -RESISTANT CELLS

We have isolated a new human head and neck carcinoma cell line (C-10E) that is highly resistant to BLM (40-fold) when compared to the parental (A-253) cell line. Consonant with BLM resistance in the C-10E cell line, we found that this cell line accumulated 2- to 3-fold less BLM A2 than A-253 cells. Kinetic analyses of BLM A2 association revealed a decreased Vmax for C-10E cells with little change in Ka. Furthermore, the BLM-resistant cell line (C-10E) metabolized BLM A2 to a greater extent than its sensitive counterpart (A-253). Thus, compared to A-253 cells, the C-10E cells exhibited both decreased cellular association and increased metabolism of BLM. Synergistic cytotoxicity was seen when BLM was combined with either E-64 or leupeptin, cysteine proteinase inhibitors known to block BLM metabolism in vitro. E-64 inhibited the metabolism of BLM A2 in both C-10E and A-253 cells, and cellular accumulation of radiolabeled BLM A2 was increased by leupeptin or E-64 in only A-253 cells. These results suggest that both inhibition of drug metabolism and increased drug accumulation contribute to this synergism.

Prenyltransferase Inhibitors as Radiosensitizers

Radiation therapy is frequently used in the treatment of a number of different tumors. However, the effectiveness of radiotherapy is limited by the ability of normal tissues adjacent to tumors to tolerate radiation in the doses required to kill or sterilize tumor cells. This limitation is compounded by the presence in tumors of radiation-resistant cells that may arise as a result of environmental factors, such as hypoxic regions in tumors, the expression of growth factors that can reduce radiation sensitivity, or tumor cell intrinsic radiation resistance that may be imparted through the activation of certain oncogenes. Ras oncogenes in particular may contribute to radiation resistance, because they have been shown to increase radiation resistance in many experimental systems, and are mutated in an estimated 30% of all human tumors. Basic fibroblast growth factor (bFGF) has also been implicated in increased radiation resistance and is over-expressed in certain tumors, particularly glioblastomas.