Showing papers by "Jackson B. Gibbs published in 1995"

A Peptidomimetic Inhibitor of Farnesyl:Protein Transferase Blocks the Anchorage-dependent and -independent Growth of Human Tumor Cell Lines

Abstract: Farnesyl protein transferase (FPTase) catalyzes the first of a series of posttranslational modifications of Ras required for full biological activity. Peptidomimetic inhibitors of FPTase have been designed that selectively block farnesylation in vivo and in vitro. These inhibitors prevent Ras processing and membrane localization and are effective in reversing the transformed phenotype of Rat1-v-ras cells but not that of cells transformed by v-raf or v-mos. We have tested the effect of the FPTase inhibitor L-744,832 (FTI) on the anchorage-dependent and -independent growth of human tumor cell lines. The growth of over 70% of all tumor cell lines tested was inhibited by 2-20 microM of the FTI, whereas the anchorage-dependent growth of nontransformed epithelial cells was less sensitive to the effects of the compound. No correlation was observed between response to drug and the origin of the tumor cell or whether it contained mutationally activated ras. In fact, cell lines with wild-type ras and active protein tyrosine kinases in which the transformed phenotype may depend on upstream activation of the ras pathway were especially sensitive to the drug. To define the important targets of FTI action, the mechanism of cellular drug resistance was examined. It was not a function of altered drug accumulation or of FPTase insensitivity since, in all cell lines tested, FPTase activity was readily inhibited within 1 h of treatment with the inhibitor. Furthermore, the general pattern of inhibition of cellular protein farnesylation and the specific inhibition of lamin B processing were the same in sensitive and resistant cells. In addition, functional activation of Ras was inhibited to the same degree in sensitive and resistant cell lines. However, the FTI inhibited the epidermal growth factor-induced activation of mitogen-activated protein kinases in sensitive cells but not in two resistant cell lines. These data suggest that the drug does inhibit ras function and that resistance in some cells is associated with the presence of Ras-independent pathways for mitogen-activated protein kinase activation by tyrosine kinases. We conclude that FPTase inhibitors are potent antitumor agents with activity against many types of human cancer cell lines, including those with wild-type ras.

Development of inhibitors of protein farnesylation as potential chemotherapeutic agents

Abstract: Protein prenylation, adding either the 15-carbon isoprenoid farnesy1 or the 20-carbon isoprenoid geranylgeranyl to cysteine residue(s) at or near the C-termini of proteins, is a recently identified post-translational modification that localizes some proteins to a membrane compartment. One of the most intensely studied prenylated proteins is Ras, a low molecular weight GTP-binding protein that plays an important role in the regulation of cell proliferation. Proteins encoded by ras genes with oncogenic mutations are capable of tranforming cells in culture. Such mutate ras genes are frequently found in a wide variety of human tumors. Localization of the Ras oncoprotein to the cytoplasmic face of the plasma membrane via farnesylation is essential for efficient cell transforming ability. Thus, inhibition of the Ras farnesylation reaction is a possible anti-cancer strategy. Several strategies have been employed to inhibit Ras farnesylation, including inhibition of isoprenoid biosynthesis and inhibition of the enzyme which catalyzes the farnesylation reaction, farnesyl-protein transferase (FPTase). Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme. A reductase, the rate limiting enzyme in isoprenoid biosynthesis, inhibit Ras farnesylation and block the growth of ras-transformed cells. However, antiproliferative effects do not result from speicific inhibition of Ras farnesylation; they are also observed in cells transformed by raf, which is independent of Ras farensylation. A more specific approach to inhibiting Ras farnesylation is to inhibit FPTase. Using randon screeing of natural products and a rational design approach, a variety of compounds that specifically inhibit FPTase have been isolated. Several of these compounds were found to block the farnesylation of Ras proteins in cell culture and were able to block the anchorage-independent growth of ras-transformed cells and human tumor cell lines. FPTase inhibitors also blocked the morphologic alteration associated with ras-induced transformation of mammalian cells. In contrast, these compounds did not affect the growth or morphology of cells transformed by the raf or mos oncogenes, which do not require farnesylation to achieve biological activity. Furthermore, these compounds suppressed the growth of tumors arising from ras-transformed cells in nude mice in the absence of systemic toxicity. Control tumors formed by raf- or mos- transformed cells were not affected by these compounds. These studies suggest that FPTase inhibitors might be safe and effective chemotherapeutic agents.

src-homology 2 (SH2) domain ligation as an allosteric regulator: modulation of phosphoinositide-specific phospholipase C gamma 1 structure and activity.

Abstract: Phosphoinositide-specific phospholipase C gamma 1 (PI-PLC gamma 1) catalyses the hydrolysis of PtdIns(4,5)P2 to generate the second messengers diacylglycerol and Ins(1,4,5)P3. PI-PLC gamma 1, an src-homology 2/3 (SH2/SH3)-domain-containing enzyme, is activated in response to growth-factor-induced tyrosine phosphorylation, and, in vivo, is translocated from the cytosol to the particulate cell fraction. Here we report the bacterial expression of rat brain PI-PLC gamma 1 under the control of the T7 promoter. Production of the active enzyme in amounts suitable for structure-function analysis depended on coupling the translation of PLC gamma 1 to the expression of the phage-phi 10 coat protein. Purification of the enzyme was facilitated by the presence of a three-amino-acid C-terminal antibody epitope tag (Glu-Glu-Phe) engineered into the cloned PLC gamma 1. Examination of the specific activity, pH-rate profile, [Ca2+]-dependence and substrate specificity of bacterially expressed PLC gamma indicated that it had kinetic properties similar to those of PLC gamma isolated from bovine brain. The substrate specificity was dependent on [Ca2+]: at low [Ca2+] (1-10 microM) PtdIns(4,5)P2 was a better substrate than PtdIns. Addition of phosphotyrosine-containing peptides (12-mers) with the cognate sequence of the high-affinity binding site for PLC gamma 1 on the activated epidermal-growth-factor (EGF) receptor (Tyr-992) increased enzyme activity (up to 85%) in vitro. Cognate non-phosphorylated peptides had no effect on activity. When c.d. spectroscopy was used to monitor the effect of added phosphotyrosine-containing peptide on the structure of recombinant PLC gamma 1, significant spectral shifts, indicative of a conformational change, were observed upon complexation with the EGF-receptor phosphotyrosine-containing 12-residue peptide (Tyr*-992). How SH2 domains from PLC gamma 1 can mediate structural rearrangements and modulate enzymic activity on their ligation by growth-factor receptors is discussed.

New fungal metabolites as potential antihypercholesterolemics and anticancer agents

Abstract: Several potent inhibitors of squalence synthetase have been discovered. Zaragozic acid A is produced by several fungi; zaragozic acid B is produced by several strains of Sporormiella intermedia; zaragozic acids C, E, and F are produced by Leptodontidium elatius; zaragozic acids D and D2 are produced by Amauroascus niger. L-731,120 and L-731,128 are minor components and coproduced with zaragozic acids A and B, respectively. Viridiofungins A, B, and C are produced by Trichoderma viride. Viridiofungin A is also produced by an unidentified sterile fungus. Several of the zaragozic acids are also potent inhibitors of farnesyl-protein transferase (FPTase). Inhibitors of FPTase may act as potential anticancer drugs. Chaetomellic acids A and B are produced by a fungus, Chaetomella acutiseta, while fusidienol is produced by Fusidium griseum. All three compounds are potent inhibitors of FPTase. Our experiences suggest that many novel inhibitors of both squalene synthase and FPTase are produced within a diverse phylo...

Actinoplanic acids A and B as novel inhibitors of farnesyl-protein transferase

Abstract: Actinoplanic acids A and B are macrocyclic polycarboxylic acids that are potent reversible inhibitors of farnesyl-protein transferase. Actinoplanic acids A and B were isolated from Actinoplanes sp. MA 7066 while actinoplanic acid B was isolated from both MA 7066 and Streptomyces sp. MA 7099. Actinoplanic acids A and B are competitive with respect to farnesyl diphosphate and are selective inhibitors of farnesyl-protein transferase because they do not inhibit geranylgeranyl-protein transferase type 1 or squalene synthase. MA 7066 is believed to be a novel species of actinomycetes while MA 7099 is believed to be a novel strain of Streptomyces violaceusniger on the basis of morphological, biochemical and chemotaxonomic characteristics as well as its production of actinoplanic acids.

Inhibitors of geranylgeranyl-protein transferase

Abstract: The compounds disclosed are analogs of the CAAX motif of proteins that can be modified by geranylgeranylation in vivo that selectively inhibit the geranylgeranylation of several proteins. The relatively poor activity of the compounds against the farnesyl protein transferase, which modifies several proteins important in cellular replication, allows the use of the compounds of the instant invention to treat diseases which are regulated by the function of a geranylgeranylated protein, such as certain cancers and inflammatory diseases. Further contained in this invention are chemotherapeutic compositions containing these geranylgeranyl protein transferase type I inhibitors and methods for their production.

Peptide inhibitors of farnesyl-protein transferase

Abstract: The present invention is directed to tetrapeptides which inhibit farnesyl-protein transferase (FPTase) and the farnesylation of the oncogene protein Ras. The invention is further directed to a method of treating cancer with tetrapeptide inhibitors of farnesyl protein transferase, as well as pharmaceutical formulations useful for this method of treatment.