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J B Gibbs

Publications and source records attributed to J B Gibbs.

At least 37 records · Page 2Linked to original sources
PubMed1997

Farnesyltransferase inhibitors versus Ras inhibitors.

Over the past few years, the idea that farnesyl-protein transferase (FPTase) inhibitors might be effective antiproliferative/antitumor agents has been realized in studies of cultured cells and in rodent models of cancer. Most of the studies with FPTase inhibitors have focused on inhibiting the growth of ras-transformed cells in vitro or the growth of ras-dependent tumors in mice. More recently, it has been recognized that the antiproliferative effect of FPTase inhibitors may extend beyond ras-driven tumors. It now seems likely that the ability of FPTase inhibitors to reverse the malignant phenotype results, at least in part, from inhibiting the farnesylation of proteins other than Ras.

Alkyl and Aryl Transferases↗
PubMed1997

Kinase-deficient Pak1 mutants inhibit Ras transformation of Rat-1 fibroblasts.

Among the mechanisms by which the Ras oncogene induces cellular transformation, Ras activates the mitogen-activated protein kinase (MAPK or ERK) cascade and a related cascade leading to activation of Jun kinase (JNK or SAPK). JNK is additionally regulated by the Ras-related G proteins Rac and Cdc42. Ras also regulates the actin cytoskeleton through an incompletely elucidated Rac-dependent mechanism. A candidate for the physiological effector for both JNK and actin regulation by Rac and Cdc42 is the serine/threonine kinase Pak (p65pak). We show here that expression of a catalytically inactive mutant Pak, Pak1(R299), inhibits Ras transformation of Rat-1 fibroblasts but not of NIH 3T3 cells. Typically, 90 to 95% fewer transformed colonies were observed in cotransfection assays with Rat-1 cells. Pak1(R299) did not inhibit transformation by the Raf oncogene, indicating that inhibition was specific for Ras. Furthermore, Rat-1 cell lines expressing Pak1(R299) were highly resistant to Ras transformation, while cells expressing wild-type Pak1 were efficiently transformed by Ras. Pak1(L83,L86,R299), a mutant that fails to bind either Rac or Cdc42, also inhibited Ras transformation. Rac and Ras activation of JNK was inhibited by Pak1(R299) but not by Pak1(L83,L86,R299). Ras activation of ERK was inhibited by both Pak1(R299) and Pak1(L83,L86,R299), while neither mutant inhibited Raf activation of ERK. These results suggest that Pak1 interacts with components essential for Ras transformation and that inhibition can be uncoupled from JNK but not ERK signaling.

Animals↗
PubMed1997

The potential of farnesyltransferase inhibitors as cancer chemotherapeutics.

Mutant ras oncogenes and alterations in the mitogenic signaling pathways that they regulate are associated with a wide variety of solid tumors and leukemias for which existing chemotherapeutics have limited utility. Of the possible approaches to inhibit Ras function, much attention has focused on a posttranslational modification, farnesylation, which is required for the subcellular localization of Ras to the plasma membrane and is critical to Ras cell-transforming activity. Inhibitors of the enzyme that catalyzes Ras farnesylation, farnesyl-protein transferase (FPTase), have been developed. These compounds inhibit the tumorigenic phenotypes of ras-transformed cells and human tumor cells in cell culture and in animal models. Moreover, FPTase inhibitors have not demonstrated toxicity to normal cells in culture or to normal tissues in mice. FPTase inhibitors are among the first small molecule compounds designed from studies of oncogenes that might serve as novel cancer chemotherapeutics.

Alkyl and Aryl Transferases↗
PubMed1996

Selection of potent inhibitors of farnesyl-protein transferase from a synthetic tetrapeptide combinatorial library.

Inhibitors of farnesyl-protein transferase (FPTase) show promise as anticancer agents. Based on the sequence of the protein substrates of FPTase (the CAAX sequence), potent and selective peptidomimetic inhibitors have been developed; these compounds share with the peptide substrate a free thiol and a C-terminal carboxylate. We have used a synthetic tetrapeptide combinatorial library to screen for new leads devoid of these features: the peptides were C-terminally amidated, and no free thiol was included in the combinatorial building blocks. To compensate for this negative bias, an expanded set of 68 amino acids was used, including both L and D as well as many non-coded residues. Sixteen individual tetrapeptides derived from the consensus were synthesized and tested; all were active, showing IC50 values ranging from low micromolar to low nanomolar. The most active peptide, D-tryptophan-D-methionine-D-4-chlorophenylalanine-L-gamma- carboxyglutamic acid (Ki = 2 nM), is also very selective showing little inhibitory activity against the related enzyme geranylgeranyl-protein transferase type I (IC50 > 50 microM). In contrast to CAAX-based peptidomimetics, D-tryptophan-D-methionine-D-4-chlorophenylalanine-L-gamma-carboxyglut amic acid appeared to mimic the isoprenoid substrate farnesyl diphosphate as determined by kinetic and physical measurements. D-Tryptophan-Dmethionine-D-4-chlorophenylalanine-L-gamma- carboxyglutamic acid was a competitive inhibitor of FPTase with respect to farnesyl diphosphate substrate and uncompetitive with respect to CAAX substrate. Furthermore, we demonstrated that FPTase undergoes ligand dependent conformational changes in its circular dichroism spectrum and that D-tryptophan-D-methionine-D-4-chlorophenylalanine-L-gamma- carboxyglutamic acid induced a conformational change identical to that observed with farnesyl diphosphate ligand.

Alkyl and Aryl Transferases↗
PubMed1996

Farnesyltransferase inhibitors and anti-Ras therapy.

The oncoprotein encoded by mutant ras genes is initially synthesized as a cytoplasmic precursor which requires posttranslational processing to attain biological activity; farnesylation of the cysteine residue present in the CaaX motif located at the carboxy-terminus of all Ras proteins is the critical modification. Once farnesylated and further modified, the mature Ras protein is inserted into the cell's plasma membrane where it participates in the signal transduction pathways that control cell growth and differentiation. The farnesylation reaction that modifies Ras and other cellular proteins having an appropriate CaaX motif is catalyzed by a housekeeping enzyme termed farnesyl-protein transferase (FPTase). Inhibitors of this enzyme have been prepared by several laboratories in an effort to identify compounds that would block Ras-induced cell transformation and thereby function as Ras-specific anticancer agents. A variety of natural products and synthetic organic compounds were found to block farnesylation of Ras proteins in vitro. Some of these compounds exhibit antiproliferative activity in cell culture, block the morphological alterations associated with Ras-transformation, and can block the growth of Ras-transformed cell lines in tumor colony-forming assays. By contrast, these compounds do not affect the growth or morphology of cells transformed by the Raf or Mos oncoproteins, which do not require farnesylation to achieve biological activity. The efficacy and lack of toxicity observed with FPTase inhibitors in an animal tumor model suggest that specific FPTase inhibitors may be useful for the treatment of some types of cancer.

Alkyl and Aryl Transferases↗
PubMed1995

A peptidomimetic inhibitor of farnesyl:protein transferase blocks the anchorage-dependent and -independent growth of human tumor cell lines.

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.

Alkyl and Aryl Transferases↗
PubMed1995

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

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.

Allosteric Regulation↗
PubMed1995

Development of inhibitors of protein farnesylation as potential chemotherapeutic agents.

Protein prenylation, adding either the 15-carbon isoprenoid farnesyl 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 transforming cells in culture. Such mutated 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 specific inhibition of Ras farnesylation; they are also observed in cells transformed by raf, which is independent of Ras farnesylation. A more specific approach to inhibiting Ras farnesylation is to inhibit FPTase. Using random screening of natural products and a rational design approach, a variety of compounds that specifically inhibit FPTase have been isolated.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

cDNA cloning and expression of rat and human protein geranylgeranyltransferase type-I.

Protein geranylgeranyltransferase type-I (GGTase-I) transfers a geranylgeranyl group to the cysteine residue of candidate proteins containing a carboxyl-terminal CAAX (C, cysteine; A, aliphatic amino acid; X, any amino acid) motif in which the "X" residue is leucine. The enzyme is composed of a 48-kilodalton alpha subunit and a 43-kilodalton beta subunit. Peptides isolated from the alpha subunit of GGTase-I were shown to be identical with the alpha subunit of a related enzyme, protein farnesyltransferase. Overlapping cDNA clones containing the complete coding sequence for the beta subunit of GGTase-I were obtained from rat and human cDNA libraries. The cDNA clones from both species each predicted a protein of 377 amino acids with molecular masses of 42.4 kilodaltons (human) and 42.5 kilodaltons (rat). Amino acid sequence comparison suggests that the protein encoded by the Saccharomyces cerevisiae gene CDC43 is the yeast counterpart of the mammalian GGTase-I beta subunit. Co-expression of the GGTase-I beta subunit cDNA together with the alpha subunit of protein farnesyltransferase in Escherichia coli produced recombinant GGTase-I with electrophoretic and enzymatic properties indistinguishable from native GGTase-I.

Alkyl and Aryl Transferases↗
PubMed1994

Ras regulatory interactions: novel targets for anti-cancer intervention?

Advances in the understanding of Ras oncoprotein function suggest novel points for anti-tumor intervention. First, upstream-acting guanine nucleotide exchange factors and SH2/SH3 domain-containing adaptor proteins that link Ras with growth factor receptor tyrosine kinases have recently been characterized. Second, work on downstream-acting Ras effector functions including the Ras GTPase-activating protein (p120GAP) and the Raf kinase has revealed direct biochemical interactions that are functionally required for oncogenic Ras signalling. We summarize progress in these areas and discuss the potential for novel applications to anti-cancer chemotherapy.

Alkyl and Aryl Transferases↗