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2.
Anal Biochem ; 290(1): 126-37, 2001 Mar 01.
Article in English | MEDLINE | ID: mdl-11180946

ABSTRACT

Cellular transformation by Ras oncoproteins requires the posttranslation modification of farnesylation in a reaction catalyzed by farnesyl protein transferase (FPTase). Thus, inhibitors of FPTase have been developed as potential anticancer agents. However, recent studies with selective inhibitors of FPTase have shown that Ki4B-Ras retains its ability to transform cells by undergoing alternative prenylation by the related geranylgeranyl protein transferase I (GGPTase-I) in human tumor cells. We have developed a high-performance liquid chromatography/mass spectrometry assay for the detection and quantitation of the different processing states of Ki4B-Ras isolated from PSN-1 cells (a human pancreatic cell line with an activating Gly12 to Arg mutation) treated with the prenyltransferase inhibitor, L-778,123. Recently tested in the clinic, L-778,123 is a potent inhibitor of FPTase (in vitro IC50 = 2 nM) with some activity against GGPTase-I (in vitro IC50 = 98 nM). We find primarily farnesylated-Ki4B-Ras in vehicle-treated PSN-1 cells, a mixture of farnesylated- and geranylgeranylated-Ki4B-Ras in cells treated with nanomolar concentrations of L-778,123, and a mixture of unprocessed, farnesylated, and geranylgeranylated-Ki4B-Ras in cells treated with micromolar concentrations of compound. Of importance, this technique does not require metabolic labeling and may be used as a pharmacodynamic assay for Ki4B-Ras processing in mouse models.


Subject(s)
Dimethylallyltranstransferase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Gas Chromatography-Mass Spectrometry/methods , Imidazoles/pharmacology , Proto-Oncogene Proteins p21(ras)/analysis , Tumor Cells, Cultured/drug effects , Alkyl and Aryl Transferases/antagonists & inhibitors , Alkyl and Aryl Transferases/metabolism , Farnesyltranstransferase , Humans , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/enzymology , Protein Prenylation , Recombinant Proteins/metabolism , Tumor Cells, Cultured/enzymology
3.
Cancer Res ; 60(10): 2680-8, 2000 May 15.
Article in English | MEDLINE | ID: mdl-10825141

ABSTRACT

For Ras oncoproteins to transform mammalian cells, they must be posttranslationally modified with a farnesyl group in a reaction catalyzed by the enzyme farnesyl:protein transferase (FPTase). Inhibitors of FPTase have therefore been developed as potential anticancer agents. These compounds reverse many of the malignant phenotypes of Ras-transformed cells in culture and inhibit the growth of tumor xenografts in nude mice. Furthermore, the FPTase inhibitor (FTI) L-744,832 causes tumor regression in mouse mammary tumor virus (MMTV)-v-Ha-ras transgenic mice and tumor stasis in MMTV-N-ras mice. Although these data support the further development of FTIs, it should be noted that Ki-ras is the ras gene most frequently mutated in human cancers. Moreover, Ki-RasB binds more tightly to FPTase than either Ha- or N-Ras, and thus higher concentrations of FTIs that are competitive with the protein substrate may be required to inhibit Ki-Ras processing. Given the unique biochemical and biological features of Ki-RasB, it is important to evaluate the efficacy of FTIs or any other modulator of oncogenic Ras function in model systems expressing this Ras oncoprotein. We have developed strains of transgenic mice carrying the human Ki-rasB cDNA with an activating mutation (G12V) under the control of the MMTV enhancer/promoter. The predominant pathological feature that develops in these mice is the stochastic appearance of mammary adenocarcinomas. High levels of the Ki-rasB transgene RNA are detected in these tumors. Treatment of MMTV-Ki-rasB mice with L-744,832 caused inhibition of tumor growth in the absence of systemic toxicity. Although FPTase activity was inhibited in tumors from the treated mice, unprocessed Ki-RasB was not detected. These results demonstrate the utility of the MMTV-Ki-rasB transgenic mice for testing potential anticancer agents. Additionally, the data suggest that although the FTI L-744,832 can inhibit tumor growth in this model, Ki-Ras may not be the sole mediator of the biological effects of the FTI.


Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Antineoplastic Agents/therapeutic use , Enzyme Inhibitors/therapeutic use , Genes, ras , Growth Inhibitors/therapeutic use , Mammary Neoplasms, Animal/drug therapy , Mammary Tumor Virus, Mouse , Methionine/analogs & derivatives , Animals , Disease Models, Animal , Farnesyltranstransferase , Female , Humans , Methionine/therapeutic use , Mice , Mice, Transgenic , Phenotype , Transgenes
5.
J Med Chem ; 42(17): 3356-68, 1999 Aug 26.
Article in English | MEDLINE | ID: mdl-10464022

ABSTRACT

Inhibitors of farnesyl protein transferase (FPTase) based upon a pseudotripeptide template are described that comprise an imidazole group substituted with a hydrophobic substituent. (1, 5)-Disubstitution of the imidazole group is shown to be the optimal array that leads to potent and selective inhibitors of FPTase. A variety of aryl and isoprenyl substituents are shown to afford effective inhibitors, and the mechanism by which these compounds inhibit FPTase has been investigated. The biochemical behavior of these compounds suggests that they bind to FPTase at the site usually occupied by the protein substrate. In experiments in cell culture, the methyl ester prodrugs of these inhibitors are cell permeant and potently inhibit the posttranslational modification of H-Ras protein. Additionally, these molecules revert the phenotype of ras transformed cells as evidenced by their ability to slow the growth of ras transformed cell lines in soft agar. One of the inhibitors, as its methyl prodrug, was evaluated in two in vivo models of tumor growth. The compound selectively inhibited the growth of tumors derived from H-ras transformed cells, in nude mice, and caused the regression of preexisting tumors in an H-ras transgenic animal model.


Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Antineoplastic Agents/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Imidazoles/chemical synthesis , 3T3 Cells , Alkyl and Aryl Transferases/metabolism , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Binding Sites , Cell Line, Transformed , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Imidazoles/chemistry , Imidazoles/pharmacology , Mice , Mice, Nude , Mice, Transgenic , Neoplasm Transplantation , Prodrugs/chemical synthesis , Prodrugs/chemistry , Prodrugs/pharmacology , Protein Processing, Post-Translational/drug effects , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Structure-Activity Relationship
6.
J Med Chem ; 41(14): 2651-6, 1998 Jul 02.
Article in English | MEDLINE | ID: mdl-9651171

ABSTRACT

Inhibitors of Ras protein farnesyltransferase are described which are reduced pseudopeptides related to the C-terminal tetrapeptide of the Ras protein that signals farnesylation. Reduction of the carbonyl groups linking the first three residues of the tetrapeptide leads to active inhibitors which are chemically unstable. Stability can be restored by alkylating the central amine of the tetrapeptide. Studies of the SAR of these alkylated pseudopeptides with concomitant modification of the side chain of the third residue led to 2(S)-(2(S)-¿[2(S)-(2(R)-amino-3-mercaptopropylamino)-3(S)- methylpentyl]naphthalen-1-ylmethylamino¿acetylamino)-4 -methylsulfany lbutyric acid (11), a subnanomolar inhibitor. The methyl ester (10) of this compound exhibited submicromolar activity in the processing assay and selectively inhibited anchorage-independent growth of Rat1 cells transformed by v-ras at 2.5-5 microM.


Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Esters/chemical synthesis , Molecular Mimicry , Naphthalenes/chemical synthesis , Oligopeptides/chemistry , Prodrugs/chemical synthesis , 3T3 Cells , Animals , Cell Division/drug effects , Cell Line, Transformed , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Esters/chemistry , Esters/pharmacology , Farnesyltranstransferase , Mice , Naphthalenes/chemistry , Naphthalenes/pharmacology , Oncogene Protein p21(ras)/antagonists & inhibitors , Prodrugs/chemistry , Prodrugs/pharmacology , Protein Processing, Post-Translational/drug effects , Rats , Structure-Activity Relationship
7.
Mol Cell Biol ; 18(1): 85-92, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9418856

ABSTRACT

The farnesyltransferase inhibitor L-744,832 selectively blocks the transformed phenotype of cultured cells expressing a mutated H-ras gene and induces dramatic regression of mammary and salivary carcinomas in mouse mammary tumor virus (MMTV)-v-Ha-ras transgenic mice. To better understand how the farnesyltransferase inhibitors might be used in the treatment of human tumors, we have further explored the mechanisms by which L-744,832 induces tumor regression in a variety of transgenic mouse tumor models. We assessed whether L-744,832 induces apoptosis or alterations in cell cycle distribution and found that the tumor regression in MMTV-v-Ha-ras mice could be attributed entirely to elevation of apoptosis levels. In contrast, treatment with doxorubicin, which induces apoptosis in many tumor types, had a minimal effect on apoptosis in these tumors and resulted in a less dramatic tumor response. To determine whether functional p53 is required for L-744,832-induced apoptosis and the resultant tumor regression, MMTV-v-Ha-ras mice were interbred with p53(-/-) mice. Tumors in ras/p53(-/-) mice treated with L-744,832 regressed as efficiently as MMTV-v-Ha-ras tumors, although this response was found to be mediated by both the induction of apoptosis and an increase in G1 with a corresponding decrease in the S-phase fraction. MMTV-v-Ha-ras mice were also interbred with MMTV-c-myc mice to determine whether ras/myc tumors, which possess high levels of spontaneous apoptosis, have the potential to regress through a further increase in apoptosis levels. The ras/myc tumors were found to respond nearly as efficiently to L-744,832 treatment as the MMTV-v-Ha-ras tumors, although no induction of apoptosis was observed. Rather, the tumor regression in the ras/myc mice was found to be mediated by a large reduction in the S-phase fraction. In contrast, treatment of transgenic mice harboring an activated MMTV-c-neu gene did not result in tumor regression. These results demonstrate that a farnesyltransferase inhibitor can induce regression of v-Ha-ras-bearing tumors by multiple mechanisms, including the activation of a suppressed apoptotic pathway, which is largely p53 independent, or by cell cycle alterations, depending upon the presence of various other oncogenic genetic alterations.


Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Carcinoma/genetics , Enzyme Inhibitors/pharmacology , Mammary Neoplasms, Experimental/genetics , Methionine/analogs & derivatives , Salivary Gland Neoplasms/genetics , Animals , Antineoplastic Agents/therapeutic use , Carcinoma/drug therapy , Carcinoma/pathology , Cell Cycle/drug effects , Enzyme Inhibitors/therapeutic use , Farnesyltranstransferase , Female , Genes, ras , Humans , Mammary Neoplasms, Experimental/drug therapy , Mammary Neoplasms, Experimental/pathology , Mammary Tumor Virus, Mouse , Methionine/pharmacology , Methionine/therapeutic use , Mice , Mice, Transgenic , Salivary Gland Neoplasms/drug therapy , Salivary Gland Neoplasms/pathology
8.
Bioorg Med Chem Lett ; 8(23): 3311-6, 1998 Dec 01.
Article in English | MEDLINE | ID: mdl-9873725

ABSTRACT

The structure-activity relationship of a series of non-thiol CaaX analogs, which are inhibitors of farnesyltransferase, is described. These inhibitors contain a substituted phenyl group at the N terminus, which may occupy a novel binding domain on the Ras protein.


Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Farnesyltranstransferase , Structure-Activity Relationship
9.
J Biol Chem ; 271(49): 31306-11, 1996 Dec 06.
Article in English | MEDLINE | ID: mdl-8940136

ABSTRACT

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.


Subject(s)
Alkyl and Aryl Transferases , Antineoplastic Agents/chemical synthesis , Oligopeptides/chemical synthesis , Transferases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Circular Dichroism , Gene Library , Oligopeptides/pharmacology
11.
J Med Chem ; 38(20): 3967-71, 1995 Sep 29.
Article in English | MEDLINE | ID: mdl-7562930

ABSTRACT

A series of pseudodipeptide amides are described that inhibit Ras protein farnesyltransferase (PFTase). These inhibitors are truncated versions of the C-terminal tetrapeptide (CAAX motif) of Ras that serves as the signal sequence for PFTase-catalyzed protein farnesylation. In contrast to CAAX peptidomimetics previously reported, these inhibitors do not have a C-terminal carboxyl moiety, yet they inhibit farnesylation in vitro at < 100 nM. Despite the absence of the X residue in the CAAX motif, which normally directs prenylation specificity, these pseudodipeptides are greater than 100-fold selective for PFTase over type 1 protein geranylgeranyltransferase.


Subject(s)
Alkyl and Aryl Transferases , Enzyme Inhibitors/pharmacology , Transferases/antagonists & inhibitors , 3T3 Cells , Amides/pharmacology , Animals , Mice , Peptides/pharmacology , Structure-Activity Relationship , ras Proteins/metabolism
12.
Protein Sci ; 4(4): 681-8, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7613466

ABSTRACT

Farnesyl-protein transferase (FPTase) catalyzes the posttranslational farnesylation of the cysteine residue located in the carboxyl-terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for the membrane association and cell-transforming activities of ras. Inhibitors of FPTase have been demonstrated to inhibit ras-dependent cell transformation and thus represent a potential therapeutic strategy for the treatment of human cancers. The FPTase-bound conformation of a tetrapeptide inhibitor, CVWM, and a novel pseudopeptide inhibitor, L-739,787, have been determined by NMR spectroscopy. Distance constraints were derived from two-dimensional transferred nuclear Overhauser effect experiments. Ligand competition experiments identified the NOEs that originate from the active-site conformation. Structures were calculated with the combination of distance geometry and restrained energy minimization. Both peptide backbones are shown to adopt nonideal reverse-turn conformations most closely approximating a type III beta-turn. These results provide a basis for understanding the spatial arrangements necessary for inhibitor binding and selectivity and may aid in the design of therapeutic agents.


Subject(s)
Alkyl and Aryl Transferases , Amides/chemistry , Oligopeptides/chemistry , Protein Conformation , Transferases/antagonists & inhibitors , Amides/metabolism , Amides/pharmacology , Amino Acid Sequence , Computer Graphics , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Molecular Structure , Oligopeptides/metabolism , Oligopeptides/pharmacology , Protein Binding , Protein Prenylation , Recombinant Proteins/chemistry , Transferases/chemistry , Transferases/metabolism
14.
Bioorg Med Chem ; 2(9): 939-47, 1994 Sep.
Article in English | MEDLINE | ID: mdl-7712129

ABSTRACT

Replacement of the central amino methylene linkage of C[psi CH2NH]A[psi CH2NH]AX tetrapeptide inhibitors with carbon tethers led to compounds with potency in the nanomolar range. Some of the more potent olefinic compounds inhibit Ras processing in intact v-ras transformed NIH 3T3 cells with IC50 values in the 0.1 to 1 microM range, and inhibit selectively the anchorage-independent growth of H-ras transformed Rat1 cells at 10 microM.


Subject(s)
Alkyl and Aryl Transferases , Oligopeptides/chemical synthesis , Oligopeptides/pharmacology , Transferases/antagonists & inhibitors , 3T3 Cells , Amino Acid Sequence , Animals , Cell Transformation, Viral , Genes, ras , Mice , Molecular Sequence Data , Molecular Structure , Stereoisomerism , Structure-Activity Relationship
15.
Proc Natl Acad Sci U S A ; 91(19): 9141-5, 1994 Sep 13.
Article in English | MEDLINE | ID: mdl-8090782

ABSTRACT

The posttranslational addition of a farnesyl moiety to the Ras oncoprotein is essential for its transforming activity. Cell-active inhibitors of the enzyme that catalyzes this reaction, protein farnesyltransferase, have been shown to selectively block ras-dependent transformation of cells in culture. Here we describe the protein farnesyltransferase inhibitor 2(S)-[2(S)-[2(R)-amino-3-mercapto]propylamino-3(S)-methyl] pentyloxy-3-phenylpropionylmethioninesulfone methyl ester (L-739,749), which suppressed the anchorage-independent growth of Rat1 cells transformed with viral H-ras and the human pancreatic adenocarcinoma cell line PSN-1, which harbors altered K-ras, myc, and p53 genes. This compound also suppressed the growth of tumors arising from ras-transformed Rat1 cells in nude mice by 66%. Under the same conditions, doxorubicin inhibited tumor growth by 33%. Control tumors formed by v-raf- or v-mos-transformed Rat1 cells were unaffected by L-739,749. Furthermore, mice treated with L-739,749 exhibited no evidence of systemic toxicity. This is a demonstration of antitumor activity in vivo using a synthetic small molecule inhibitor of protein farnesyltransferase.


Subject(s)
Alkyl and Aryl Transferases , Cell Transformation, Viral , Genes, ras , Oligopeptides/pharmacology , Transferases/antagonists & inhibitors , Animals , Genes, mos , Mice , Mice, Nude
16.
J Med Chem ; 37(6): 725-32, 1994 Mar 18.
Article in English | MEDLINE | ID: mdl-8145221

ABSTRACT

Inhibitors of Ras farnesyl-protein transferase are described. These are reduced pseudopeptides related to the C-terminal tetrapeptide of the Ras protein that signals farnesylation. Deletion of the carbonyl groups between the first two residues of the tetrapeptides either preserves or improves activity, depending on the peptide sequence. The most potent in vitro enzyme inhibitor described (IC50 = 5 nM) is Cys [psi CH2NH]Ile[psi CH2NH]Phe-Met (3). To obtain compounds able to suppress Ras farnesylation in cell culture, further structural modification to include a homoserine lactone prodrug was required. Compound 18 (Cys[psi CH2NH]Ile[psi CH2NH]Ile-homoserine lactone) reduced the extent of Ras farnesylation by 50% in NIH3T3 fibroblasts in culture at a concentration of 50 microM. Structure-activity studies also led to 12 (Cys[psi CH2NH]Val-Ile-Leu), a potent and selective inhibitor of a related enzyme, the type-I geranylgeranyl protein transferase.


Subject(s)
Alkyl and Aryl Transferases , Dipeptides/chemical synthesis , Dipeptides/pharmacology , Oligopeptides/chemical synthesis , Protein Prenylation/drug effects , Transferases/antagonists & inhibitors , Amino Acid Sequence , Animals , Cattle , Cells, Cultured , Farnesyltranstransferase , Molecular Sequence Data , Oligopeptides/pharmacology , Stereoisomerism , Structure-Activity Relationship
17.
Biochemistry ; 32(32): 8341-7, 1993 Aug 17.
Article in English | MEDLINE | ID: mdl-8347630

ABSTRACT

The catalytic utilization of dimethylallyl, geranyl, farnesyl, and geranylgeranyl diphosphates in the reaction catalyzed by recombinant human farnesyl:protein transferase (hFPTase) has been examined in the presence of three different protein substrates, Ras-CVLS, Ras-CVIM, and Ras-CAIL. hFPTase catalyzed both farnesylation and geranylation of Ras-CVLS and of Ras-CVIM but not of Ras-CAIL. Geranylgeranylation was observed, but only when Ras-CVIM was the acceptor substrate. Steady-state initial velocity and dead-end inhibitor studies indicate that hFPTase-catalyzed geranylation, like bovine FPTase-catalyzed farnesylation, proceeds through a random order, sequential mechanism. Surprisingly, however, Michaelis constants for a given protein acceptor substrate varied depending upon which isoprenoid diphosphate was used as the donor substrate, showing that these substrates do not bind independently to the enzyme (under catalytic conditions). In addition, at very high concentrations of Ras-CVIM, substrate inhibition was observed in the presence of both FPP and GPP. Isotope partitioning studies showed that, at high concentrations of Ras-CVIM, more than 80% of the bound farnesyl diphosphate (FPP) can be trapped as product, suggesting that the binary complex is catalytically competent and that the ternary complex proceeds to product faster than it releases FPP. The release rate of FPP from the binary complex was calculated to be 0.05 s-1, which is only about eight times greater than kcat. Thus, the binding of FPP to the enzyme in the presence of the protein substrate is not an equilibrium situation.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Alkyl and Aryl Transferases , Polyisoprenyl Phosphates/metabolism , Transferases/metabolism , Computer Simulation , Escherichia coli , Humans , Kinetics , Recombinant Proteins/metabolism , Substrate Specificity , Tritium
18.
Science ; 260(5116): 1934-7, 1993 Jun 25.
Article in English | MEDLINE | ID: mdl-8316833

ABSTRACT

To acquire transforming potential, the precursor of the Ras oncoprotein must undergo farnesylation of the cysteine residue located in a carboxyl-terminal tetrapeptide. Inhibitors of the enzyme that catalyzes this modification, farnesyl protein transferase (FPTase), have therefore been suggested as anticancer agents for tumors in which Ras contributes to transformation. The tetrapeptide analog L-731,735 is a potent and selective inhibitor of FPTase in vitro. A prodrug of this compound, L-731,734, inhibited Ras processing in cells transformed with v-ras. L-731,734 decreased the ability of v-ras-transformed cells to form colonies in soft agar but had no effect on the efficiency of colony formation of cells transformed by either the v-raf or v-mos oncogenes. The results demonstrate selective inhibition of ras-dependent cell transformation with a synthetic organic inhibitor of FPTase.


Subject(s)
Alkyl and Aryl Transferases , Antineoplastic Agents/pharmacology , Cell Transformation, Neoplastic/drug effects , Dipeptides/pharmacology , Genes, ras , Oncogene Proteins/metabolism , Protein Prenylation/drug effects , Transferases/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Cell Division/drug effects , Cell Line , Dipeptides/chemistry , Drug Design , Farnesyltranstransferase , Rats
19.
J Biol Chem ; 268(11): 7617-20, 1993 Apr 15.
Article in English | MEDLINE | ID: mdl-8463291

ABSTRACT

The ras oncogene product, Ras, is synthesized in vivo as a precursor protein that requires post-translational processing to become biologically active and to be capable of transforming mammalian cells. Farnesylation appears to be a critical modification of Ras, and thus inhibitors of the farnesyl-protein transferase (FPTase) that catalyzes this reaction may block ras-dependent tumorigenesis. Three structural classes of FPTase inhibitors were identified: (alpha-hydroxyfarnesyl)phosphonic acid, chaetomellic acids, and zaragozic acids. By comparison, these compounds were weaker inhibitors of geranylgeranyl-protein transferases. Each of these inhibitors was competitive with respect to farnesyl diphosphate in the FPTase reaction. All compounds were assayed for inhibition of Ras processing in Ha-ras-transformed NIH3T3 fibroblasts. Ras processing was inhibited by 1 microM (alpha-hydroxyfarnesyl)phosphonic acid. Neither chaetomellic acid nor zaragozic acid were active in this assay. These results are the first demonstration that a small organic chemical selected for inhibition of FPTase can inhibit Ras processing in vivo.


Subject(s)
Alkyl and Aryl Transferases , Bridged Bicyclo Compounds, Heterocyclic , Bridged Bicyclo Compounds/pharmacology , Farnesol/analogs & derivatives , Genes, ras , Maleates/pharmacology , Organophosphonates , Organophosphorus Compounds/pharmacology , Protein Processing, Post-Translational/drug effects , Transferases/antagonists & inhibitors , Tricarboxylic Acids/pharmacology , 3T3 Cells , Animals , Brain/enzymology , Cattle , Cell Line, Transformed , Farnesol/pharmacology , Gene Expression Regulation/drug effects , Kinetics , Mice , Transferases/genetics
20.
Biochemistry ; 31(15): 3800-7, 1992 Apr 21.
Article in English | MEDLINE | ID: mdl-1567835

ABSTRACT

The steady-state kinetic mechanism of bovine brain farnesyl:protein transferase (FPTase) has been determined using a series of initial velocity studies, including both dead-end substrate and product inhibitor experiments. Reciprocal plots of the initial velocity data intersected on the 1/[s] axis, indicating that a ternary complex forms (sequential mechanism) and suggesting that the binding of one substrate does not affect the binding of the other. The order of substrate addition was probed by determining the patterns of dead-end substrate and product inhibition. Two nonhydrolyzable analogues of farnesyl diphosphate, (alpha-hydroxyfarnesyl)phosphonic acid (1) and [[(farnesylmethyl)hydroxyphosphinyl]methyl]phosphonic acid (2), were both shown to be competitive inhibitors of farnesyl diphosphate and noncompetitive inhibitors of Ras-CVLS. Four nonsubstrate tetrapeptides, CV[D-L]S, CVLS-NH2, N-acetyl-L-penicillamine-VIM, and CIFM, were all shown to be noncompetitive inhibitors of farnesyl diphosphate and competitive inhibitors of Ras-CVLS. These data are consistent with random order of substrate addition. Product inhibition patterns corroborated the results found with the dead-end substrate inhibitors. We conclude that bovine brain FPTase proceeds through a random order sequential mechanism. Determination of steady-state parameters for several physiological Ras-CaaX variants showed that amino acid changes affected the values of KM, but not those of kcat, suggesting that the catalytic efficiencies (kcat/KM) of Ras-CaaX substrates depend largely upon their relative binding affinity for FPTase.


Subject(s)
Alkyl and Aryl Transferases , Transferases/metabolism , Animals , Brain/enzymology , Catalysis , Cattle , Chromatography, Thin Layer , Electrophoresis, Polyacrylamide Gel , Kinetics , Oligopeptides/pharmacology , Polyisoprenyl Phosphates/antagonists & inhibitors , Polyisoprenyl Phosphates/pharmacology , Sesquiterpenes , Substrate Specificity
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