Novel Tricyclic Inhibitors of Farnesyl Protein Transferase (original) (raw)
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Purification of ras farnesyl:Protein transferase
Methods, 1990
We describe a method for the purification of farnesyl:protein transferase, an enzyme that transfers a farnesyl group from farnesyl pyrophosphate to a COOH-terminal cysteine in ras proteins, nuclear lamin B, and the 7 subunit of bovine transducin. The enzyme is purified to homogeneity from rat brain cytosol through use of an affinity chromatography step based on the enzyme's ability to specifically bind to a hexapeptide containing the consensus sequence for farnesylation. The purification procedure is reproducible and enables the isolation of microgram amounts of purified enzyme from 50 rat brains. Two methods for assaying enzymatic activity are also described. One assay measures the transfer of [3H]farnesyl from [3H]farnesyl pyrophosphate to recombinant H-ras, and the other measures the transfer of [3H]farnesyl to a biotinylated peptide containing the Cys-AAX COOH-terminal sequence of K-rasB.
Structural homology among mammalian and Saccharomyces cerevisiae isoprenyl-protein transferases
1991
Farnesyl-protein transferase (FTase) purified from rat or bovine brain is an a/#? heterodimer, comprised of subunits having relative molecular masses of approximately 47 (a) and 46 kDa (#?). In the yeast Saccharomyces cerevisiae, two unlinked genes, RAMlIDPRl (R A M I) and RAM2, are required for FTase activity. To explore the relationship between the mammalian and yeast enzymes, we initiated cloning and immunological analyses. cDNA clones encoding the 329-amino acid COOH-terminal domain of bovine FTase a-subunit were isolated. Comparison of the amino acid sequences deduced from the a-subunit cDNA and the RAM2 gene revealed 30% identity and 58% similarity, suggesting that the RAM2 gene product encodes a subunit for the yeast FTase analogous to the bovine FTase a-subunit. Antisera raised against the RAMl gene product reacted specifically with the #?-subunit of bovine FTase, suggesting that the RAMl gene product is analogous to the bovine FTase #?-subunit. Whereas a raml mutation specifically inhibits FTase, mutations in the CDC43 and BET2 genes, both of which are homologous to R A M l , specifically inhibit geranylgeranyl-protein transferase (GGTase) type I and GGTase-11, respectively. In contrast, a ram2 mutation impairs both FTase and GGTase-I, but has little effect on GGTase-11. Antisera that specifically recognized the bovine FTase a-subunit precipitated both bovine FTase and GGTase-I activity, but not GGTase-I1 activity. Together, these results indicate that for both yeast and mammalian cells, FTase, GGTase-I, and GGTase-I1 are comprised of different but homologous #?-subunits and that the a-subunits of FTase and GGTase-I share common features not shared by GGTase-11. Site-specific farnesylation or geranylgeranylation of cellular polypeptides a t a COOH-terminal cysteine residue is a functionally essential post-translational modification (see Ref. 1). Protein acceptor substrates in mammalian cells for farnesylation include the cell-transforming 20-kDa GTPase Ras, nuclear lamin B, and the y-subunit of retinal transducin. Substrates for geranylgeranylation include some 20-kDa
Specific isoprenoid modification is required for function of normal, but not oncogenic, Ras protein
Molecular and Cellular Biology, 1992
While the Ras C-terminal CAAX sequence signals modification by a 15-carbon farnesyl isoprenoid, the majority of isoprenylated proteins in mammalian cells are modified instead by a 20-carbon geranylgeranyl moiety. To determine the structural and functional basis for modification of proteins by a specific isoprenoid group, we have generated chimeric Ras proteins containing C-terminal CAAX sequences (CVLL and CAIL) from geranylgeranyl-modified proteins and a chimeric Krev-1 protein containing the H-Ras C-terminal CAAX sequence (CVLS). Our results demonstrate that both oncogenic Ras transforming activity and Krev-1 antagonism of Ras transforming activity can be promoted by either farnesyl or geranylgeranyl modification. Similarly, geranylgeranyl-modified normal Ras [Ras(WT)CVLL], when overexpressed, exhibited the same level of transforming activity as the authentic farnesyl-modified normal Ras protein. Therefore, farnesyl and geranylgeranyl moieties are functionally interchangeable for ...
Nature Protocols, 2011
The importance of the post-translational lipid modifications farnesylation and geranylgeranylation in protein localization and function coupled with the critical role of prenylated proteins in malignant transformation has prompted interest in their biology and the development of farnesyl transferase and geranylgeranyl transferase inhibitors (FTIs and GGTIs) as chemical probes and anticancer agents. The ability to measure protein prenylation before and after FTI and GGTI treatment is important to understanding and interpreting the effects of these agents on signal transduction pathways and cellular phenotypes, as well as to the use of prenylation as a biomarker. Here we describe protocols to measure the degree of protein prenylation by farnesyl transferase or geranylgeranyl transferase in vitro, in cultured cells and in tumors from animals and humans. The assays use [ 3 H]farnesyl diphosphate and [ 3 H]geranylgeranyl diphosphate, electrophoretic mobility shift, membrane association using subcellular fractionation or immunofluorescence of intact cells, [ 3 H]mevalonic acid labeling, followed by immunoprecipitation and SDS-PAGE, and in vitro transcription, translation and prenylation in reticulocyte lysates. These protocols require from 1 day (enzyme assays) to up to 3 months (autoradiography of [ 3 H]-labeled proteins).
Proceedings of the National Academy of Sciences, 1991
ras proteins undergo posttranslational modification by a 15-carbon farnesyl isoprenoid at a cysteine within a defined COOH-terminal amino acid motif; i.e., Cys-Ali-Ali-Ser/Met (where Ali represents an aliphatic residue). In other low molecular mass GTP-binding proteins, cysteines are modified by 20-carbon geranylgeranyl groups within a Cys-Afi-Ali-Leu motif. We changed the terminal Ser-189 of Ha-ras p21 to Leu-189 by site-directed mutagenesis and found that the protein was modified by [3Hlgeranylgeranyl instead of [3H]farnesyl in an in vitro assay. Gel-permeation chromatography of [3H]mevalonate-labeled hydrocarbons released from Immunoprecipitated ras proteins overexpressed in COS cells indicated that'Ha-ras p21(Leu-189) was also a substrate for 20-carbon isoprenyl modification in vivo. Additional steps in Ha-ras p21 processing, normally initiated by farnesylation, appear to be supported by geranylgeranylation, based on metabolic labeling of Ha-ras p21(Leu-189) with [3H~palmitate and its subcellular localization in a particulate fraction from COS cells. These observations indicate that the amino acid occupying the terminal position (Xaa) in the Cys-Ali-Ali-Xaa motif constitutes a key structural feature by which Ha-ras p21 and other proteins with ras-like COOH-terminal isoprenylation sites are distinguished as substrates for farnesylor geranylgeranyltransferases.
Cancer research, 2001
Farnesyl:protein transferase (FPTase) inhibitors (FTIs) were originally developed as potential anticancer agents targeting the ras oncogene and are currently in clinical trials. Whereas FTIs inhibit the farnesylation of Ha-Ras, they do not completely inhibit the prenylation of Ki-Ras, the allele most frequently mutated in human cancers. Whereas farnesylation of Ki-Ras is blocked by FTIs, Ki-Ras remains prenylated in FTI-treated cells because of its modification by the related prenyltransferase, geranylgeranyl:protein transferase type I (GGPTase-I). Hence, cells transformed with Ki-ras tend to be more resistant to FTIs than Ha-ras-transformed cells. To determine whether Ki-ras-transformed cells can be targeted by combining an FTI with a GGPTase-I inhibitor (GGTI), we evaluated potent, selective FTIs, GGTIs, and dual prenylation inhibitors (DPIs) that have both FTI and GGTI activity. We find that in human PSN-1 pancreatic tumor cells, which harbor oncogenic Ki-ras, and in other tumor ...
Cancer research, 2001
R115777 [(B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)-methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone] is a potent and selective inhibitor of farnesyl protein transferase with significant antitumor effects in vivo subsequent to oral administration in mice. In vitro, using isolated human farnesyl protein transferase, R115777 competitively inhibited the farnesylation of lamin B and K-RasB peptide substrates, with IC50s of 0.86 nM and 7.9 nM, respectively. In a panel of 53 human tumor cell lines tested for growth inhibition, approximately 75% were found to be sensitive to R115777. The majority of sensitive cell lines had a wild-type ras gene. Tumor cell lines bearing H-ras or N-ras mutations were among the most sensitive of the cell lines tested, with responses observed at nanomolar concentrations of R115777. Tumor cell lines bearing mutant K-ras genes required higher concentrations for inhibition of cell growth, with 50% of the cell lines resistant to R115777 up to co...
Prenylation of Rab8 GTPase by type I and type II geranylgeranyl transferases
The Biochemical journal, 1998
Rab GTPases are post-translationally modified by addition of geranylgeranyl moieties to carboxyl-terminal cysteine residues. For Rab proteins ending with xxCC xCxC and CCxx motifs this modification is catalysed by geranylgeranyltransferase type II (GGTaseII), and is entirely dependent on the Rab substrate being bound to Rab escort protein (REP). Several Rab proteins contain carboxyl-terminal CaaL prenylation motifs typical of members of the Rho family, which are modified in a REP-independent manner by geranylgeranyltransferase type I (GGTaseI). The present studies show that one such Rab protein (Rab8), which ends with a CVLL motif, is uniquely able to serve as a substrate for either REP/GGTaseII or GGTaseI in cell-free assays. The modification of Rab8 by GGTaseI did not require REP, indicating that a REP-induced conformational change is not essential for exposure of the Rab carboxyl-terminal cysteine prenylation site. To determine whether one enzyme plays a predominant role in Rab8 ...
The Journal of Organic Chemistry, 2000
The posttranslational addition of a farnesyl moiety to the Ras oncoprotein is essential for its membrane localization and is required for both its biological activity and ability to induce malignant transformation. We describe the design and synthesis of a farnesyl pyrophosphate (FPP) analogue, 8-anilinogeranyl pyrophosphate 3 (AGPP), in which the ω-terminal isoprene unit of the farnesyl group has been replaced with an aniline functionality. The key steps in the synthesis are the reductive amination of the R, -unsaturated aldehyde 5 to form the lipid analogue 6, and the subsequent conversion of the allylic alcohol 7 to the chloride 8 via Ph 3 PCl 2 followed by displacement with [(n-Bu) 4 N] 3 HP 2 O 7 to give AGPP (3). AGPP is a substrate for protein farnesyltransferase (FTase) and is transferred to Ras by FTase with the same kinetics as the natural substrate, FPP. AGPP is highly selective, showing little inhibitory activity against either geranylgeranyl-protein transferase type I (GGTase I) (K i ) 0.06 µM, IC 50 ) 20 µM) or squalene synthase (IC 50 ) 1000 µM). AGPP is the first efficiently transferable analogue of FPP to be modified at the ω-terminus that provides a platform from which additional analogues can be made to probe the biological function of protein farnesylation. AGPP is the first example of a class of compounds that are alternate substrates for protein isoprenylation that are not inhibitors of squalene synthase.