Novel reverse-turn mimics inhibit farnesyl transferase (original) (raw)
Preparation of a clinically investigated ras farnesyl transferase inhibitor
Journal of Heterocyclic Chemistry, 2003
The synthesis of ras farnesyl-protein transferase inhibitor 1 is described on a multi-kilogram scale. Retrosynthetic analysis reveals chloromethylimidazole 2 and a piperazinone 3 as viable precursors. The 1,5disubstituted imidazole system was regioselectively assembled via an improved Marckwald imidazole synthesis. A new imidazole dethionation procedure has been developed to convert the Marckwald mercaptoimidazole product to the desired imidazole. This methodology was found to be tolerant of a variety of functional groups providing good to excellent yields of 1,5-disubstituted imidazoles. A new Mitsunobu cyclization strategy was developed to prepare the arylpiperazinone fragment 3.
Farnesyl Diphosphate-Based Inhibitors of Ras Farnesyl Protein Transferase
Journal of Medicinal Chemistry, 1995
The rational design, synthesis, and biological activity of farnesyl diphosphate (FPP)-based inhibitors of the enzyme Ras farnesyl protein transferase (FPT) is described. Compound 3, wherein a ,&carboxylic phosphonic acid type pyrophosphate (PPI surrogate is connected to the hydrophobic farnesyl group by a n amide linker, was found to be a potent (150(FPT) = 75 nM) and selective inhibitor of FPT, as evidenced by its inferior activity against squalene synthetase (15dSS) = 516 pM) and mevalonate kinase (150(MK) = '200 pM). A systematic structureactivity relationship study involving modifications of the farnesyl group, the amide linker, and the PP surrogate of 3 was undertaken. Both the carboxylic and phosphonic acid groups of the P-carboxylic phosphonic acid PP surrogate are essential for activity, since deletion of either group results in 50-2600-fold loss in activity (6-9, 1 5 0 = 4.6-220 pM). The farnesyl group also displays very stringent requirements and does not tolerate one carbon homologation (12, 1 5 0 = 17.7 pM), substitution by a dodecyl fragment (14,150 = 9 pM), or introduction of a n extra methyl group at the allylic position (18,150 = 55 ,LAM). Modifications around the amide linker group of 3 were more forgiving, as evidenced by the activity ofN-methyl analog (21,150 = 0.53 pM), the one carbon atom shorter farnesoic acid-derived retroamide analog (32,150 = 250 nM), and the exact retroamide analog (49, 1 5 0 = 50 nM). FPP analogs such as 3, 32, and 49 are novel, potent, selective, small-sized, nonpeptidic inhibitors of FPT that may find utility as antitumor agents.
Hydroxamic Acid-Based Bisubstrate Analog Inhibitors of Ras Farnesyl Protein Transferase
Journal of Medicinal Chemistry, 1996
The rational design, synthesis, and activity of novel, hydroxamic acid-based, collective bisubstrate analog inhibitors of farnesyl protein transferase (FPT) is described. This class of compounds differ structurally from the conventional FPT inhibitors by being non-sulfhydryl and by being bisubstrate based rather than peptide or FPP derived inhibitors. Whereas replacement of the sulfhydryl group of tetrapeptide CVLS (I 50) 1 µM) by an N-methylhydroxamic acid had a deleterious effect (10, I 50 > 360 µM), moderate inhibition was realized with 16 (I 50) 42.5 µM), a bisubstrate analog involving anchorage of farnesyl and tripeptide groups by a hydroxamic acid-embedded linker. Starting from 16, a 1 order of magnitude improvement in in vitro potency was obtained by optimization of the linker (20, I 50) 4.35 µM). An additional 13-fold enhancement was achieved by substituting the tripeptide moiety VLS in 20 by VVM (23, I 50) 0.33 µM). The dependence of these inhibitors on their peptide and farnesyl subunits is suggestive of their bisubstrate nature. Compound 23 (I 50) 0.33 µM) is 2 orders of magnitude better in activity compared to the initial lead 16 (I 50) 42.5 µM) and is effective in blocking prenylation of protein in whole cells including p21 ras .
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...
Targeting of K-Ras 4B by S-trans,trans-farnesyl thiosalicylic acid
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1999
Ras proteins regulate cell growth, differentiation and apoptosis. Their activities depend on their anchorage to the inner surface of the plasma membrane, which is promoted by their common carboxy-terminal S-farnesylcysteine and either a stretch of lysine residues (K-Ras 4B) or S-palmitoyl moieties (H-Ras, N-Ras and K-Ras 4A). We previously demonstrated dislodgment of H-Ras from EJ cell membranes by S-trans,trans-farnesylthiosalicylic acid (FTS), and proposed that FTS disrupts the interactions between the S-prenyl moiety of Ras and the membrane anchorage domains. In support of this hypothesis, we now show that FTS, which is not a farnesyltransferase inhibitor, inhibits growth of NIH3T3 cells transformed by the non-palmitoylated K-Ras 4B(12V) or by its farnesylated, but unmethylated, K-Ras 4B(12) CVYM mutant. The growth-inhibitory effects of FTS followed the dislodgment and accelerated degradation of K-Ras 4B(12V), leading in turn to a decrease in its amount in the cells and inhibition of MAPK activity. FTS did not affect the rate of degradation of the K-Ras 4B, SVIM mutant which is not modified post-translationally, suggesting that only farnesylated Ras isoforms are substrates for facilitated degradation. The putative Ras-recognition sites (within domains in the cell membrane) appear to tolerate both C 15 and C 20 S-prenyl moeities, since geranylgeranyl thiosalicylic acid mimicked the growth-inhibitory effects of FTS in K-Ras 4B(12V)-transformed cells and FTS inhibited the growth of cells transformed by the geranylgeranylated K-Ras 4B(12V) CVIL isoform. The results suggest that FTS acts as a domain-targeted compound that disrupts Ras^membrane interactions. The fact that FTS can target K-Ras 4B(12V), which is insensitive to inhibition by farnesyltransfarase inhibitors, suggests that FTS may target Ras (and other prenylated proteins important for transformed cell growth) in an efficient manner that speaks well for its potential as an anticancer therapeutic agent. ß 1999 Elsevier Science B.V. All rights reserved.
Journal of Peptide Research, 1999
Conformation of a novel tetrapeptide inhibitor NH2-d-Trp-d-Met-Phe(pCl)-Gla-NH2 bound to farensyl-protein transferase. Abstract: Farnesyl-protein transferase (FPTase) catalyzes the posttranslational farnesylation of the cysteine residue located in the C-terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for membrane association and celltransforming activities of ras. Inhibitors of FPTase have been demonstrated to display antitumor activity in both tissue culture and animal models, and thus represent a potential therapeutic strategy for the treatment of human cancers. A synthetic tetrapeptide library, which included an expanded set of 68 L-, Dand noncoded amino acids, has been screened for inhibitors of FPTase activity. The tetrapeptide, NH 2 -D-Trp-D-Met-L-Phe(pCl)-L-Gla-NH 2 was shown to be competitive with the isoprenyl cosubstrate, farnesyl diphosphate (FPP) but not with the peptide substrate, the C-terminal tetrapeptide of the Ras protein. The FPTase-bound conformation of the inhibitor, NH 2 -D-Trp-D-Met-L-Phe(pCl)-L-Gla-NH 2 was determined by NMR spectroscopy. Distance constraints were derived from two-dimensional transferred nuclear Overhauser effect (TRNOE) experiments. Ligand competition experiments identi®ed the NOEs that originate from the activesite conformation of the inhibitor. Structures were calculated using a combination of distance geometry and restrained energy minimization. The peptide backbone is shown to adopt a reverseturn conformation most closely approximating a type II' b-turn. The resolved conformation of the inhibitor represents a distinctly different structural motif from that determined for Rascompetitive inhibitors. Knowledge of the bound conformation of this novel inhibitor provides a template and future direction for the design of new classes of FPTase antagonists. Abbreviations: FPP, farnesyl diphosphate; FPTase, farnesylprotein transferase; TRNOE, transferred nuclear Overhauser effect.
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.
Inhibition of farnesyltransferase with A-176120, a novel and potent farnesyl pyrophosphate analogue
European Journal of Cancer, 2000
Farnesylation of Ras is required for its transforming activity in human cancer and the reaction is catalysed by the enzyme farnesyltransferase. Recently, we discovered a novel chemical series of potent farnesyl pyrophosphate (FPP) analogues which selectively inhibited farnesyltransferase. Our most potent compound to date in this series, A-176120, selectively inhibited farnesyltransferase activity (IC 50 1.2AE0.3 nM) over the closely related enzymes geranylgeranyltransferase I (GGTaseI) (IC 50 423AE1.8 nM), geranylgeranyltransferase II (GGTaseII) (IC 50 3000 nM) and squalene synthase (SSase) (IC 50 >10 000 nM). A-176120 inhibited ras processing in H-ras-transformed NIH3T3 cells and HCT116 K-ras-mutated cells (ED 50 1.6 and 0.5 mM, respectively). The antiangiogenic potential of A-176120 was demonstrated by a decrease in Ras processing, cell proliferation and capillary structure formation of human umbilical vein endothelial cells (HUVEC), and a decrease in the secretion of vascular endothelial growth factor (VEGF) from HCT116 cells. In vivo, A-176120 reduced H-ras NIH3T3 tumour growth and extended the lifespan of nude mice inoculated with H-or K-ras-transformed NIH3T3 cells. A-176120 also had an additive eect in combination with cyclophosphamide in nude mice inoculated with K-ras NIH3T3 transformed cells. Overall, our results demonstrate that A-176120 is a potent FPP mimetic with both antitumour and anti-angiogenic properties.