©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Novel Tricyclic Inhibitors of Farnesyl Protein Transferase
BIOCHEMICAL CHARACTERIZATION AND INHIBITION OF Ras MODIFICATION IN TRANSFECTED Cos CELLS (*)

(Received for publication, May 23, 1995; and in revised form, October 16, 1995)

W. Robert Bishop (§) Richard Bond Joanne Petrin Lynn Wang Robert Patton Ronald Doll George Njoroge Joseph Catino Jerome Schwartz William Windsor Rosalinda Syto Jeffrey Schwartz Donna Carr Linda James Paul Kirschmeier

From the Departments of Molecular Pharmacology, Structural Chemistry, Fermentation, Synthetic Chemistry, and Tumor Biology, Schering-Plough Research Institute, Kenilworth, New Jersey 07033

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Ras protooncogenes encode 21-kDa membrane-associated guanine nucleotide-binding proteins, which play a critical role in control of cellular proliferation and differentiation. Oncogenic, activated forms of Ras proteins are associated with a broad range of human cancers. The elucidation of the post-translational modifications that occur at the carboxyl terminus of Ras and the demonstration that farnesylation of Ras by farnesyl protein transferase is essential for Ras-induced cellular transformation has opened up a new and promising approach to the development of anti-Ras therapeutics. We report here a novel series of potent farnesyl protein transferase (FPT) inhibitors, represented by SCH 44342. This compound inhibits both rat brain and recombinant human FPT with an IC of approximately 250 nM, while it is only weakly active against rat brain geranylgeranyl protein transferase-1 (IC > 114 µM). FPT inhibition has been observed using both Ha-Ras protein and Ki-Ras-derived peptide substrates in two different assay formats. SCH 44342 and its analogs also inhibit farnesylation of Ras in Cos cells transiently expressing [Val]Ha-Ras with IC values in the low micromolar range. At these concentrations they do not inhibit sterol biosynthesis or geranylgeranylation of protein. In addition, we observed that Cos cells undergo pronounced morphological changes upon overexpression of [Val]activated forms of Ha-Ras containing COOH-terminal sequences allowing farnesylation (CVLS) or geranylgeranylation (CVLL) but not upon overexpression of activated Ras lacking the isoprenylated Cys (SVLS). Ras-induced morphological changes can be partially reverted with lovastatin. Importantly, SCH 44342 can block morphological changes induced by [Val]Ha-Ras-CVLS but not [Val]Ha-Ras-CVLL. Recently, a number of other FPT inhibitors have been reported. Most of the compounds reported to have cell-based activity are peptidomimetic analogs of the CAAX substrate. Our FPT inhibitors are novel in that although they compete with Ras protein in kinetic experiments they are entirely nonpeptidic in nature, they do not have oxidizable sulfhydryl functions, and they are active in cells at low micromolar concentrations.


INTRODUCTION

Oncogenic forms of Ras proteins are associated with a broad range of human cancers including an estimated 90% of all colon cancers(1) . Ras proteins undergo a complex series of post-translational processing events, which have been defined over the past several years(2, 3) . The initial post-translational event is the transfer of the 15-carbon isoprene farnesyl from farnesyl pyrophosphate to a Cys residue (Cys in Ha-Ras) in the conserved carboxyl-terminal ``CAAX'' motif (where ``A'' is an aliphatic residue) present in all Ras proteins(4, 5) . Studies employing site-directed mutagenesis (6, 7) or inhibitors of hydroxymethylglutaryl-CoA reductase(8) , the rate-limiting enzyme in isoprenoid biosynthesis, demonstrated that isoprenylation is required for Ras proteins to become membrane-associated and to induce cellular transformation. The farnesyl protein transferase (FPT) (^1)that catalyzes this reaction has been purified (9) and cDNA clones for its alpha and beta subunits isolated(10, 11, 12) .

A number of other cellular proteins are also isoprenylated on a Cys residue near their COOH terminus(13, 14) . These include other substrates for FPT, such as the nuclear lamins(15) . However, the majority of cellular isoprenylated proteins are modified with geranylgeranyl, a 20-carbon isoprene. Two distinct geranylgeranyl protein transferases (GGPT I and II) have been identified (16, 17) and cDNA clones for their alpha and beta subunits isolated(18, 19) . GGPT I and FPT share a common alpha subunit(18, 20) .

The primary determinant for recognition of protein substrates by the isoprenyl transferases is the substrate's carboxyl-terminal amino acid sequence. Proteins ending in Cys-X-X-Ser (or Met) are preferred substrates for FPT, while proteins terminating in Cys-X-X-Leu are preferred substrates for GGPT I (21, 22) . Substitution of leucine for serine at the COOH terminus of the Ha-Ras CAAX box (Ser Leu) makes this protein a substrate for geranylgeranylation (rather than farnesylation) both in vitro and in cells(23) . The different substrate specificities of FPT and GGPT-1 are likely mediated by their distinct beta subunits. GGPT II utilizes protein substrates terminating in Cys-Cys or Cys-X-Cys(17, 24) .

A number of inhibitors of FPT have been reported over the past several years(25) . The design of CAAX peptidomimetics (26, 27, 28, 29) has resulted in potent and selective FPT inhibitors capable of blocking Ras processing in cells. These compounds have shown considerable promise as antitumor agents based on their ability to inhibit cellular transformation induced by oncogenic Ras proteins (26, 27) and the growth of Ras-dependent tumors in nude mice(30) .

We report here the discovery of a novel series of nonpeptide tricyclic FPT inhibitors active at submicromolar concentrations. SCH 44342 (Fig. 1) is a representative compound in this series. Although it is entirely nonpeptidic in nature and lacks a sulfhydryl function, SCH 44342 appears to compete with the Ras-CAAX substrate for binding to FPT. The biochemical characterization of these inhibitors and their effects on Cos cells transiently overexpressing activated Ha-Ras are described. Additional cell-based biological evaluation of these compounds will be published separately. (^2)These compounds are novel inhibitors of Ras farnesylation, which, due to their potent activity in cell-based assays, should serve as useful tools in understanding Ras signal transduction pathways.


Figure 1: Structure of SCH 44342. SCH 44342 is a representative compound in the tricyclic series of non-peptide inhibitors of FPT. These compounds appear to be competitive with the Ras p21 substrate.




EXPERIMENTAL PROCEDURES

Expression Constructs for Mutant Ras Proteins

Amino acids 186-189 (Cys-Val-Leu-Ser (CVLS)) of Ha-Ras serve as a recognition motif for FPT. A mutant form of Ras, which is a substrate for GGPT I, was constructed using site-directed mutagenesis, by converting Ser to Leu (Ras-CVLL). A second mutant form was constructed in which Cys is changed to Ser (Ras-SVLS); this protein is not isoprenylated. Coding sequences for full-length Ha-Ras were subcloned into the BamHI site of the pSelect vector (ProMega) and grown in Escherichia coli JM109. In all cases the Ras sequences contained an activating Gly Val mutation. Single-stranded DNA was isolated from cells after superinfection with the helper phage, R408. The single-stranded DNA contained the anticoding strand for Ras. Two mutagenic oligonucleotides designed to introduce the Ser Leu (AAGTGTGTGCTCCTGTGAGGATCCTC) and Cys Ser mutations (ATGAGCTGCAAGTCCGTGCTCTCCTAG) were synthesized and mutagenesis was done according to the manufacturer's (Promega) instructions. Mutations were confirmed by DNA sequencing.

E. coli Expression and Purification of Ha-Ras p21 Proteins

cDNAs encoding [Val]Ha-Ras-CVLS and [Val]Ha-Ras-CVLL were subcloned into the E. coli expression vector QE-9 (Qiagen) at a BamHI site. These constructs encode an additional 11 amino acids at their NH(2) terminus, including a stretch of 6 consecutive histidines (His(6)-Ha-Ras-CVLS and His(6)-Ha-Ras-CVLL). Orientation and reading frame were confirmed by DNA sequencing. Vectors were introduced into the SURE strain of E. coli (Stratagene), and cultures were grown overnight in LB broth. Isopropyl-1-thio-betaD-galactopyranoside was added to a final concentration of 1 mM for 1 h prior to harvesting cells by centrifugation. Cells were resuspended in 200 ml of Buffer A (60 mM Tris-HCl, pH 7.5, 10 mM MgCl, and 10 mM beta-mercaptoethanol) and the protease inhibitors PMSF, leupeptin, and benzamidine were added at 0.2 mM, 100 µg/ml, and 10 mM respectively. The suspension was passed through a Microfluidizer (model 110S) at 18,500 p.s.i. to break the cells, and the insoluble debris was removed by centrifugation at 15,000 rpm in an SS-34 rotor (Sorvall) for 20 min at 4 °C. The cleared lysate was passed over a Ni chelate affinity resin. The column was washed with Buffer A and then with Buffer A containing 1 M NaCl until the A was < 0.1 OD units followed by another wash with 5 column volumes of Buffer A. Ras proteins were eluted with Buffer A containing 250 mM imidazole. Fractions containing the eluted protein were passed over PD10 gel filtration columns (Bio-Rad) in Buffer A to remove the imidazole. The final yield of protein is approximately 20 mg of Ras/liter of culture.

Expression of Recombinant Human FPT

cDNA clones for human FPT alpha and beta subunits were obtained from the ATCC (ATCC 63225 and 63226, respectively). The 5` end of the beta clone was completed by ligating DNA into the BbsI site following its amplification by polymerase chain reaction from human brain cortex cDNA (Quick-Clone from Clontech). The FPT alpha cDNA was cut out with BamHI and PpuMI and ligated into p2-BAC (Invitrogen) cut with BamHI and NcoI to produce pA2BN. The completed beta cDNA was cut out with NotI and ApaI and ligated into pA2BN (cut with the same enzymes) to produce pABC-2B. This construct was used to produce recombinant baculovirus according to Summers et al.(31) . For FPT production, log phase Sf(9) cells (2 times 10^6/ml) were infected at a multiplicity of infection of 1.5 in a 10-liter Biolafitte tank and cultured for 3 days at 28 °C and 140 rpm in SF900-II medium (Life Technologies, Inc.). Cells were harvested by centrifugation at 12,000 times g for 10 min at 4 °C.

For E. coli expression, the FPT alpha and beta coding sequences were expressed co-cistronically from the trc promoter of the pTrcHisA vector (Invitrogen) lacking the His(6) tag. The alpha sequence was ligated between the NcoI and BglII sites of pTrcHisA to produce pTrcFTA. After the addition of a ribosome binding site immediately upstream of the FPT beta initiator ATG, this sequence was ligated between the EcoRI and HindIII sites of pTrcFTA to produce pTrcFTAB. Topp 1 E. coli cells (Stratagene) were transformed with pTrcFTAB and grown at 30 °C in M9 medium + 0.16% yeast extract. When the cells reached an OD of 0.5, FPT expression was induced by the addition of 0.1 mM isopropyl-1-thio-betaD-galactopyranoside. After 4 h the cells were harvested, resuspended in 1/100 volumes of homogenization buffer and lysed in a Microfluidizer at 18,500 p.s.i. Insoluble debris was removed by centrifugation at 12,000 times g. The alpha and beta subunits were also expressed in E. coli DH5a cells (Life Technologies, Inc.) from separate plasmids.

A truncated form of the FPT alpha subunit (Delta 2-41) was produced by deleting the region between the EagI and NcoI sites in pTrcFTAB. The EagI overhang was degraded with mung bean nuclease and the NcoI overhang was filled in with the Klenow fragment of E. coli DNA polymerase I and the two blunt ends were ligated to produce pTRAB.

Purification of FPT

For initial studies, both FPT and GGPT-1 were partially purified from rat brain by ammonium sulfate fractionation followed by Q-Sepharose anion exchange chromatography essentially as described(16) .

Soluble Sf(9)-derived human FPT was extracted at 4 °C from a 300-g cell pellet by homogenization in 1200 ml of 50 mM Tris pH 7.5, 2 mM dithiothreitol (DTT), 1 mM EDTA, 1 mM EGTA, 0.1 mM leupeptin, and 0.2 mM PMSF followed by cell lysis using a Microfluidizer at 18,500 p.s.i. Insoluble debris was removed by centrifugation for 1 h at 4 °C and the supernatant (8.3 g total protein) was loaded onto a 1-liter QAE-Sepharose Fast Flow column (Pharmacia Biotech Inc.) equilibrated in 20 mM Tris, pH 7.5, 0.1 M NaCl, 20 µM ZnCl(2), 2 mM DTT (Buffer A). The column was washed with 10 bed volumes of Buffer A, and FPT was eluted (1.1 g total protein) with a linear gradient of 0.1-0.6 M NaCl in Buffer A. An additional chromatography step using a smaller (180 ml) QAE-Sepharose column and a 0.15-0.5 M NaCl gradient in Buffer A provided further purification and excellent recovery. Fractions containing FPT activity (370 mg of total protein) were adjusted to 0.75 M NaCl and loaded onto a phenyl-Sepharose column in the absence of ZnCl(2). Elution was performed with a linear gradient from 0.75 M to 0 M NaCl in 5 mM Tris, pH 7.1, 1 mM DTT. At this step, peak FPT fractions (50 mg of total protein) were greater than 90% pure and could be further enriched by size exclusion chromatography. NH(2)-terminal amino acid sequencing of the alpha subunit identified the expected sequence (Ala-Ala-Thr-Glu-Gly) lacking the initiator methionine. The NH(2) terminus of the beta subunit appeared to be blocked, since no sequence could be obtained. Protein concentrations of purified FPT were obtained by amino acid analysis.

A much lower expression of soluble human FPT was obtained in E. coli. Purification from this system using the same method described above resulted in a partially (approx50%) purified sample.

In Vitro Enzyme Assays of FPT and GGPT-1

FPT activity was determined by measuring transfer of [^3H]farnesyl from [^3H]farnesyl pyrophosphate to trichloroacetic acid-precipitable His(6)-Ha-Ras-CVLS similar to the method described by Reiss et al.(9) . Typical reaction mixtures (100 µl total) contained 40 mM Hepes, pH 7.5, 20 mM MgCl(2), 5 mM DTT, 90-250 nM [^3H]farnesyl pyrophosphate (DuPont NEN; 20 Ci/mmol), 25-50 ng (approximately 2.5-5 nM) of purified enzyme or 10 µl of partially purified Q-Sepharose-derived FPT, the indicated concentration of SCH 44342 or dimethyl sulfoxide (Me(2)SO) vehicle control (5%, v/v, final), and 5 µg (approximately 2.5 µM) Ha-Ras-CVLS. After a 30-min incubation at room temperature reactions were stopped with 0.5 ml of 4% sodium dodecyl sulfate (SDS) followed by 0.5 ml of cold 30% trichloroacetic acid. Samples were allowed to sit on ice for 45 min, and precipitated protein was collected on GF/C filter paper mats using a Brandel cell harvester. Filter mats were washed once with 6% trichloroacetic acid, 2% SDS and radioactivity was measured in a Wallac 1204 Betaplate BS liquid scintillation counter. Percent inhibition was calculated relative to the Me(2)SO vehicle control.

Alternatively, FPT assays were done using a scintillation proximity assay kit following the protocol described by the manufacturer (Amersham Corp.) except that a biotinylated substrate peptide containing the Ki-Ras carboxyl-terminal sequence was used. The Ki-Ras peptide (Analytical Biotechnology Services, Boston, MA) has the sequence biotin-KKSKTKCVIM and was typically used at 50-100 nM. Farnesyl pyrophosphate was typically present at 90-100 nM. Other reaction conditions were as described above for the trichloroacetic acid precipitation assay. Kinetic parameters were determined by nonlinear regression analysis using the program k.Cat (BioMetallics, Inc.).

The GGPT-1 assay was essentially identical to the trichloroacetic acid precipitation assay for FPT described above, with two exceptions; [^3H]geranylgeranyl pyrophosphate (220-250 nM) replaced farnesyl pyrophosphate as the isoprenoid donor, and Ha-Ras-CVLL (3.6 µg/100 µl reaction) was the protein acceptor, similar to the method reported by Casey et al.(32) .

Transient Expression and Processing of Val-Ha-Ras-CVLS and Val-Ha-Ras-CVLL in Cos Monkey Kidney Cells

cDNAs encoding Ras with the three carboxyl-terminal sequences (-CVLS, -CVLL, and -SVLS) were subcloned into the mammalian expression vector pSV-Sport (Life Technologies, Inc.), which contains both the SV40 promoter sequences and the SV40 origin of replication. Cos-7 monkey kidney cells were transfected by electroporation with 23 µg of the various plasmids. Following electroporation, cells were plated into six-well tissue culture dishes containing 1.5 ml of Dulbecco's-modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum and the appropriate FPT inhibitors. After 24 h, medium was removed and fresh medium containing the appropriate drugs was re-added.

48 h after electroporation, cells were examined microscopically to monitor morphological changes and photographed. Cells were then washed twice with 1 ml of cold phosphate-buffered saline (PBS) and scraped from the dish into 1 ml of buffer containing 25 mM Tris, pH 8.0, 1 mM EDTA, 1 mM PMSF, 50 µM leupeptin, and 0.1 µM pepstatin. Cells were lysed by homogenization, and debris was removed by centrifugation at 2000 times g for 10 min.

Protein was precipitated from lysates by addition of ice-cold trichloroacetic acid and redissolved in 100 µl of SDS-electrophoresis sample buffer. Samples (5-10 µl) were loaded onto 14% polyacrylamide gels (Novex, Inc.) and electrophoresed until the tracking dye neared the bottom of the gel. Resolved proteins were electroblotted onto nitrocellulose membranes. Membranes were blocked by incubation overnight at 4 °C in PBS containing 2.5% dried milk and 0.5% Tween 20 and then incubated with a Ras-specific monoclonal antibody (Y13-259) in PBS containing 1% fetal calf serum for 1 h at room temperature. After washing, membranes were incubated for 1 h at room temperature with a 1:5000 dilution of rabbit anti-rat IgG conjugated to horseradish peroxidase in PBS containing 1% fetal calf serum. Ras proteins were visualized using a colorimetric peroxidase reagent (4-chloro-1-naphthol; Bio-Rad).

In some experiments Cos cells were labeled with [S]methionine by washing twice with methionine-free DMEM followed by growth for 1 h in this medium containing 5% dialyzed fetal bovine serum to deplete endogenous methionine. Subsequently, cells were labeled in this medium (in the presence or absence of FPT inhibitor) containing 100 µCi/ml [S]methionine (1000 Ci/mmol, Amersham) for 24 h. Chase experiments were performed for various times in DMEM containing 10% fetal bovine serum and 5 mM unlabeled methionine. Cells were lysed by sonication in 400 µl of 100 mM Tris, pH 8.3, 0.5% SDS, 2 mM EDTA, 100 µg/ml leupeptin, and 1 mM PMSF. Deoxycholate and Nonidet P-40 were added to a final concentration of 0.5%, and aprotinin was added to 1 mg/ml final. Lysates were incubated with 50 µl (50 µg) of Y13-259 agarose (Oncogene Science) for 18 h at 4 °C. The pelleted agarose beads were washed four times with buffer (1 ml each), and the final pellet resuspended in 40 µl of SDS-polyacrylamide gel electrophoresis buffer. Processed and unprocessed Ras were separated as described above, and nitrocellulose membranes were exposed to x-ray film for 1-4 days prior to development.

Incorporation of [^14C]Acetate into Sterols

NIH 3T3 cells were plated into six-well cluster dishes in DMEM containing 10% calf serum. The following day the medium was changed to DMEM containing 0.5% calf serum, 50 µCi/ml 2-[^14C]acetate (DuPont NEN, 57 mCi/mmol) and the indicated concentration of SCH 44342. After 18 h, cells were washed once with PBS, then scraped into 2 ml of cold methanol. Cells were extracted according to the method of Bligh and Dyer(33) , and a portion of the CHCl(3) phase was subjected to mild alkaline deacylation in methanolic NaOH(34) . The final chloroform phase was analyzed by thin layer chromatography on silica gel 60 F-254 plates developed in petroleum ether/diethyl ether/acetic acid (80:20:1). TLC plates were autoradiographed, and spots corresponding to sterol standard (R(F) ranged from 0.10 to 0.19) were scraped and quantified by liquid scintillation spectrometry.


RESULTS

Expression and Characterization of Human FPT

Previous reports have described expression of FPT in both E. coli(12) and baculovirus/Sf9 (35) expression systems. We expressed the subunits of human FPT in E. coli both from separate plasmids and using a co-cistronic expression plasmid. In either case, the expressed FPT was highly (>95%) insoluble. We also coexpressed the beta subunit with an amino-terminal truncated form of the alpha subunit (Delta41alpha). Expression of soluble FPT enzymatic activity in E. coli was 10-30-fold higher with this construct, and we estimate expression to be about 1 mg of soluble FPT/liter. Most of our work has focused on full-length human FPT expressed in Sf9 cells from the polyhedrin and P10 promoters of a dual expression transfer vector. Using this system we have achieved purification to >95% purity of 5-10 mg of FPT/liter of Sf9 cells. This is similar to the levels reported by Chen et al.(35) .

Highly purified FPT from Sf9 cells typically displayed V(max) values of 132-150 nmol/h/mg in the trichloroacetic acid precipitation assay, corresponding to k values of 0.0038-0.0042/s. This is somewhat lower than the value reported by Omer et al.(12) for E. coli expressed enzyme (0.0096-0.013/s). This enzyme had a mean K(m)(app) for His(6)-Ha-Ras-CVLS of 10.9 ± 2.0 µM. This relatively high K(m) is similar to that reported by others(9, 36) . Omer et al.(12) recently reported K(m) values for Ras-CVLS in the 300-400 nM range. They suggested that higher values may reflect the presence of forms of Ras lacking a complete carboxyl terminus in the E. coli-derived preparations(36) . In the scintillation proximity assay, the K(m)(app) were 11.1 nM for farnesyl pyrophosphate and between 150 and 220 nM for the Ki-Ras peptide. This value for the Ki-Ras peptide is similar to previously reported values for Saccharomyces cerevisiae RAS1 protein terminating in the Ki-Ras CAAX sequence (CVIM)(12) . IC determinations for FPT inhibitors were performed at nonsaturating concentrations of the protein/peptide substrate.

SCH 44342 Inhibits Farnesylation of Ras in Vitro

During the course of our efforts to develop inhibitors of Ras farnesyl transferase, we discovered a series of compounds active at nanomolar to low micromolar concentrations. These compounds possess a substituted tricyclic ring system and are represented by SCH 44342 (Fig. 1). SCH 44342 inhibited rat brain FPT with an IC value of 0.25 µM in the experiment shown (Fig. 2). The IC values determined for this compound, and other tricyclics were very similar versus recombinant human or rat brain-derived FPT and in either of the assay formats described. SCH 44342 inhibits human FPT with an IC of 0.28 ± 0.03 µM. These compounds are structurally related to tricyclic compounds possessing histamine H1 and platelet activating factor antagonist activity(37) ; however, the FPT inhibitory activity is clearly separable from these other activities. For example, a related tricyclic antihistamine cyproheptadine lacks significant FPT activity (IC > 500 µM).


Figure 2: Inhibition of isoprenyl protein transferases by SCH 44342. FPT and GGPT-1 assays were performed using the trichloroacetic acid precipitation method described under ``Experimental Procedures'' with Ras-CVLS or Ras-CVLL as isoprene acceptor, respectively. Enzymes used in this experiment were partially purified from rat brain as described. The concentration of farnesyl pyrophosphate employed was 250 nM. Increasing concentrations of SCH 44342 were included in the assay, and data are calculated as percent inhibition relative to the 5% Me(2)SO control. box, IC = 0.25 µM; , IC > 114 µM.



To evaluate the selectivity of these compounds, we examined their inhibitory activity against rat brain geranylgeranyl transferase I, measured by incorporation of [^3H]geranylgeranyl into Ha-Ras-CVLL. Most of the tricyclic FPT inhibitors tested were weakly active or inactive as GGPT-1 inhibitors. For example, SCH 44342 has little or no inhibitory activity against GGPT-1 up to 50 µg/ml (114 µM) (Fig. 2). Therefore, this compound displays at least 400-fold selectivity in vitro for inhibition of FPT. Other analogs in the series display a minimum of 3,000-fold selectivity.

We performed kinetic analysis of FPT inhibition by SCH 44342. Fig. 3shows the results of an experiment in which the concentration of the Ki-Ras peptide was varied in the scintillation proximity assay and the farnesyl pyrophosphate concentration was kept constant. The mechanism of inhibition of highly purified SF9-derived human FPT appears to be primarily competitive with respect to the farnesyl acceptor. Similar results were observed using the trichloroacetic acid precipitation assay format (with full-length Ha-Ras substrate) and rat brain enzyme (data not shown). From plots of the slope of the double-reciprocal lines versus inhibitor concentration, the K(i) value for SCH 44342 inhibition of FPT was calculated to be 0.24 µM in the experiment shown (ranged from 0.10 to 0.24 µM in three independent experiments). Experiments where the farnesyl pyrophosphate concentration was varied at a fixed concentration of Ha-Ras-CVLS in the trichloroacetic acid assay displayed a complex kinetic pattern, clearly not competitive with the isoprenoid donor (data not shown).


Figure 3: SCH 44342 competes with Ras-CVLS for binding to FPT. FPT assays were performed using recombinant human FPT expressed in Sf9 cells and the scintillation proximity assay method described under ``Experimental Procedures.'' The concentration of the Ki-Ras-derived peptide (biotin-KKSKTKCVIM) was varied, while the farnesyl pyrophosphate concentration was held constant at 90 nM. Double-reciprocal plots of 1/reaction rate versus 1/[Ras-CVLS] are plotted at various concentrations of SCH 44342. The concentrations of SCH 44342 used were 0 (bullet), 0.12 (circle), and 0.4 () µM. Curve fitting was done by nonlinear regression analysis.



SCH 44342 Blocks Processing of Ras-CVLS in Cos Cells

To examine the effect of SCH 44342 on Ras processing in whole cells, we used Cos-7 monkey kidney cells transiently expressing either Ha-Ras-[Val]CVLS or Ha-Ras-[Val]CVLL. Processed and unprocessed Ras proteins were resolved by SDS-polyacrylamide gel electrophoresis, detected by immunoblotting and quantitated by densitometry. Endogenous Cos cell Ras protein is undetectable by this method; therefore, the signal arises only from newly synthesized Ras. The faster migrating immunoreactive band represents mature fully processed Ras, while the slower migrating form is unprocessed protein. In the experiment shown, Me(2)SO-treated control Cos cells express primarily mature Ras-CVLS protein (Fig. 4). We have consistently found that in control Cos cells not all of the overexpressed Ras protein is isoprenylated, the extent of processing ranging from 50 to approximately 90%, most likely due to high expression levels exceeding the capacity of the processing machinery. When Cos cells are treated with lovastatin (20 µM) for the 48 h following electroporation, all of the Ras-CVLS accumulates as unprocessed upper band (data not shown). Treatment of Cos cells with SCH 44342 (1-20 µM) results in a dose-dependent inhibition of processing. At 5 µM greater than 95% of the overexpressed Ras is present as precursor (lane 3). The IC value for SCH 44342 inhibition of Ras processing was 3.0 µM in this experiment (range 1.0-3.0 µM in three experiments).


Figure 4: SCH 44342 inhibits Ras-CVLS processing in Cos cells in a dose-dependent manner. Cos cells were electroporated and then cultured in the presence of either 0.5% Me(2)SO or the indicated concentration of SCH 44342. Cells were harvested after 2 days and lysed, and Ras was detected by immunoblotting. The amount of processed and unprocessed Ras was quantified by densitometry.



To confirm the identity of the two electrophoretic forms of Ras, we used Triton X-114 partitioning of cell lysates followed by immunoblotting.Using this method, isoprenylated proteins partition into the detergent phase, while unprocessed proteins are recovered almost exclusively in the aqueous phase(39) . In cells treated with either 20 µM lovastatin or 5 µg/ml (11.5 µM), SCH 44342 nearly all of the overexpressed Ras is recovered in the aqueous phase as the slower migrating protein (data not shown). These results further demonstrate that SCH 44342 inhibits farnesylation of Ras-CVLS in cells. Similar experiments were performed in Cos cells expressing [Val]Ha-Ras-CVLL. At concentrations up to 11.5 µM (the highest concentration tested), SCH 44342 did not inhibit Ras-CVLL processing, indicating that the in vitro specificity we observe carries over into the cell-based processing assay.

[S]Methionine labeling experiments followed by Ras immunoprecipitations were performed to examine the fate of Ras precursor when it accumulates in the presence of SCH 44342 (data not shown). These studies indicated that both mature Ras (pulse-chase done in the absence of SCH 44342) and precursor Ras (pulse-chase done in the presence of SCH 44342) turnover with similar t of 16 and 17 h, respectively. In addition, when precursor Ras was pulse-labeled in the presence of SCH 44342 and drug was removed during the chase period conversion of precursor to mature Ras was observed in addition to turnover, suggesting that at least some of the precursor that accumulates in the presence of an FPT inhibitor can serve as a pool of substrate when inhibitor levels drop.

Farnesylation Inhibitors Block Disordered Growth of Cos Cells Induced by Ras-CVLS

Cos cells typically have a flat morphology with little overgrowth of the monolayer at confluence. This morphology is retained by cells overexpressing a Val form of Ras-SVLS, a form of Ras which is not isoprenylated (Fig. 5A). In contrast, Cos cells overexpressing Val forms of Ras-CVLS or Ras-CVLL become more rounded and refractile and overgrow the monolayer, reminiscent of the phenotype observed in Ras-transformed cells (Fig. 5, B and C). Treatment of Cos cells that are overexpressing Val -Ras-CVLS or Ras-CVLL with 20 µM lovastatin reduces the morphological changes induced by either farnesylated or geranylgeranylated Ras (Fig. 6, B and C). This concentration of lovastatin, however, is somewhat cytotoxic, as indicated by detachment of cells from the dish and induction of a more spindle-shaped morphology in the remaining cells.


Figure 5: Morphological changes induced by expression of Val-Ha-Ras-CVLS or CVLL (but not SVLS) in Cos cells. Logarithmically growing Cos cells were electroporated with the vector pSV Sport containing one of three forms of Ha-Ras: A, Ras-SVLS (nonprenylated); B, Ras-CVLS (farnesylated); C, Ras-CVLL (geranylgeranylated). In these experiments the cells were incubated with 0.5% Me(2)SO as vehicle control.




Figure 6: Lovastatin partially reverts the morphological change induced by Ras-CVLS or Ras-CVLL. Cos cells were electroporated as in Fig. 5. A, cells expressing nonprenylated Ras protein, Ras-SVLS. B, cells expressing Ras-CVLS, grown in the presence of 20 µM lovastatin. C, cells expressing geranylgeranylated Ras (Ras CVLL) grown in the presence of 20 µM lovastatin.



In contrast, SCH 44342 suppresses the morphological changes observed in cells overexpressing [Val]Ras-CVLS but not in cells overexpressing [Val]Ras-CVLL (Fig. 7, B and C). Ras-CVLS-overexpressing cells grown in the presence of 20 µg/ml (45 µM) SCH 44342 appear morphologically similar to the Ras-SVLS or untransfected controls. Suppression of Ras-CVLS morphological effects was also observed at both 5 and 2 µg/ml (11.5 and 4.6 µM, respectively); at 0.5 µg/ml suppression was incomplete. In agreement with the specificity observed in the enzyme assays and the Cos processing assays, Ras-CVLL-overexpressing cells treated with 20 µg/ml SCH 44342 appear indistinguishable from their Me(2)SO-treated counterparts.


Figure 7: SCH 44342 reverts the morphological change induced by Ras-CVLS, but not Ras-CVLL in Cos cells. Cos cells were electroporated as in Fig. 5. A, cells expressing the nonprenylated Ras-SVLS control. B, cells expressing farnesylated Ras-CVLS grown in the presence of 20 µg/ml SCH 44342. C, cells expressing geranylgeranylated Ras-CVLL grown in the presence of 20 µg/ml SCH 44342.



It is important to note that SCH 44342 and its analogs had no apparent cytotoxic effects on Cos cells as the concentrations employed. This is indicated by a lack of inhibitory effect on the amount of total immunoreactive Ha-Ras-CVLS protein synthesized (Fig. 4). In fact, we have often observed an increase in the amount of total immunoreactive Ras recovered from drug-treated cells; the reason for this is not known. A lack of cytotoxicity is also indicated by the finding that up to 30 µM SCH 44342 has no effect on incorporation of [^14C]acetate into sterols in NIH 3T3 fibroblasts (Fig. 8). This concentration is 10 times the IC for inhibition of Ha-Ras processing. Similar results have been seen with related tricyclic inhibitors and in other cell types (Cos-7 and Rat-6 fibroblasts). At higher concentrations a reduction in [^14C]acetate incorporation into sterols was observed (about 50% at 50 µM SCH 44342).


Figure 8: Effect of SCH 44342 on incorporation of 2-[^14C]acetate into sterols. NIH 3T3 cells were labeled with 2-[^14C]acetate as described under ``Experimental Procedures.'' Cells were extracted; extracts were subject to mild alkaline methanolysis and analyzed for incorporation of [^14C]acetate into sterols by TLC. Data shown are mean cpm ± range of duplicate determinations. Similar results were obtained in three experiments.




DISCUSSION

Inhibitors of the function of oncogenic Ras proteins may have utility in the treatment of human cancers and of benign disorders in which Ras is implicated(2) . The elucidation of the pathway by which Ras is posttranslationally modified and the isolation and cloning of the enzymes responsible for this modification have opened up a new and promising approach to the development of anti-Ras therapeutics.

This report describes a novel, potent class of Ras farnesyl transferase inhibitors that inhibit enzymatic activity in vitro with submicromolar potency and block Ha-Ras processing in cells in the low micromolar range. Our results suggest that SCH 44342 inhibits FPT primarily by competition with the acceptor protein. These compounds do not compete with Ras-CVLL, an engineered form of Ras possessing a single amino acid change (Ser Leu), for binding to GGPT-1. This selectivity indicates that the compounds most likely interact with the distinct beta subunit of FPT, which is thought to be responsible for recognition of the acceptor peptide, rather than with the alpha subunit, which is common to both transferases(18, 20) . It will be of great interest to perform molecular modeling of these compounds with respect to CAAX peptides and to directly examine the physical interaction of these inhibitors with FPT. Preliminary experiments indicate that direct binding of the tricyclic inhibitors to the purified enzyme can be monitored by a decrease in intrinsic fluorescence of FPT. (^3)

One prediction that arises from the competitive mechanism is that the potency of these compounds in culture or in vivo may be modulated by the Ras expression level. The ability of these and other FPT inhibitors to block processing and transformation by oncogenic Ras proteins may also be affected by the specific isoform (Ha, Ki, or N) of Ras expressed. As noted above and previously reported(40) , Ki-Ras and Ki-Ras-derived peptides are higher affinity substrates than Ha-Ras and competitive inhibitors may be less effective against the higher affinity substrate when expressed at comparable levels. In our in vitro assays, we observe similar potency using either Ki-Ras peptide or Ha-Ras protein as farnesyl acceptor since both substrates were present at or below their K(m). We are currently exploring the relative efficacy of these compounds for inhibition of Ha- and Ki-Ras processing and function in intact cells.

An early approach to the prevention of Ras isoprenylation was to use inhibitors of isoprene biosynthesis, such as lovastatin, to block farnesyl pyrophosphate biosynthesis(8) . In addition to serving as a farnesyl donor for protein modification, farnesyl pyrophosphate is a precursor to geranylgeranyl pyrophosphate, sterols, and other isoprenoids. Therefore, blocking farnesyl pyrophosphate biosynthesis will have numerous repercussions on cellular metabolism. This can be seen, for example, in our Cos cell results where lovastatin was equally effective at blocking processing and morphological changes induced by Ras-CVLL and Ras-CVLS. In contrast, the tricyclic FPT inhibitors have no effect on either processing of or cellular responses to Ras-CVLL, indicating that the in vitro selectivity carries over into our cell-based assays. By depleting intracellular pools of isoprene units, lovastatin and other inhibitors of hydroxymethylglutaryl-CoA reductase also inhibit cellular sterol biosynthesis. Experiments using [^14C]acetate to biosynthetically label cellular sterol pools showed that SCH 44342 and other tricyclic FPT inhibitors do not inhibit sterol biosynthesis at concentrations up to 30 µM in Cos cells, providing another indication of the selectivity and lack of cytotoxicity of this class of compounds.

Recently, a number of potent peptidomimetic FPT inhibitors have been reported that are capable of blocking Ras processing in cells(26, 27, 28, 29) . SCH 44342 and analogs are novel in that they are entirely nonpeptidic in nature and lack a free sulfhydryl moiety. These features may impart favorable stability and pharmacokinetic properties on these compounds. This may be reflected in the observation that the IC values for tricyclic inhibitors in cell-based assays of Ras processing are in the range of 5-20 times greater then their IC values in the enzymatic assays. This is in contrast to many of the published peptidomimetics which, despite having greater intrinsic potency in some cases, have ICs in cell-based assays that are 200-2,000 times higher(26, 27, 28) . In addition, in some reports reducing agent is employed during cell-based assays to maintain the free sulfhydryl form of the peptidomimetic(26) . This is not the case with the the tricyclic compounds described here.

A number of questions remain concerning the effects of FPT inhibition by SCH 44342 and its analogs on cellular function. What will be the effect on other farnesylated cellular proteins including nuclear lamin B and prelamin A? Will it prove possible to selectively interfere with oncogenic (versus normal) Ras function and what are the implications of inhibition of cellular Ras? SCH 44342 analogs and other FPT inhibitors (see, e.g., (41) and (42) ) seem to be relatively free of adverse effects on normal cellular function. The lack of cytotoxicity of SCH 44342 is indicated by (i) morphological examination of cells, (ii) the level of expression of protein from transfected cDNAs, and (iii) the rate of cellular sterol biosynthesis. In addition SCH 44342 and analogs have no adverse effects on the ability of subconfluent fibroblasts to grow on plastic^2 or the ability of platelet-derived growth factor to activate MAP kinase in NIH 3T3 cells. (^4)Similar results have been observed by James et al.(42) using the Genentech benzodiazepene FPT inhibitor, BZA-5B. Critical to our understanding of the biological consequences of FPT inhibition is definition of the by-pass mechanism by which normal cell functions are spared.

Additional questions remain concerning the pharmacokinetic properties that will be necessary to achieve antitumor efficacy with FPT inhibitors like SCH 44342. The S-Ras labeling studies described here indicated that precursor Ras accumulates in Cos cells grown in the presence of SCH 44342 and that both the mature and precursor forms of Ras turnover with similar t values (16 and 17 h, respectively), suggesting that turnover is unaffected by prenylation in the Cos overexpression system. This is in contrast to an earlier report that unprocessed forms of Ras may be relatively unstable, since they were not found to accumulate substantially in the presence of another FPT inhibitor(43) . The accumulation of precursor has two implications. First, if a lower affinity alternative processing pathway (e.g. geranylgeranylation) exists(40) , expansion of the precursor pools in the presence of an FPT inhibitor may make this pathway more efficient. Second, our pulse-chase results suggest precursor that accumulates in the presence of an FPT inhibitor can subsequently be farnesylated upon removal of the block. Repko and Maltese (44) also found accumulation of nonisoprenylated precursor proteins that could subsequently be isoprenylated in cells grown in the presence of lovastatin. These findings suggest that levels of FPT inhibitors in cell culture and in tumors must be maintained in order to prevent later processing of accumulated Ras. The reversible, cytostatic nature of these compounds will pose additional challenges in finding clinical application for FPT inhibitors.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Pharmacology, Schering-Plough Research Institute, 2015 Galloping Hill Rd., Kenilworth, NJ 07033. Tel.: 908-298-3050; Fax: 908-298-3918.

(^1)
The abbreviations used are: FPT, farnesyl protein transferase; GGPT, geranylgeranyl protein transferase; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; PBS, phosphate-buffered saline.

(^2)
P. Kirschmeier, L. Armstrong, L. James, D. Carr, F. Zhang, R. Doll, G. Njoroge, M. Sinensky, M. Dalton, and W. R. Bishop, manuscript in preparation.

(^3)
W. Windsor, unpublished data.

(^4)
W. R. Bishop, D. Carr, L. James, E. Frank, and P. Kirschmeier, unpublished results.


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