(Received for publication, May 23, 1995; and in revised form, October 16, 1995)
From the
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.
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) (
)that catalyzes this reaction has
been purified (9) and cDNA clones for its
and
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 and
subunits isolated(18, 19) . GGPT I and FPT share a
common
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
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. ()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.
For E. coli expression, the FPT and
coding sequences were
expressed co-cistronically from the trc promoter of the
pTrcHisA vector (Invitrogen) lacking the His
tag. The
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
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-
D-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
g. The
and
subunits were also expressed in E.
coli DH5a cells (Life Technologies, Inc.) from separate plasmids.
A truncated form of the FPT subunit (
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.
Soluble
Sf-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 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
. 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
-terminal amino acid
sequencing of the
subunit identified the expected sequence
(Ala-Ala-Thr-Glu-Gly) lacking the initiator methionine. The NH
terminus of the
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
(50%) purified sample.
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;
[H]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) .
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 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.
Highly purified FPT from Sf9 cells
typically displayed V 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
(app) for His
-Ha-Ras-CVLS of 10.9
± 2.0 µM. This relatively high K
is similar to that reported by others(9, 36) .
Omer et al.(12) recently reported K
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
(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.
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% MeSO
control.
, 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
[H]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 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 (), 0.12 (
), and 0.4 (
) µM. Curve
fitting was done by nonlinear regression
analysis.
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% MeSO 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.
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
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
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 [C]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 [
C]acetate
incorporation into sterols was observed (about 50% at 50 µM SCH 44342).
Figure 8:
Effect of SCH 44342 on incorporation of
2-[C]acetate into sterols. NIH 3T3 cells were
labeled with 2-[
C]acetate as described under
``Experimental Procedures.'' Cells were extracted; extracts
were subject to mild alkaline methanolysis and analyzed for
incorporation of [
C]acetate into sterols by TLC.
Data shown are mean cpm ± range of duplicate determinations.
Similar results were obtained in three
experiments.
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
subunit of FPT, which is thought to be
responsible for recognition of the acceptor peptide, rather than with
the
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. (
)
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. 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
[C]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 IC
s 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 or the ability
of platelet-derived growth factor to activate MAP kinase in NIH 3T3
cells. (
)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.