(Received for publication, August 29, 1995; and in revised form, September 15, 1995)
From the
Prenylation of the carboxyl-terminal CAAX (C, cysteine; A, aliphatic acid; and X, any amino acid) of Ras is
required for its biological activity. We have designed a CAAX peptidomimetic, GGTI-287, which is 10 times more potent toward
inhibiting geranylgeranyltransferase I (GGTase I) in vitro (IC = 5 nM) than our previously
reported farnesyltransferase inhibitor, FTI-276. In whole cells, the
methyl ester derivative of GGTI-287, GGTI-286, was 25-fold more potent
(IC
= 2 µM) than the corresponding
methyl ester of FTI-276, FTI-277, toward inhibiting the processing of
the geranylgeranylated protein Rap1A. Furthermore, GGTI-286 is highly
selective for geranylgeranylation over farnesylation since it inhibited
the processing of farnesylated H-Ras only at much higher concentrations
(IC
> 30 µM). While the processing of
H-Ras was very sensitive to inhibition by FTI-277 (IC
= 100 nM), that of K-Ras4B was highly resistant
(IC
= 10 µM). In contrast, we found
the processing of K-Ras4B to be much more sensitive to GGTI-286
(IC
= 2 µM). Furthermore, oncogenic
K-Ras4B stimulation of mitogen-activated protein (MAP) kinase was
inhibited potently by GGTI-286 (IC
= 1
µM) but weakly by FTI-277 (IC
= 30
µM). Significant inhibition of oncogenic K-Ras4B
stimulation of MAP kinase by GGTI-286 occurred at concentrations
(1-3 µM) that did not inhibit oncogenic H-Ras
stimulation of MAP kinase. The data presented in this study provide the
first demonstration of selective disruption of oncogenic K-Ras4B
processing and signaling by a CAAX peptidomimetic. The higher
sensitivity of K-Ras4B toward a GGTase I inhibitor has a tremendous
impact on future research directions targeting Ras in anticancer
therapy.
Ras is a small guanine nucleotide binding protein that cycles
between its active (GTP-bound) and inactive (GDP-bound) forms to
transduce growth and differentiation signals from receptor tyrosine
kinases to the nucleus(1, 2) . Binding of epidermal
and platelet-derived growth factors to their receptor tyrosine kinases
results in autophosphorylation and recruitment of key signaling
proteins to the receptor. Among these proteins are the Ras exchange
factors that activate Ras by catalyzing the exchange of GDP for GTP.
GTP-bound Ras activates a cascade of mitogen-activated protein (MAP) ()kinases by recruiting Raf to the plasma membrane. Raf, a
serine/threonine kinase, phosphorylates MAP kinase kinase, which in
turn activates MAP kinase by phosphorylating it on threonine and
tyrosine. Hyperphosphorylated MAP kinase translocates to the nucleus
where it phosphorylates transcription factors that are involved in the
regulation of growth-related genes. The growth signal is terminated
when Ras hydrolyzes GTP to GDP(1, 2, 3) .
However, mutations that lock Ras in its GTP-bound form result in an
uninterrupted growth signal and are believed to contribute to the
development of more than one-third of human
cancers(4, 5) .
In order for Ras to transduce its
normal and oncogenic signal it must be anchored to the plasma membrane,
which is accomplished by post-translational modifications that increase
its hydrophobicity (6, 7, 8) . A key step in
this process is catalyzed by farnesyltransferase (FTase), an enzyme
that transfers farnesyl from farnesylpyrophosphate, a cholesterol
biosynthesis intermediate, to the cysteine of the carboxyl-terminal
CAAX of Ras (C, cysteine, A, aliphatic amino acid; X, serine or threonine)(9, 10) . A closely
related enzyme, geranylgeranyltransferase I (GGTase I), attaches the
lipid geranylgeranyl to the cysteine of the CAAX box of
proteins, where X is leucine(11, 12) . FTase
and GGTase I are /
heterodimers that share the
subunit(13, 14) . Cross-linking experiments suggested
that both substrates (farnesylpyrophosphate and Ras CAAX)
interact with the
subunit of FTase(15, 16) .
Although GGTase I prefers leucine at the X position, its
substrate specificity was shown to overlap with that of FTase in
vitro(17) . Furthermore, GGTase I was also able to
transfer farnesyl to a leucine terminating peptide(18) .
Because farnesylation of Ras is required for its oncogenic activity,
we (19, 20, 21, 22) and others (23, 24, 25, 26, 27) have
designed potent inhibitors of FTase as potential anticancer drugs.
These inhibitors are CAAX peptidomimetics, which show great
selectivity for FTase over GGTase I in vitro and selectively
block the processing of farnesylated but not geranylgeranylated
proteins in whole cells(22) . Furthermore, FTase inhibitors can
selectively block oncogenic Ras signaling and reverse malignant
phenotype at concentrations that do not affect normal
cells(24, 25) . However, mammalian cells express four
types of Ras proteins (H-, N-, KA-, and KB-Ras) among which K-Ras4B is
the most frequently mutated form of Ras in human
cancers(4, 5) . Although several laboratories have
demonstrated potent inhibition of oncogenic H-Ras processing and
signaling(26, 28) , this disruption has not been shown
with K-Ras4B. Hence, a drawback of the previous studies is the use of
H-Ras and not K-Ras4B as a target for the development of these
inhibitors. Recently, we have shown that a potent inhibitor of FTase
disrupts K-Ras4B processing but only at very high concentrations that
also inhibited the processing of geranylgeranylated
proteins(29) . This suggested that K-Ras4B may be
geranylgeranylated, particularly in cells where FTase is inhibited.
Consistent with this possibility is the recent observation that K-Ras4B
can be geranylgeranylated in vitro, but its Kfor GGTase I is 7 times higher than its K
for FTase (30) . GGTase I
CAAX-based inhibitors that can block geranylgeranylation
processing have not been reported. In the present study, we have
designed a CAAX peptidomimetic that selectively inhibits
GGTase I and demonstrate that oncogenic K-Ras4B processing and
signaling are disrupted at concentrations that affect
geranylgeranylation but not farnesylation processing.
The carboxyl-terminal CAAX tetrapeptide of Ras is a
substrate for FTase and serves as a target for designing inhibitors of
this enzyme with potential anticancer activity(23) . We have
recently made a highly potent (IC = 500
pM) inhibitor of FTase, FTI-276 (Fig. 1)(29) .
Its cell-permeable methyl ester FTI-277 inhibited H-Ras processing in
whole cells with an IC
of 100 nM(29) .
Furthermore, FTI-276 is highly selective (100-fold) for FTase over
GGTase I (Table 1). Although oncogenic H-Ras processing and
signaling were exquisitely sensitive to FTI-277, those of K-Ras4B were
highly resistant. However, at high concentrations of FTI-277, when the
processing of the geranylgeranylated Rap1A protein was inhibited,
K-Ras4B processing was also inhibited(29) . We, therefore, set
out to determine whether a GGTase I-selective inhibitor would disrupt
K-Ras4B processing and signaling. Our approach involved replacing the
central ``AA'' of CAAX tetrapeptides by a
hydrophobic spacer and incorporating a leucine residue in the
carboxyl-terminal position to optimize recognition by GGTase I. We
herein report a CAAL peptidomimetic, GGTI-287, where reduced cysteine
is linked to leucine by 2-phenyl-4-aminobenzoic acid (Fig. 1).
The phenyl substituent was designed to occupy the hydrophobic dipeptide AA binding pocket that must be present in the enzyme. GGTI-287
potently inhibited GGTase I in vitro (IC
=
5 nM) and was selective toward inhibiting GGTase I over FTase
(IC
= 25 nM) (Table 1). Thus, the
substitution of methionine in FTI-276 by a leucine in GGTI-287 (Fig. 1) increased the potency toward GGTase I by approximately
10-fold (Table 1). More importantly, it reversed the selectivity
from a FTase to a GGTase I-specific inhibitor by a factor of 500 (Table 1). To determine whether this selectivity is respected in
whole cells, we have synthesized the cell-permeable methyl ester
derivative of GGTI-287, GGTI-286 (Fig. 1), and treated NIH 3T3
cells, which overexpress oncogenic H-Ras-CVLS(31) , with
GGTI-286 (0-30 µM). Cell lysates were
electrophoresed on SDS-PAGE and immunoblotted with an anti-Ras antibody
as described under ``Experimental Procedures.'' Fig. 2shows that accumulation of unprocessed H-Ras did not occur
until 30 µM GGTI-286. Therefore, GGTI-286 is not a good
inhibitor of H-Ras processing in whole cells. However, GGTI-286 was a
very potent inhibitor of the processing of the geranylgeranylated Rap1A
protein (IC
= 2 µM) (Fig. 2).
Thus, GGTI-286 is more than 15-fold selective for inhibition of
geranylgeranylation over farnesylation processing (Table 1).
These data are in direct contrast to the FTase-specific inhibitor
FTI-277, which inhibited H-Ras and Rap1A processing with
IC
s of 100 nM and 50 µM,
respectively (Fig. 2). Thus, GGTI-286 is 25-fold more potent
than FTI-277 at inhibiting geranylgeranylation in whole cells (Table 1).
Figure 1: CAAX peptidomimetic structures. Structures of FTI-276/277 and GGTI-287/286 are shown.
Figure 2: Disruption of H-Ras and Rap1A processing. NIH 3T3 cells that overexpress oncogenic H-Ras were treated with various concentrations of FTI-277 (0-50 µM) or GGTI-286 (0-30 µM). The cells were lysed, and the lysates were electrophoresed on SDS-PAGE and immunoblotted with either anti-Ras or anti-Rap1A antibodies as described under ``Experimental Procedures.'' U and P designate unprocessed and processed forms of the proteins. Data are representative of three independent experiments.
We next evaluated the ability of GGTI-286 to
inhibit the processing and signaling of oncogenic K-Ras4B. NIH 3T3
cells, which overexpress oncogenic K-Ras4B(32) , were treated
with either GGTI-286 (0-30 µM) or FTI-277
(0-30 µM), and the lysates were immunoblotted with
an anti-Ras antibody as described under ``Experimental
Procedures.'' Fig. 3shows that GGTI-286 inhibited potently
the processing of K-Ras4B with an IC of 2 µM.
The ability of GGTI-286 to inhibit the processing of K-Ras4B was much
closer to its ability to inhibit the processing of geranylgeranylated
Rap1A (IC
= 2 µM) than that of
farnesylated H-Ras (IC
> 30 µM) ( Fig. 2and Table 1). This suggested that K-Ras4B might be
geranylgeranylated. Consistent with this is the fact that K-Ras4B
processing was very resistant to the FTase-specific inhibitor FTI-277
(IC
= 10 µM) (Fig. 3).
Furthermore, GGTI-286 inhibited K-Ras4B processing at concentrations
(1-3 µM) (Fig. 3) that had no effect on the
processing of farnesylated H-Ras (Fig. 2). These results are not
consistent with the work of Casey et al.(7) , who used
[
H]mevalonic acid to label cellular proteins and
provided evidence for a farnesylated K-Ras4B based on high pressure
liquid chromatography of the radiolabeled prenyl group. However, the
mass of the prenyl group in these studies was not determined.
Figure 3: Disruption of K-Ras4B processing. NIH 3T3 cells that overexpress oncogenic K-Ras4B were treated with FTI-277 or GGTI-286 (0-30 µM). The cells were lysed and the lysates were electrophoresed on SDS-PAGE and immunoblotted with anti-Ras antibodies as described under ``Experimental Procedures.'' U and P designate unprocessed and processed forms of Ras. The data are representative of three independent experiments.
To
determine whether inhibition of K-Ras4B processing by GGTI-286 results
in disruption of oncogenic signaling, we evaluated the ability of
GGTI-286 to antagonize oncogenic K-Ras4B constitutive activation of MAP
kinase. Activated MAP kinase is hyperphosphorylated and migrates slower
than hypophosphorylated (inactive) MAP kinase on
SDS-PAGE(26, 29) . Fig. 4shows that
K-Ras4B-transformed cells contained mainly activated MAP kinase.
Treatment of these cells with the FTase-specific inhibitor FTI-277
(0-30 µM) did not inhibit MAP kinase activation
until 30 µM (Fig. 4). In contrast, GGTI-286
inhibited MAP kinase activation with an IC of 1
µM, and the block was complete at 10 µM.
Thus, GGTI-286 blocked oncogenic K-Ras4B MAP kinase activation at a
concentration (10 µM) where FTI-277 had no effect. In
contrast, oncogenic H-Ras activation of MAP kinase was inhibited only
slightly by GGTI-286 whereas FTI-277 completely blocked this activation
at 3 µM (Fig. 4). Furthermore, GGTI-286 blocked
K-Ras4B activation of MAP kinase at a concentration (10
µM) that had little effect on H-Ras activation of MAP
kinase (Fig. 4). It should be noted that GGTI-286 was not toxic
to cells at concentrations as high as 10 µM. However, at
higher concentrations (30 µM), GGTI-286 did show some
signs of toxicity as reflected by a rounded morphology of the cells.
Thus, GGTI-286 was not toxic at concentrations (10 µM)
that resulted in complete inhibition of MAP kinase activation.
Figure 4: Inhibition of oncogenic activation of MAP kinase. NIH 3T3 cells that overexpress either oncogenic H-Ras or K-Ras4B were treated with either FTI-277 or GGTI-286 (0-30 µM). The cells were lysed, and the lysates were electrophoresed on SDS-PAGE and immunoblotted with an anti-MAP kinase antibody. P-MAPK designates hyperphosphorylated MAP kinase. The data are representative of three independent experiments.
Recently, we have demonstrated that the FTase-specific inhibitor
FTI-277 inhibits oncogenic H-Ras processing and signaling (29) and blocks in vivo tumor growth of
H-Ras-transformed NIH 3T3 cells and a human lung carcinoma that
expresses a K-Ras mutation(33) . However, processing of K-Ras4B
was inhibited by FTI-277 only at high concentrations similar to those
needed to inhibit the processing of the geranylgeranylated protein
Rap1A(29) . In the present study, we have described the design
of a geranylgeranylation-specific inhibitor and its effects on
oncogenic K-Ras4B processing and signaling. Our results demonstrate
that oncogenic K-Ras4B processing and constitutive activation of MAP
kinase are potently inhibited by a GGTase I-selective inhibitor
(GGTI-286) but are resistant to one selective for FTase (FTI-277). This
is in direct contrast to the processing and signaling of oncogenic
H-Ras, which was very sensitive to FTI-277 and highly resistant to
GGTI-286. The resistance of K-Ras4B to disruption by FTase inhibitors
could be explained by the 50-fold higher affinity of K-Ras4B for FTase
compared with H-Ras(30) . Our current data strongly suggest,
however, that K-Ras4B may be resistant to FTase inhibition because it
is post-translationally processed by a geranylgeranyl rather than a
farnesyl group. This is consistent with the recent observation that in vitro K-Ras4B can be geranylgeranylated by GGTase
I(30) . Although this previous work shows that K-Ras4B is a 7
times better substrate in vitro for FTase (K = 0.2 µM) than GGTase I (K
= 1.5 µM)(30) , our data suggest
that, in cultured cells, K-Ras4B is geranylgeranylated. This is
supported by the fact that GGTI-286 inhibited oncogenic K-Ras4B
processing and MAP kinase activation at concentrations (1 and 3
µM) that did not affect farnesylation-dependent
processing.
The results presented in this study are critical to the further design and development of inhibitors of Ras prenylation as potential anticancer agents. The results identify the GGTase I-specific inhibitor GGTI-286 as a small molecule capable of antagonizing selectively oncogenic K-Ras4B (not H-Ras) signaling. This is a key finding since K-Ras4B is the most frequently identified mutated Ras in human cancers, and its function has been resistant to FTase inhibitors. Furthermore, we have recently shown that a GGTase I inhibitor selectively suppressed activated DRas1 in Drosophila without side effects demonstrating the utility of these Ras CAAX peptidomimetics in whole animals(34) . Finally, the availability of K-Ras4B-selective inhibitors (i.e. GGTI-286) in addition to H-Ras-selective inhibitors (i.e. FTI-277) will enhance our understanding of the distinctive roles of these two forms of Ras in normal and oncogenic signaling.