From the Departments of Cancer Research and
Medicinal Chemistry, Merck Research Laboratories,
West Point, Pennsylvania 19486
Received for publication, January 12, 2001, and in revised form, March 23, 2001
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ABSTRACT |
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We have identified and characterized potent and
specific inhibitors of geranylgeranyl-protein transferase type I
(GGPTase I), as well as dual inhibitors of GGPTase I and
farnesyl-protein transferase. Many of these inhibitors require the
presence of phosphate anions for maximum activity against GGPTase I
in vitro. Inhibitors with a strong anion dependence were
competitive with geranylgeranyl pyrophosphate (GGPP), rather than with
the peptide substrate, which had served as the original template
for inhibitor design. One of the most effective anions was ATP, which
at low millimolar concentrations increased the potency of GGPTase I
inhibitors up to several hundred-fold. In the case of clinical
candidate L-778,123, this increase in potency was shown to
result from two major interactions: competitive binding of inhibitor
and GGPP, and competitive binding of ATP and GGPP. At 5 mM,
ATP caused an increase in the apparent Kd for the
GGPP-GGPTase I interaction from 20 pM to 4 nM,
resulting in correspondingly tighter inhibitor binding. A subset of
very potent GGPP-competitive inhibitors displayed slow tight binding to
GGPTase I with apparent on and off rates on the order of
106 M Inhibition of prenylation of Ras oncoproteins has long been
considered an attractive approach to anti-cancer therapy of
Ras-dependent tumors. Four isoforms of Ras are found in
mammalian cells, Kirsten-Ras (Ki-Ras) 4A and 4B, Harvey-Ras (Ha-Ras),
and N-Ras. Ki4B-Ras is the most frequently mutated isozyme in human
cancer, ranging from 20% up to 90% in pancreatic cancers, whereas
Ha-Ras mutations are found in a small percentage of bladder cancers.
Most oncogenic mutations in Ras result in the elimination of the GTPase
activity, locking Ras in its active, GTP-bound conformation. All Ras
proteins undergo multiple post-translational modifications on their
carboxyl termini that are required for membrane localization and
biological activity. The COOH-terminal CAAX motif (where C
is cysteine, A stands for aliphatic, and X for
any amino acid) is recognized by farnesyl-protein transferase
(FPTase),1 which transfers a
farnesyl group from farnesyl pyrophosphate (FPP) to the cysteine thiol.
This prenylation step is followed by proteolytic clipping of the three
terminal amino acids by a farnesyl-CAAX specific protease
and subsequent methylation of the cysteine residue by a carboxymethyl
transferase. Ha-Ras and N-Ras are further modified by palmitoylation
which provides additional membrane interactions. A polylysine stretch
in Ki-Ras near the COOH terminus serves the same purpose. However, the
prenylation step has been shown to be the single most critical
modification for function of all Ras proteins (reviewed in Ref. 1).
Initial efforts at inhibiting Ras function by many groups focused
exclusively on FPTase, the enzyme normally responsible for prenylation
of all four Ras isozymes. Specific inhibitors of FPTase showed dramatic
efficacy in Ha-Ras-dependent mouse xenograft tumor models
(2) and were also effective in Ki-Ras-driven mouse tumor models (3).
However, it was soon realized that FPTase inhibitors did not block
prenylation of Ki- and N-Ras isozymes because of cross-prenylation by
the related enzyme geranylgeranyl-protein transferase type I (GGPTase
I) (4-7), resulting in apparently fully functional and, in the case of
mutant Ras, transforming protein. It thus became clear that pure FPTase
inhibitors were not acting by blocking Ras signaling but by inhibiting
farnesylation of one or more as yet unidentified proteins. Blocking
Ki-Ras signaling through inhibition of both prenyltransferases remains
a viable option, however (8-10). To further explore the feasibility of preventing Ki4B-Ras signaling in vivo, we developed and
characterized potent inhibitors of GGPTase I, FPTase, and of both
enzymes (dual inhibitors).
GGPTase I was originally purified from bovine brain (11); the human
enzyme was subsequently cloned and purified (12). GGPTase I is an
All reported inhibitors of GGPTase I are either competitive with
CAAX or GGPP substrate, and many were designed based on the structure of the peptide or prenylpyrophosphate (9, 21-26). Compounds
in both categories show activity in cell-based assays. Some inhibitors
of FPTase have been observed to be more potent in the presence of
certain anions (27). Increases in potency of up to 50-fold were
observed, with phosphate and chloride being the most effective anions.
This anion effect was limited to FPP-competitive inhibitors and was not
seen with GGPTase I. We report here the identification of GGPTase
I-specific and dual inhibitors with a similar requirement for
phosphates for maximal potency. As in the case of the FPTase inhibitors
(27), the anion effect is limited to prenylpyrophosphate-competitive
compounds; however, with our current dual-specificity compounds, the
anion effect is only seen when assayed with GGPTase I. Utilizing an
equilibrium binding assay, we show that the anion effect is primarily
due to competitive binding between phosphates and GGPP, which
results in a dramatic increase in the apparent Kd
for GGPP. Consistent with this mechanism, a significant anion effect is
only observed with GGPP-competitive, but not
CAAX-competitive, inhibitors. Although the addition of
phosphates increased inhibitor potency, it did not change the mechanism
of inhibition in equilibrium binding studies. However, the unexpectedly
tight binding of GGPP in the absence of phosphates resulted in an
apparently CAAX-competitive mode of inhibition in
enzyme activity experiments.
To determine the significance of these observations for the inhibition
of GGPTase I in vivo, we evaluated the inhibition of prenylation of the GGPTase I substrate Rap1a in PSN-1 cells.
IC50 values measured in vitro in the absence of
phosphates did not predict potency of GGPTase I inhibitors in
cell-based assays. However, after accounting for anion requirement,
slow binding, and mechanism of competition, the structure-activity
relationship determined in vitro correlated well with the
inhibition of processing in vivo.
Materials--
Biotinylated Ki4B-Ras-derived peptide
b-GKKKKKKSKTKCVIM (single-letter amino acid code) was ordered from
Research Organics. [3H]GGPP was purchased from
PerkinElmer Life Sciences. Scintillation proximity assay (SPA)
beads pre-coated with streptavidin and ECF reagents were obtained from
Amersham Pharmacia Biotech. Antibodies specific for Rab6, Rap1a, and
for the unprenylated form of Rap1a were from Santa Cruz Biotechnology.
YL1/2 antibody was from Harlan Bioproducts for Science. The aspartic
acid-phenylalanine (Asp-Phe) dipeptide was from Sigma.
GGPTase I--
Human GGPTase I was expressed and purified as
described previously (28). A biotinylated version of GGPTase I was
produced by inserting the recognition site for biotin ligase,
GLNDIFEAQKIEWH, at the NH2 terminus of the alpha subunit.
Co-expression in BL21(DE3) birA pYCAC, an Escherichia coli
strain expressing the biotin ligase gene, in the presence of 50 µM biotin resulted in
NH2-terminally biotinylated GGPTase I. The enzyme
was purified by YL1/2 antibody affinity chromatography followed by
MonoQ ion exchange chromatography. Liquid chromatography-mass
spectrometry analysis confirmed that biotinylation had proceeded to
completion (>95%).
GGPTase I Assay--
GGPTase I reactions were run at
room temperature with biotinylated Ki4B-Ras derived peptide substrate
and tritiated GGPP. Reactions in 100-µl volumes were started with the
addition of peptide unless otherwise noted and contained the following
final concentrations of reagents: 80 pM GGPTase I, 100 nM [3H]GGPP, 1.6 µM
biotinylated peptide, 50 mM Hepes-NaOH, pH 7.5, 7 mM MgCl2, 10 µM
ZnCl2, 0.1% polyethylene glycol (Mr
15,000-20,000), 1 mM dithiothreitol, 4%
Me2SO. MgCl2 is not required for GGPTase activity but is essential for FPTase. MgCl2 was always
included in the GGPTase reaction to keep reaction conditions for the
two enzymes closely matched. Inhibitors and 5 mM ATP or
other anions were included as indicated in individual experiments. For
slow-binding inhibitors, GGPTase I and inhibitor were preincubated
at room temperature for 30 min in the absence of substrates. After 15 min at room temperature, reactions were stopped with the addition of
100 µl of 2 mg/ml SPA beads in 0.2 M NaPO4,
pH4, 50 mM EDTA, 0.5% bovine serum albumin, 0.05%
NaN3, and counted after an additional 60 min in a TopCount
scintillation counter (Packard Instruments).
The mechanism of inhibition by selected GGPTase I inhibitors
was determined at a fixed concentration of GGPP (100 nM) or
peptide (1.6 µM) while the concentration of the second
substrate was varied. Instead of a graphical analysis via
double-reciprocal plots, the entire raw data set was simultaneously fit
with the following equation for linear mixed type inhibition using
SigmaPlot software.
To obtain qualitative information on competition between inhibitors and
GGPP, IC50 values were determined at three concentrations of GGPP: 20, 100, and 500 nM. The slope of a plot of
log(IC50) versus log[GGPP] was used as a
measure of the extent of competition, where slopes of 1, 0, and <0
indicate competitive, non-competitive, and uncompetitive binding, respectively.
Equilibrium Binding Assay--
Equilibrium binding experiments
were performed under GGPTase I reaction conditions, except for the
absence of peptide substrate. In a typical experiment 10 nM
biotinylated GGPTase I was preincubated with 0.67 mg/ml
streptavidin SPA beads for 1-2 h at room temperature or overnight at
4C. After addition of ligands the mixtures (typically 60-µl volumes)
were incubated for 30 min, followed by determination of bound ligand on
a Packard TopCount scintillation counter. Multiple readings were taken
over time to assure that equilibrium had been reached.
For the determination of the Kd of the GGPTase
I-GGPP interaction, lower enzyme concentrations in correspondingly larger volumes were used. Final concentrations in 15-ml volumes were 15 pM GGPTase I, 5 mM ATP unless otherwise
indicated, and variable concentrations of [3H]GGPP. After
equilibrium had been reached, free [3H]GGPP was
determined by removing 1-ml aliquots, sedimenting the beads by
centrifugation, and mixing 0.75 ml of the supernatant with 15 ml of
80% ReadySafe scintillation mixture/20% isopropanol. Free and
bead-bound radioactivity were measured in a Beckman scintillation counter. Parallel controls of [3H]GGPP and
[3H]biotin of known specific activity were carried along
to correct for loss of GGPP due to nonspecific adsorption and to
determine counting efficiencies of free and bound ligand. Recovered
GGPP at the end of the experiment (free plus bound) was generally in the 30-70% range.
Data from equilibrium binding experiments were fit with an equation
describing the binding of two ligands, one of which is labeled, to two
distinct sites on a protein.
In the case of a single binding site for two competing ligands,
Equation 2 simplifies to Equation 3.
Rap1a Processing--
Inhibition of Rap1a geranylgeranylation
was measured in the PSN-1 human pancreatic tumor cell line. Cells were
treated for 24 h with inhibitor, and lysates were resolved using
10-20% SDS-polyacrylamide gel electrophoresis gradient gels (Novex
Inc) and then analyzed by Western blotting using a polyclonal antibody
specific for Rap1a (Santa Cruz Biotechnology Inc., Rap1/Krev-1 antibody
121). Blots were developed using an alkaline-phosphatase conjugated
anti-rabbit IgG (Cappel Inc), developed with a fluorescent detection
reagent (ECF, Amersham Pharmacia Biotech) and scanned on a
PhosphorImager (Storm®, Molecular Dynamics). Unprenylated
Rap1a was distinguished from prenylated Rap1a by virtue of their
different electrophoretic mobilities. The percentage of unprenylated
Rap1a relative to total Rap1a was determined by peak integration using
Imagequant® software (Molecular Dynamics), and
EC50 values were derived from dose-response curves with a
four-parameter curve-fitting equation using SigmaPlot® software.
Anion Dependence of Inhibition--
We have characterized the
potency and specificity of a large number of prenyl-protein transferase
inhibitors. The structures of several GGPTase I, FPTase, and
dual-specificity (GGPTase I and FPTase) inhibitors are shown in Fig.
1. GGPTase I IC50 values for
these compounds were determined with a 15-amino acid Ki-Ras-derived peptide substrate with the COOH-terminal CAAX sequence CVIM.
IC50 values measured under various conditions are
summarized in Table I. The
dual-specificity inhibitor and clinical candidate,
L-778,123 (compound 1), showed a 50-fold
preference for inhibition of FPTase over GGPTase I. The most potent
GGPTase I inhibitors, exemplified by compounds 2 and
4, resulted from the addition of hydrophobic groups to
either end of the basic structure of compound 1. Similarly,
the most specific GGPTase I inhibitors, including compound
5, are characterized by the presence of hydrophobic groups
at both ends of the molecule. More detailed structure-activity
relationships will be presented elsewhere.
Many compounds showed little or no inhibition of GGPTase I when assayed
in Hepes buffer despite the fact that they appeared to inhibit
processing of GGPTase I substrates in cell-based assays. For example,
compound 1 showed negligible activity against GGPTase I when
assayed in 50 mM Hepes buffer but it inhibited Rap1a
processing in PSN-1 cells with an EC50 of 6.8 µM. Unexpectedly, the addition of several anions to the
in vitro reaction resulted in a decrease of the
IC50 values for compound 1 from greater than 50 µM to less than 100 nM (Fig.
2). No inhibition of the reaction by the
anions themselves was seen except as noted below. Different anions
lowered the IC50 value of compound 1 to a
similar extent, though the required concentrations varied widely. ATP
and dithiophosphate were the most effective anions (with respect to the
concentration needed), but many phosphate and sulfate-based anions,
ranging from nucleotides to glycerophosphate, could substitute. On the
other hand, several commonly used buffer anions, such as chloride and
acetate, were completely ineffective at concentrations up to 50 mM. The rank order for the tested anions was as follows: dithiophosphate ~ ATP ~ dNTP > thiosulfate ~ tripolyphosphate ~ pyrophosphate ~ sulfate ~ GTP > phosphate ~ glycerophosphate
Since our standard assay makes use of a biotinylated peptide substrate,
we confirmed the anion effect with full-length proteins as substrates.
ATP caused similar shifts in potency of compound 1 (~1000-fold) and several other compounds when assayed with either
full-length Ki-Ras or RhoB as substrates. The absolute IC50
values varied, however, due to the differences in Km values of the protein substrates (data not shown). The extent of the
gain in potency in the presence of ATP and other phosphates varied
widely with different inhibitors, but was most dramatic for compounds
1 and 5 and closely related structures (Table I
and data not shown).
Kinetic Analysis of Inhibition--
Substrate competition
experiments showed that our GGPTase I inhibitors spanned the entire
range of GGPP-competitive to mixed type to CAAX-competitive
compounds, despite the fact that the design of these compounds had
evolved from the CAAX peptide sequence. Interestingly, the
anion dependence of these inhibitors correlated with their mechanism of
inhibition. Compounds that showed a pronounced shift in
IC50 upon addition of ATP, such as compounds 1 and 5, were found to be GGPP-competitive (determined in the
presence of ATP). On the other hand, compounds with less than 3-fold
shifts, such as compounds 6 and 7, consistently were non- or uncompetitive with respect to GGPP (Table I). This relationship is shown for a large number of compounds in qualitative fashion in Fig. 3. For this analysis the
ratio of IC50 values determined in the absence and presence
of 5 mM ATP is plotted against a qualitative measure of
GGPP competition in the presence of ATP (see "Experimental
Procedures"). In general, the more competitive with GGPP, the more
dependent on anions for potency the compounds were found to be. A
marked exception was the hydroxyphosphonate 8, a GGPP
analog, for which the potency was reduced by phosphates (Table I).
A detailed kinetic analysis was performed for a subset of the above
compounds and is shown in Fig. 4 for
compound 1. For this analysis the entire data set was fit
simultaneously with a general equation for linear mixed type inhibition
(see "Experimental Procedures"). In the presence of phosphates (5 mM ATP), compound 1 is competitive with GGPP and
noncompetitive with CAAX peptide (Fig. 4, A and
B). On the other side of the spectrum, closely related
compound 7 showed CAAX-competitive behavior while
being un- or noncompetitive versus GGPP. Several other
compounds were partly competitive with GGPP, displaying mixed type
inhibition versus GGPP with finite ratios of
Ki to Kii (data not shown).
Unexpectedly, in the absence of phosphates, the mechanism of inhibition
of compound 1 changed to competitive with respect to
CAAX peptide. Inhibition with respect to GGPP appeared to be mixed type, albeit the data at low GGPP concentrations were not reliable enough to rule out uncompetitive inhibition (Fig. 4, C and D). Similarly, all fully or partially
GGPP-competitive compounds became non-competitive inhibitors with
respect to GGPP in the absence of ATP. The omission of phosphates thus
induces an apparent change in the mechanism of inhibition,
from GGPP- to CAAX-competitive. Based on the results from
binding experiments, it appears, however, that kinetic uncoupling,
rather than a true switch in mechanism, are responsible for this
phenomenon (see below).
Remarkably, when assayed against FPTase, none of the above compounds
showed a dependence on anions (less than 3-fold shifts in
IC50 values). Furthermore, with the exception of a farnesyl homolog of compound 8, all inhibitors displayed a
CAAX-competitive mechanism of inhibition independent of the
presence of ATP or inorganic phosphate (data not shown).
In summary, kinetic analyses of inhibition of GGPTase I, but not
FPTase, showed a phosphate-dependent partial or complete competition between our inhibitors and GGPP, suggesting an overlap of
the respective binding sites.
Equilibrium Binding--
In an attempt to better understand the
underlying interactions of inhibitors, substrates, and anions with
GGPTase I, we designed a binding assay with an immobilized form of
GGPTase I. GGPTase I with a biotinylation tag (b-GGPTase I) was
expressed and biotinylated in E. coli. Complete
biotinylation was confirmed by liquid chromatography-mass spectrometry.
b-GGPTase I was then coupled to streptavidin-coated scintillation
proximity beads, and binding of tritium-labeled ligands was measured in
a standard SPA format. The availability of tritiated forms of GGPP and
compounds 1, 3, and 7 allowed pairwise
competition experiments among inhibitors, substrates, and anions.
Since initial experiments indicated that the Kd for
GGPP in the absence of phosphates may be much lower than suggested by
the nanomolar Km value, we first performed
equilibrium binding experiments with [3H]GGPP at very low
enzyme concentration. The concentrations of free and bound
[3H]GGPP were measured separately since a substantial
fraction of GGPP was bound to enzyme under our assay conditions. Due to
the tendency of the hydrophobic prenyl group to adsorb to vessel walls and pipette tips, the measured concentrations of free GGPP were always
significantly lower than calculated. In two independent experiments,
the equilibrium binding constant for GGPP was found to be remarkably
low, 15-30 pM in the absence of phosphates, but nearly
200-fold higher, 2-6 nM, in the presence of 5 mM ATP (Fig. 5A).
To determine the mechanism by which ATP raises the apparent Kd of GGPP, we measured bound [3H]GGPP
as a function of ATP concentration (Fig. 5B). The nearly complete displacement of GGPP at high ATP indicates that the two ligands bind competitively to GGPTase I. The data could be fitted well
with the equation for a two-ligand/single-site receptor binding model,
consistent with a single specific binding site for ATP, rather than
multiple nonspecific ionic interactions, and suggestive of competitive
binding to the pyrophosphate binding site of GGPP. The curve fit
yielded a surprisingly low Kd(app) for the ATP-GGPTase I interaction of 50-100 µM. Despite
being competitive with one of the substrates, ATP did not inhibit the
reaction. This was not unexpected since the association rate of GGPP is limited only by diffusion and the catalytic rate of the GGPTase I
reaction is slow (kcat = 0.07 s
For competition experiments between inhibitors and GGPP, the binding of
tritium-labeled inhibitor to GGPTase I was determined in the presence
of varying concentrations of cold GGPP. Fig. 5C shows the
effect of GGPP on compound 1 binding to GGPTase I in the
presence of ATP. Simultaneous curve fitting of all data points with a
"two-ligand/two binding sites" model yielded a
Kd for compound 1 of 30 nM.
In the absence of ATP, the Kd value rose to 190 nM (data not shown). The interaction factor,
The equilibrium binding analysis was subsequently expanded to a large
number of GGPTase I inhibitors by measuring displacement of
[3H]GGPP, with results consistent with those described
above. All inhibitors that showed a GGPP-competitive mechanism in
kinetics experiments were found to compete with [3H]GGPP
in the binding assay, with EC50 values that correlated well
with their IC50 values in the enzyme assay, while
CAAX-competitive inhibitors were unable to displace
[3H]GGPP. The presence or absence of phosphate did not
affect the qualitative outcome of these binding studies, even though
the EC50 values for [3H]GGPP displacement in
the absence of ATP were dramatically higher, as expected from the
picomolar Kd of GGPP under those conditions.
The equilibrium binding data suggest a model in which phosphates
increase the potency of GGPP-competitive inhibitors by displacing GGPP
from the pyrophosphate-binding site, but without impeding binding of
the inhibitor itself. In fact, ATP can facilitate inhibitor binding to
the enzyme even in the absence of GGPP, at least in the case of
compound 1. As shown in Fig. 5D, the apparent Kd of compound 1 decreases with
increasing ATP concentration. Simultaneous curve fitting of the entire
data set with a "two-ligand/two binding sites" model yielded
Kd values for compound 1 of 220 nM and for ATP of 35 µM, consistent with the
equilibrium binding constants determined above. The interaction factor
The hydroxyphosphonate 8 did not follow the pattern of
GGPP-competitive inhibitors, where potency increases with anions (Table
I). This initially puzzling result is consistent with the above model,
in that the phosphonate head group presumably binds in the
pyrophosphate site now occupied by ATP. In contrast to the situation
with compound 1, this would result in competitive rather
than synergistic binding with phosphates.
Slow Binding Inhibition--
In some of the binding experiments
described above, the final equilibria established rather slowly.
Although the association of GGPP with GGPTase I proceeded with nearly
diffusion-limited on rates of ~109
M Correlation with Cell-based Activity--
All of the in
vitro phenomena described above were found to have in
vivo correlates, i.e. anion effect, GGPP
versus CAAX competition, and slow binding had to
be taken into account to optimally predict potency of GGPTase I
inhibitors in cell-based assays. Fig. 7
demonstrates the improvement in the correlation between in
vitro IC50 and cell-based activity. GGPTase I activity
in PSN-1 cells was measured via the inhibition of geranylgeranylation
of Rap1a, a substrate for GGPTase I but not FPTase. Inhibition of Rap1a
prenylation by nearly 70 compounds showed a complete lack of
correlation with the in vitro potency as determined under
the initial assay conditions in the absence of phosphates (Fig. 7,
left panel). GGPTase I IC50 values determined in the presence of 5 mM ATP and after
preincubation of enzyme and inhibitor result in a dramatically improved
correlation (Fig. 7, middle panel). A separation
of inhibitors into GGPP versus CAAX-competitive
categories improved the correlation further. Linear regression of the
data for the two groups of compounds indicated a 30-fold difference in
cell-based versus biochemical potency (Fig. 7,
middle panel). After correcting the
IC50 values of CAAX-competitive compounds by a
factor of 30, an acceptable correlation with a slope of close to 1 was
obtained. The need for the latter correction likely reflects
differences in the effective substrate concentrations in cells and
in vitro. Differences in cell uptake of compounds are not
taken into account in the data shown in Fig. 7 and may be responsible
for the remaining scatter in the in vitro/in vivo
correlation.
Prenylation and membrane localization of Ki-Ras are essential for
its oncogenic potential. In vivo Ki-Ras is normally
predominantly farnesylated on its COOH-terminal cysteine, but in the
presence of FPTase inhibitors the 21-kDa protein is prenylated by
GGPTase I. Geranylgeranylated Ki4B-Ras is biologically active, implying that inhibition of both enzymes would be required to down-regulate Ki-Ras signaling. Interestingly, however, inhibition of either FPTase
or GGPTase I alone can result in tumor growth inhibition in nude mouse
tumor models, although it is not clear which prenylated protein(s) is
responsible for the observed efficacy. In the case of FPTase,
inhibition of RhoB farnesylation, followed by geranylgeranylation, has
been suggested as the critical step (29, 30). Inhibition of
farnesylation subsequently leads to induction of p21 and a cell cycle
block at G1 (31) or G2-M (Ref. 32 and
unpublished data). The G2-M block may be the result of a
block of prenylation of the mitotic kinesin CENP-E (33). G1
cell cycle arrests, including hypophosphorylation of retinoblastoma and
effects on cyclin-dependent kinase inhibitors, were
observed after treatment of cells with specific GGPTase I inhibitors
(32, 34-36). Nevertheless, inhibition of either prenyltransferase
alone does not block signaling via Ki-Ras. Since we wished to examine
the effects of inhibition of Ki-Ras in vivo and to conduct
tumor efficacy studies with combinations of FPTase and GGPTase I
inhibitors, we synthesized and evaluated specific GGPTase I inhibitors
and dual inhibitors.
While analyzing FPTase inhibitors for their ability to inhibit
prenylation of Ki-Ras in cell-based assays, we found that some compounds with little activity against GGPTase I were capable of
completely blocking farnesylation and geranylgeranylation in cells.
This apparent paradox led to the discovery that we were underestimating
the potency of GGPTase I inhibitors in vitro because of a
specific anion requirement for maximum inhibition. Many different phosphate-based anions could satisfy this anion requirement, including inorganic phosphates, nucleotides, and phospholipids. The fact that
some anions were effective at significantly lower concentrations than
others immediately suggested that this effect was not due to
nonspecific ionic interactions but rather to a direct effect on
inhibitor or substrate binding.
Despite being closely related and derived from the same basic
structural motif, our GGPTase I inhibitors separated into
CAAX-competitive and GGPP-competitive classes. Only the
latter compounds showed a significant gain in potency in the presence
of anions, suggesting that the anion effect was directly related to the
GGPP-competitive mode of inhibition. A similar phenomena has been
reported for certain inhibitors of FPTase, which also became much more
potent in the presence anions (27). Increases in potency of up to
50-fold were observed in the presence of phosphate. As is the case for the GGPTase I inhibitors described here, this anion effect was limited
to prenylpyrophosphate-competitive inhibitors. In contrast, however,
the anion effect was only seen with FPTase and not with GGPTase I. Furthermore, the rank order of anions differed substantially from that
described in the present report for GGPTase I inhibitors. For example,
chloride, one of the best anions for FPTase, was completely ineffective
for GGPTase I inhibitors.
What is the basis of the anion effect? Scholten and co-workers proposed
a model in which anions would compete with the pyrophosphate moiety of
FPP for binding to the enzyme. To test this hypothesis for the case of
GGPTase I, we developed an equilibrium binding assay to directly
measure binding of GGPP and other ligands. Using carefully controlled
conditions with picomolar enzyme concentrations, we found that GGPP
binds to GGPTase I with a much lower Kd than
expected, 15-30 pM in the absence of phosphates. Previous reports had found Kd values in the low nanomolar
range (3-16 nM), a result that may be explained at least
in part by the presence of phosphate in some of the buffer systems used
(15, 20, 37). We show that phosphates behave as competitive ligands with respect to GGPP. Although our data do not prove that the two
ligands bind to physically overlapping sites, anion binding to the
pyrophosphate site on GGPTase I would be the simplest explanation. ATP,
our anion of choice, raises the apparent Kd of the GGPP-enzyme interaction ~200-fold into the low nanomolar range at
physiological concentration.
Equilibrium binding experiments were also used to analyze the extent to
which GGPTase I inhibitors compete with GGPP binding. In the presence
of phosphates (i.e. ATP), the results were entirely consistent with the data from enzyme assays. GGPP-competitive inhibitors such as compound 1 were able to displace GGPP completely without significant formation of the ternary
enzyme-GGPP-inhibitor (E-GGPP-I) complex
(interaction factor In the absence of phosphate, equilibrium binding and enzyme assays gave
apparently contradictory results, in that compounds that competed with
GGPP in binding studies behaved as CAAX-competitive inhibitors in enzyme assays. This discrepancy has probably two underlying reasons: (i) kinetic uncoupling in the enzymatic assays at
high concentrations of GGPP (in contrast to equilibrium binding experiments where GGPP concentrations could be lowered into the low
picomolar range to show competition by less potent inhibitors, steady-state reactions needed to be run in the mid-nanomolar range), and (ii) the ability of most, if not all, GGPP-competitive compounds to
form the energetically unfavorable ternary
E-GGPP-I complex at high enough
concentrations (i.e. interaction factor Several potent GGPP-competitive inhibitors displayed slow binding to
the enzyme even in the absence of GGPP. On-rates 2 orders of magnitude
below diffusion limited on rates were observed, and enzyme-inhibitor
complexes showed half-lives of up to 1 h. Synergistic inhibition
of enzymes in the presence of anions has been reported in several cases
and has been discussed in the context of FPTase inhibitors (27). The
observation of slow tight binding in combination with anion dependence
furthermore suggests the formation of inhibition complexes that may be
mimicking the transition state of GGPTase I. For FPTase, as well as for
the related reactions catalyzed by squalene synthase, isopentenyl
pyrophosphate isomerase, and prenyltransferase, the reaction mechanism
has been shown or proposed to proceed through an allylic
carbonium-pyrophosphate intermediate (38-43). Nitrogen-containing
phosphates and phosphonates have been reported to be transition state
analogs of isopentenyl-pyrophosphate isomerase (44, 45). Most of our
slow-binding GGPTase I inhibitors have at least one electrophilic
nitrogen that may play the role of the carbonium cation, whereas the
anion (ATP) would take the place of the pyrophosphate leaving group.
We have identified closely related compounds with either GGPP or
CAAX-competitive mode of inhibition in vitro.
This difference in mechanism was reflected in cell-based assays, where
GGPP and CAAX-competitive compounds fall into different
categories, resulting in the need for a correction factor when
predicting cell potency from in vitro IC50
values. This is not entirely surprising since the effective
concentrations ([S]/Km) of the two GGPTase I
substrates differed ~10-fold for experimental reasons; GGPP was used
at 100 nM (20-fold over Km), and the
CAAX peptide was used at 1.6 µM (2-fold over
Km). After correction of the IC50 values
of CAAX-competitive compounds by a factor of 30, enzyme
assays predicted cell potency fairly reliably, implying that the
effective concentrations of GGPP and Rap1a in cells are in fact very similar.
Interestingly, none of the dual inhibitors in this study showed a
significant anion-dependence when assayed with FPTase. Although the two
prenyltransferases are analogous in terms of function and general
reaction mechanism, their differences obviously extend beyond the
different substrate preferences. Crystal structures of the
enzyme-inhibitor-anion complexes will eventually shed light on some of
these idiosyncrasies and should help further refine inhibitor
specificity and potency. We show here that relative subtle structural
changes affect the mechanism of inhibition of GGPTase I, which may
positively impact drug development. The rank order of inhibition of
prenylation of various GGPTase I protein substrates depends on their
relative Km values. Since this rank order is
affected differently by CAAX and GGPP-competitive inhibitors, it should allow for the design of inhibitors with different
substrate profiles within the same structural class.
Compound 1 (L-778,123) is a dual-specificity inhibitor with
a 50-fold preference for FPTase over GGPTase I in enzyme assays. In
cell culture this compound shows a 35-fold preference for FPTase, as
measured by the inhibition of prenylation of Rap1a and FPTase substrate
HDJ2. Inhibition of geranylgeranylation of Rap1a in PSN-1 cells is
detectable at low micromolar concentrations. Preliminary pharmacodynamic analysis of Phase I clinical data shows that
L-778,123 partially inhibits Rap1a prenylation in patients
at the highest dose tested.3
The potential consequences and therapeutic benefits of inhibition of
both FPTase and GGPTase I in the clinic are still uncertain; however,
we have created a solid basis for selecting compounds for further development.
1
s
1 and 10
3
s
1, respectively. Slow binding and the anion
requirement suggest that these inhibitors may act as transition state
analogs. After accounting for anion requirement, slow binding, and
mechanism of competition, the structure-activity relationship
determined in vitro correlated well with the inhibition of
processing of GGPTase I substrate Rap1a in vivo.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
heterodimer with the same
subunit as FPTase. The enzyme
transfers the prenyl moiety of geranylgeranyl pyrophosphate (GGPP) to
the cysteine of carboxyl-terminal CAAX sequences, with a
preference for leucine in the X position. It is also capable of prenylating the COOH-terminal CVIM sequence of Ki-Ras, albeit with a
Km 500-fold higher than that of FPTase (13). The
enzyme mechanism of mammalian GGPTase I has been characterized with
enzyme purified from bovine brain as well as from recombinant sources
(14, 15). The reaction was reported to follow a random Bi Bi pathway
with steady-state kinetics, although GGPP binding is the kinetically
preferred first step. The order of addition of substrates is reflected
in their relative Km values of low nanomolar for
GGPP and micromolar for peptide substrate, although the absolute
numbers vary dramatically with the particular CAAX sequence
and length of the peptide substrate (7, 13, 15, 16). The reaction
mechanism thus appears identical to that of FPTase (17). On the other
hand, the yeast enzymes yGGPTase I and yFPTase have been reported to
proceed through an ordered Bi Bi mechanism (18, 19). Interestingly, and
in contrast to FPTase, GGPTase I does not require Mg2+ for
GGPP binding or catalysis. However, Zn2+ is required for
peptide binding and catalysis (15, 20).
EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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I is the inhibitor, S is the varied substrate,
Ki and Kii are the binding
constants for the interaction of I with enzyme (E) and with
the E-S complex, respectively. For a competitive inhibitor,
we expect that Kii
(Eq. 1)
Ki, whereas the reverse would be true for an uncompetitive inhibitor.
E is the protein, S and X are
the labeled and unlabeled ligands, respectively,
Ks and Kx are the binding
constants for the E-S and the E-X
interactions, respectively, and
(Eq. 2)
is the interaction factor between
the two binding sites. A value of
of <1, 1, >1, and
1
indicates synergistic, independent, partly interfering, and mutually
exclusive binding of the two ligands, respectively. ES and
ESX are the labeled complexes detected in the SPA-based equilibrium binding format. In all experiments, except for the GGPP
Kd determination described above, ligand
concentrations were substantially greater than the GGPTase I
concentrations and [X]t and
[S]t were substituted for free
[X] and [S].
(Eq. 3)
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
[in a new window]
Fig. 1.
Structures of selected GGPTase
I and dual-specificity inhibitors.
In vitro potency, selectivity, and mechanism of inhibition of selected
prenyltransferase inhibitors
acetate,
carbonate, chloride, or nitrate. Based on intracellular anion
concentrations, ATP would be expected to be the major contributor to an
anion-effect in cells. We therefore decided to include 5 mM
ATP in subsequent GGPTase I assays. When used in the presence of a
slight excess of Mg2+, ATP had no effect on the reaction
rate at concentrations up to 20 mM. Mg2+ was
not required, however, for either GGPTase I activity or the anion
effect per se. In contrast to ATP and inorganic phosphate, pyrophosphate and dithiophosphate inhibited the reaction rate with
IC50 values of 1 mM, likely due to product
inhibition in the case of pyrophosphate. ATP at 5 mM did
not affect the apparent Km values for GGPP (4-8
nM, determined at 1.6 µM concentration of
peptide substrate) and CAAX peptide (0.4-0.7
µM at 100 nM GGPP).
View larger version (23K):
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Fig. 2.
Effect of various anions on the inhibition of
GGPTase I by compound 1. The log of the IC50 of
compound 1 is plotted as a function of the concentration of
the following anions added to the standard GGPTase I reaction.
Closed circle, dithiophosphate; open
circle, ATP; closed square,
thiosulfate; open square, pyrophosphate;
closed triangle, sulfate; open
triangle, phosphate; closed diamond,
glycerophosphate; open diamond, chloride.
View larger version (20K):
[in a new window]
Fig. 3.
Correlation between anion-dependence and GGPP
competition for GGPTase I inhibitors. The increase in potency in
the presence of anions (expressed as the ratio of IC50
values determined in the absence and presence of 5 mM ATP)
is plotted against a qualitative measure of GGPP competition. For the
latter the IC50 values of GGPTase I inhibitors were
determined at various GGPP concentrations in the presence of 5 mM ATP. The slope of a plot of log(IC50)
versus log[GGPP] was used as a measure of GGPP
competition, with slopes of 0 and 1 indicating noncompetitive and
competitive binding, respectively.
View larger version (36K):
[in a new window]
Fig. 4.
Kinetic analysis of GGPTase I inhibition by
compound 1. Reaction rates in the presence (A and
B) or absence (C and D) of 5 mM ATP were measured as a function of GGPP or peptide
substrate concentration at increasing concentrations of inhibitor. All
data points from a given experiment were curve-fitted simultaneously
(see "Experimental Procedures") based on a linear mixed type
reaction, where Ki and Kii are
defined with respect to the varied substrate S, according to the
following scheme.
Curve fitting yielded the following results and
constants: A, competitive (Ki, 0.004 µM; Kii, >10 µM;
Km, 0.008 µM); B,
noncompetitive (Ki, 0.09 µM;
Kii, 0.13 µM; Km,
0.7 µM); C, noncompetitive
(Ki, 12 µM; Kii, 48 µM; Km, 0.006 µM);
D, competitive (Ki, 10 µM;
Kii, 200 µM; Km,
0.6 µM). Inhibitor concentrations (in
µM) were as follows: A, 0 (closed
circles), 0.05 (open circles), 0.13 (closed triangles), 0.32 (open
triangles), 0.8 (closed squares), and
2 (open squares); B, 0, 0.02, 0.04, 0.08, 0.16, and 0.32, same order of symbols as A;
C, 0 (closed circles), 12.8 (open circles), 32 (closed
triangles), 80 (open triangles), and
200 (closed squares); D, 0, 12.5, 25, 50, and 100, same order of symbols as C.
1 at saturating GGPP and 1.6 µM peptide, data not shown).
View larger version (38K):
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Fig. 5.
Equilibrium binding competition experiments
with immobilized GGPTase I. A, plot of bound
versus free [3H]GGPP in the absence
(circles) or presence (squares) of 5 mM ATP. Inset, magnification of low GGPP
concentration range. Hyperbolic curve fit was used. B,
effect of ATP concentration on binding of [3H]GGPP to
GGPTase I (100 pM). ATP concentrations: 1200 pM
(circles), 400 pM (squares), and 120 pM (triangles). C, plot of bound
versus free tritiated compound 1 in the presence
of 5 mM ATP and varying concentrations of GGPP: none
(closed circles), 35 nM
(open circles), 150 nM
(closed triangles), 600 nM
(open triangles), and 900 nM
(closed squares). D, plot of bound
versus free tritiated compound 1 in the presence
of increasing concentrations of ATP: none (closed
circles), 1.6 µM (open
circles), 8 µM (closed
triangles), 40 µM (open
triangles), 200 µM (closed
squares), and 1000 µM (open
squares).
, was
poorly defined with a value of >50 under both conditions, but
indicative of mutually exclusive binding (
values of <1, 1, >1,
and
1 indicate cooperative, independent, interfering, and
competitive binding, respectively). In binding experiments compound
1 is thus competitive with GGPP, independent of the presence
of phosphates (see "Discussion"). Analogous experiments with
compound 3 yielded Kd values of 4 and 24 nM, and
values of 30 and 5 in the presence and absence
of ATP, respectively. For this compound simultaneous binding with GGPP is thus possible, although energetically unfavorable. In the case of
compound 7, no competition by GGPP was seen under any condition, consistent with its CAAX-competitive behavior in
GGPTase I enzyme assays (data not shown).
of 0.13 indicates cooperative binding between compound 1 and phosphates. The increased potency of compound 1 in the
presence of ATP is thus the combined result of ATP raising the apparent
Kd of GGPP 200-fold and lowering the apparent Kd for compound 1 ~7-fold.
1 s
1
(data not shown), several GGPTase I inhibitors displayed slower kinetics. Since these compounds were not available in isotopically labeled form, and association could therefore not be followed directly
in our assays, the formation and dissociation of enzyme-inhibitor complexes was monitored in subsequent short activity assays (5-min reactions). Inhibition of GGPTase I activity was determined as a
function of preincubation time of enzyme and inhibitor in the absence
of GGPP. Fig. 6 shows a typical
experiment with compound 2, in which association as well as
dissociation of preformed enzyme-inhibitor complexes were measured.
Dissociation was initiated by the addition of excess GGPP, followed by
a GGPTase I reaction at the indicated times. Curve fitting yielded on
and off rates of ~2 × 106
M
1 s
1
and 10
3 s
1,
respectively. Similarly slow association rates were observed with
several of the more potent GGPP-competitive inhibitors, but not with
potent CAAX-competitive inhibitors such as compound
6. Preincubation for at least 30 min in the absence of GGPP
was required to obtain maximal inhibition of GGPTase I. As expected for
GGPP-competitive inhibitors, preincubation in the presence of GGPP
resulted in an additional, concentration-dependent lowering
of the apparent association rates (data not shown).
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Fig. 6.
Association-dissociation time courses for
compound 2 in the presence of ATP. GGPTase I (75 pM)
and compound 2 at 0.3 nM (circles), 1 nM (squares), 3 nM
(triangles), or 10 nM (diamonds) were
preincubated in the absence of GGPP for the indicated time. At 30 min
300 nM GGPP was added to initiate dissociation of the
enzyme-inhibitor complex. Aliquots were taken at the indicated times
for activity assays (5-min reactions).
View larger version (19K):
[in a new window]
Fig. 7.
Correlation between potency of GGPTase I
inhibitors in vitro and inhibition of prenylation in
PSN-1 cells. EC50 values for inhibition of Rap1a
processing are plotted against GGPTase I IC50 values
determined in vitro. Left panel,
GGPTase I IC50 values determined in the absence of anions
and without preincubation. Middle panel, GGPTase
I IC50 values determined in the presence of 5 mM ATP and after preincubation of enzyme and inhibitors;
GGPP-competitive and CAAX-competitive compounds are shown
with circles and triangles, respectively.
Right panel, GGPTase I IC50 values
corrected for mechanism of inhibition (multiplied by factor of 30; see
"Results" for details).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
> 50). In contrast,
CAAX-competitive inhibitors, including compounds
6 and 7, did not affect GGPP binding at all.
Several compounds showed partially competitive behavior where the
ternary E-GGPP-I complex was observable but
energetically unfavorable (interaction factors of greater than 1).
These data are consistent with a model in which the inhibitor primarily
occupies the CAAX binding site but, depending on the particular compound, partly overlaps with the GGPP binding site. Inhibitor binding in the CAAX site is expected since the
design of all compounds described here (with the exception of the
phosphonate 8) evolved from the CAAX peptide
structure. In the case of FPTase, compound 1 was shown by
x-ray crystallography to bind in the CAAX pocket alongside
FPP.2 Whether or not a given
inhibitor overlaps with the GGPP binding site seems to depend on minor
structural alterations, as demonstrated by the orthogonal behavior of
closely related compounds 1 and 7. In contrast to
GGPP, ATP does not compete with inhibitor binding; instead, anion and
inhibitor binding are cooperative, at least in case of compounds
1 and 3 (interaction factor <1). The overlap
between GGPP and these inhibitors thus appears to be limited to the
prenyl binding pocket, whereas the triphosphate moiety of ATP occupies
the pyrophosphate binding site. Consistent with this model, a reverse
anion effect is observed with the GGPP analog 8. In this
case anions directly compete with inhibitor binding in the
pyrophosphate pocket.
1, but not
infinite). The resulting inhibition would be weak but CAAX-competitive, as exemplified by compound 1.
It follows from this explanation that the less favorable the formation
of the E-GGPP-I complex, the greater the expected
anion effect. This prediction is in fact borne out in that the extent
of GGPP-competition correlates with the magnitude of the anion effect
(Fig. 3). In the presence of phosphates, which compete with GGPP and
thus facilitate the formation of E-I complexes even at
higher GGPP concentrations, equilibrium binding and enzyme assays gave
consistent results.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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 Cancer Research, Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486. E-mail: hans_huber@merck.com.
¶ Current address: DuPont Pharmaceuticals Co., Wilmington, DE 19880.
Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M100325200
2 S. B. Long and L. S. Beese, manuscript in preparation.
3 R. B. Lobell, D. Liu, and N. Kohl, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: FPTase, farnesyl-protein transferase; FPP, farnesyl pyrophosphate; GGPTase I, geranylgeranyl-protein transferase type I; GGPP, geranylgeranyl pyrophosphate; SPA, scintillation proximity assay.
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