(Received for publication, October 28, 1994)
From the
Cysteine farnesylation of the carboxyl-terminal tetrapeptide
CAAX (C = Cys, A = Leu, Ile, or Val, X = Met or Ser) of the oncogene product Ras is required
for its malignant transformation activity. As a consequence
farnesyltransferase (FTase), the enzyme responsible for this lipid
modification, has become one of the most sought-after targets for
anticancer drug development. We have recently designed peptide mimics
of the COOH-terminal Cys-Val-Ile-Met of K-Ras where the
dipeptide Val-Ile was replaced by aminobenzoic acid derivatives.
Although these peptidomimetics are potent inhibitors of FTase in
vitro, they retain several undesirable peptide features that
hamper their use in vivo. We report here the design,
synthesis, and biological activity of the first non-peptide mimetics of
CAAX where the tripeptide AAX was replaced by
biphenyl derivatives. (R)-4-[N-(3-mercapto-2-aminopropyl)]amino-3`-carboxybiphenyl,
where the cysteine is linked to the biphenyl derivative through a
secondary amine, contains no amino acids, lacks peptidic features, and
has no hydrolyzable bonds. This peptidomimetic is a potent inhibitor of
FTase in vitro (IC
= 50-150
nM) and disrupts Ras processing in whole cells. Furthermore,
this non-peptide mimetic of CAAX is highly selective for FTase
(666-fold) relative to the closely related geranylgeranyltransferase I.
This selectivity is also respected in vivo since the
processing of Ras but not the geranylgeranylated Rap1A was disrupted in
whole cells. Structure activity relationship studies revealed that
FTase recognition and inhibitory potency of CAAX peptidomimetics require free thiol and carboxylate groups
separated by a hydrophobic moiety, and that precise positioning of
these functional groups must correspond to that of the parent
CAAX. The true CAAX peptidomimetic described in this
manuscript has several desirable features for further development as a
potential anticancer agent. It is not metabolically inactivated by
FTase, does not require a prodrug strategy for inhibition in
vivo, and is selective for farnesylation relative to
geranylgeranylation.
Ras is a small guanine nucleotide-binding GTPase that transduces biological information from the cell surface to the nucleus(1) . Its ability to transfer growth signals from receptor tyrosine kinases to a mitogen-activated protein kinase cascade puts it in the heart of signaling pathways that cause proliferation in normal cells and uncontrolled growth in cancer cells(2) . Indeed, mutations that lock Ras in its active, GTP-bound state lead to malignant transformation and are among the most frequently identified mutations in human cancers (1) . For example, 50% of colorectal and 95% of pancreatic human cancers have activated ras oncogenes.
Over the last decade several strategies have been
investigated, with little success, to disrupt Ras function and, hence,
to inhibit the growth of tumors with activated ras oncogenes.
The search has recently intensified with the discovery that Ras
requires lipid modification with a farnesyl group for localization to
the plasma membrane, where it plays a pivotal role in growth
signaling(3, 4, 5, 6, 7, 8, 9) .
Because farnesylation is required as well as sufficient for Ras
membrane association and transformation(10) , the enzyme that
catalyzes this lipid modification, farnesyltransferase (FTase), ()has become a major target for the design of novel
anticancer agents(11, 12) . FTase is an
and
heterodimer that transfers farnesyl from farnesylpyrophosphate, a
cholesterol biosynthesis intermediate, to the cysteine of proteins
containing the carboxyl-terminal sequence CAAX (A = aliphatic and X = any amino acid except
Leu or Ile)(13, 14) . A closely related
prenyltransferase, geranylgeranyltransferase I (GGTase I) catalyzes
cysteine geranylgeranylation of proteins ending in CAAX where X = Leu or Ile(15, 16) . Prenylation
of CAAX sequences by FTase and GGTase I is followed by
proteolysis of the tripeptide AAX and carboxymethylation of
the resulting prenylated cysteine. Since the number of
geranylgeranylated proteins in the cell far exceeds that of
farnesylated proteins(15, 16) , it is critical that
farnesylation inhibitors with potential anticancer activity be highly
selective for FTase over GGTase I to minimize side effects.
Developing Ras CAAX tetrapeptide mimics as anticancer drugs
has been the focus of several laboratories over the last two
years(11, 12) . This was prompted by the observation
that FTase recognizes and farnesylates CAAX peptides which
were also found to be potent competitive inhibitors of the enzyme
(IC = 50-200
nM)(13, 17, 18, 19, 20, 21, 22) .
Because of their peptidic nature, CAAX peptides do not inhibit
Ras processing in whole cells. To enhance their poor cellular uptake
and decrease their sensitivity to cellular proteases, several
investigators have made CAAX pseudopeptides(23, 24, 25) . Reduction
of the amino-terminal and central amide bonds of CAAX and
neutralization of the free carboxylate resulted in greater activity in
whole cells(23, 24, 25) . Although the FTase
inhibitors discussed above are potent inhibitors in vitro,
they retain several peptidic features. To avoid inherent problems of
peptides, our strategy has been to design non-peptide CAAX mimetics. Working toward this goal, we initially made potent
inhibitors of FTase where ``VI'' in CVIM, the carboxyl
terminus of K
-Ras, was replaced by 4-aminobenzoic acid
(4ABA) derivatives that served as hydrophobic spacers to link Cys to
Met (i.e. Cys-4ABA-Met)(26, 27, 28) . Similarly,
James et al.(29) used a benzodiazepine group between
cysteine and methionine.
A critical and as yet unreported goal in this area has been to construct non-peptidic inhibitors with no amide bonds in their molecular backbone. Our approach to improving the in vivo potency and hydrolytic stability of our inhibitors involved extending the hydrophobic spacer strategy to include the terminal methionine as well as the central aliphatic residues of CVIM. Here we report the design, synthesis, and selective inhibition of FTase in vitro and in vivo by the first non-peptide CAAX mimetic where 4-amino-3`-carboxybiphenyl was designed as a VIM tripeptide mimetic.
Figure 1: Ras CAAX peptidomimetics.
For Rap1A processing assays, 50 µg of cell lysates were electrophoresed as described above for Ras processing and transferred to nitrocellulose. The membranes were then probed with anti-Rap1A (Santa Cruz Biotechnology, Santa Cruz, CA). Antibody reactions were visualized using peroxidase-conjugated goat anti-rabbit IgG and ECL chemiluminescence as described above.
Figure 2: Energy-minimized structures for CVIM and 4a.
Figure 3:
FTase
and GGTase I inhibition studies. Partially purified FTase and GGTase I
were incubated with Ras CAAX peptidomimetics and their ability
to transfer [H]farnesyl to Ha-Ras-CVLS (FTase) and [
H]geranylgeranyl to
Ha-Ras-CVLL (GGTase I) was determined as described under
``Experimental Procedures.'' A, FTase inhibition by:
4a (
) and 4b (
); B, FTase (
) and GGTase
I (
) inhibition by 4a. Each curve is representative of at least
four independent experiments.
An earlier proposal (29, 34) suggested that potent inhibitory activity
toward FTase might require inhibitors to take up a turn
conformation bringing the cysteine thiol and free carboxylate in close
proximity to form a bidentate complex with Zn
.
Recently, we have provided evidence that argues against this
model(27) . Consistent with this, the biphenyl-based
non-peptide CAAX mimetic described here cannot take up this
structure (Fig. 2), leading us to conclude that potent
inhibition of FTase does not require a
turn conformation.
Figure 4:
Ras CAAX peptide and
peptidomimetic farnesylation. The transfer of
[H]farnesyl to peptides and peptidomimetic by
FTase was determined by silica G TLC as described under
``Experimental Procedures.'' FPP and F-peptide designate farnesyl pyrophosphate and farnesylated peptide,
respectively. Lane 1, FPP only; lane 2, FPP and CVLS
but no FTase; lane 3, FPP and FTase but no peptide; lanes
4-9, all contained FTase and FPP. Lane4,
VCIM; lane5, CVLS; lane6, 3; lane7, 4a; lane 8, 4b, lane 9, 4c.
Data are representative of two independent
experiments.
Figure 5: Ras and Rap1A processing. A, Ras-transformed Balb/c 3T3 cells were treated with inhibitors, the lysate was immunoprecipitated and blotted with anti-Ras antibody. B, lysates were blotted with anti-Rap1A antibody as described under ``Experimental Procedures.'' Lane 1, control; lane 2, lovastatin; lane 3, methyl ester of 3 (200 µM); lane4, 4a (100 µM); lane5, 4a (50 µM); lane6, 4a (25 µM); lane7, 4b; lane8, 4c. Data are representative of three independent experiments.
We then investigated the selectivity of our Ras farnesylation inhibitors by determining their ability to inhibit processing of Rap1A, a small G-protein that is geranylgeranylated(15, 16) . Cells were treated with lovastatin or peptidomimetics exactly as described for Ras processing experiments. Lysates were then separated by SDS-PAGE and immunoblotted with anti-Rap1A antibody as described under ``Experimental Procedures.'' Control cells contained only the geranylgeranylated Rap 1A (Fig. 5B, lane1), whereas lovastatin-treated cells contained both processed and unprocessed forms of Rap 1A, indicating, as expected, that lovastatin inhibited the processing of Rap 1A (Fig. 5B, lane2). Peptidomimetic 4a, which inhibited Ras processing, was not able to inhibit Rap 1A geranylgeranylation (Fig. 5B, lanes4-6). Peptidomimetics 4b and 4c did not inhibit Rap 1A processing (Fig. 5B, lanes7 and 8).
Thus, we have designed the first non-peptide CAAX mimetic that contains no hydrolyzable groups and lacks other peptidic features. Although we have made major structural alterations in the parent CAAX tetrapeptide, the peptidomimetic retained high potency toward inhibiting FTase. Structure activity relationship studies further revealed that the crucial structural features for FTase recognition and potent inhibition are free thiol and carboxylate groups maintained at a critical distance and orientation by a hydrophobic spacer. Furthermore, these true CAAX peptidomimetics have several desirable features as potential therapeutic agents. They are selective inhibitors for farnesylation relative to geranylgeranylation both in vitro and in vivo, they do not require a prodrug strategy for whole cell activity, and they are not metabolically inactivated by FTase. We are presently evaluating their selectivity toward antagonizing oncogenic Ras signaling and their efficacy as anticancer agents against tumors with activated ras oncogenes.