Transition state analogs for protein farnesyltransferase

Full text of DOI:10.1021/ja961214c

J. Am. Chem. Soc. 1996, 118, 8761-8762
8761
Scheme 1. Electrophilic Mechanism for Protein
Prenyltransferases
Transition State Analogs for Protein
Farnesyltransferase
Pamela B. Cassidy and C. Dale Poulter*
Department of Chemistry, UniVersity of Utah
Salt Lake City, Utah 84112
ReceiVed April 12, 1996
Addition of an isoprenoid residue to the sulfhydryl moiety
of a cysteine is an essential posttranslational modification for
many physiologically important proteins.1 Three different
enzymes are responsible for the modifications. Protein farne-
syltransferase (PFTase) and protein geranylgeranyltransferase-I
(PGGTase-I) catalyze the alkylation of C-terminal cysteines in
proteins ending in CaaX, where a is usually an aliphatic amino
acid and X is variable. The cysteine in CaaX motifs is
farnesylated when X is alanine, methionine, serine, glutamine,
or cysteine2-4 and geranylgeranylated when X is leucine or
isoleucine.3,5 CaaX proteins are further modified by proteolytic
removal of the aaX tripeptide and methylation of the new
carboxy terminus.6 Protein geranylgeranyltransferase-type-II
(PGGTase-II) modifies both cysteines in C-terminal XXCC,
XCXC, or CXCX sequences.7 These modifications enhance
the lipophilicity of the proteins and promote their association
with membranes.
The oncogene products of the Ras family of proteins (pRas)
are among the CaaX proteins bearing a farnesyl moiety.
Approximately 30% of human cancers are associated with
mutations in pRas.8 The discovery that farnesylation is required
for oncogenic forms of pRas to manifest their transforming
activity has stimulated an intense search for inhibitors of PFTase
as potential chemotherapeutic agents.9 Tetrapeptides containing
CaaX sequences are alternate substrates for the enzyme and were
the first class of inhibitors of the enzyme to be studied.10
Nonhydrolyzable peptidomimetics containing cysteine and me-
thionine residues separated by a variety of spacers were reported
shortly afterward.11-14 Some of these compounds are potent
inhibitors of PFTase in ViVo and disrupt the growth of Ras-
transformed cells. Nonhydrolyzable analogs of farnesyl diphos-
phate (FPP) such as R-(hydroxyfarnesyl)phosphonic acid are
also potent inhibitors of the enzyme.15 Other compounds such
as chaetomellic acid A and zargazoic acid were identified
through microbial screens and do not closely resemble either
substrate.16
Recent work with fluorinated analogs of FPP indicates that
alkylation of the sulfhydryl moiety in cysteine by FPP is an
electrophilic displacement, as illustrated in Scheme 1.17 While
several different structural classes of PFTase inhibitors, including
bisubstrate analogs,18 have been studied, none were designed
to duplicate the topological and electrostatic features of the tran-
sition state for the reaction. We now report the synthesis and
evaluation of L-â-farnesylaminoalanine (faA) and tetrapeptide
faAVIA (see Figure 1) as transition state analogs for the elec-
trophilic alkylation of the sulfhydryl group in cysteine by FPP.
faA was prepared by treatment of the Cbz derivative of
L-serine lactone19 with farnesylamine20 followed by removal of
the blocking group, as shown in Scheme 2. The farnesylated
amino acid was then incorporated into tetrapeptide faAVIA by
(1) Omer, C. A.; Gibbs, J. B. Mol. Microbiol. 1994, 11, 219.
(2) Omer, C. A.; Kral, A. M.; Diehl, R. E.; Prendergast, G. C.; Powers,
S.; Allen, C.M.; Gibbs, J. B.; Kohl, N. E. Biochemistry 1993, 32, 5167.
(3) Caplin, B. E.; Hettich, L. A.; Marshall, M. S. Biochim. Biophys. Acta
1994, 1205, 39.
(4) Reiss, Y.; Stradley, S. J.; Gierasch, L. M.; Brown, M. S.; Goldstein,
J. L. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 732.
(5) Yokoyama, K.; Goodwin, G. W.; Ghomashchi, F.; Glomset, J. A.;
Gelb, M. H. 1991, Proc. Natl. Acad. Sci. U.S.A. 88, 5302.
(6) Clarke, S. Annu. ReV. Biochem. 1992, 61, 355.
(7) Farnsworth, C. C.; Seabra, M. C.; Ericsson, L. H.; Gelb, M. H.;
Glomset, J. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11963.
(8) Barbacid, M. Annu. ReV. Biochem 1987, 56, 779.
(9) Bos, J. L. Cancer Res. 1989, 49, 4682.
(10) Reiss, Y.; Goldstein, J. L.; Seabra, M. C.; Casey, P. J.; Brown, M.
J. Cell 1990, 62, 81.
(11) James, G. L.; Goldstein, J. L.; Brown, M. S.; Rawson, T. E.; Somers,
T. C.; McDowell, R. S.; Crowley, C. W.; Lucas, B. K.; Levinson, A. D.;
Marsters, J. C., Jr. Science 1993, 260, 1937.
(12) Marsters, J. C., Jr.; McDowell, R. S.; Reynolds, M. E.; Oare, D.
A.; Somers, T. C.; Stanley, M. S.; Rawson, T. E.; Struble, M. E.; Burdick,
D. J.; Chan, D. S.; Duarte, C. M.; Paris, K. J.; Tom, J. Y. K.; Wan, D. T.;
Xue, Y.; Burnier J. P. Bioorg. Med. Chem. Lett. 1994, 2, 949.
(13) Wai, J. S.; Bamberger, D. L.; Fisher, T. E.; Graham, S. L.; Smith,
T. L.; Gibbs, J. B.; Mosser, S. D.; Oliff, A. I.; Pompliano, D. L.; Rands,
E.; Kohl, N. E. Bioorg. Med. Chem. Lett. 1994, 2, 939.
(14) Vogt, A.; Qian, Y.; Blaskovich, M. A.; Fossum, R. D.; Hamilton,
A. D.; Sebti, S. M. J. Biol. Chem. 1995, 270, 660.
(15) Pompliano, D. L.; Rands, E.; Schaber, M. D.; Mosser, S. D.;
Anthony, N. J.; Gibbs, J. B. Biochemistry 1992, 31, 3800.
(16) Tamanoi, F. TIBS 1993, 18, 349.
(17) Dolence, J. M.; Poulter, C. D. Proc. Natl. Acad. Sci. U.S.A. 1995,
92, 5008.
(18) Patel, D. V.; Gordon, E. M.; Schmidt, R. J.; Weller, H. N.; Young,
M. G.; Zabler, R.; Barbacid, M.; Carboni, J. M.; Gullo-Brown, J. L.;
Hunikan, L.; Ricca, C.; Robinson, S.; Seizinger, B. R.; Tuomari, A. V.;
Manne, V. J. Med. Chem. 1995, 38, 435.
(19) Arnold, L. D.; Kaluntur, T. H.; Vederas, J. C. J. Am. Chem. Soc.
1985, 107, 7105.
(20) Matsumoto, S.; Doteuchi, M.; Mizui, T.; Hirai, K. Eur. Pat. Appl.
01 194 901, 1986.
Figure 1.
S0002-7863(96)01214-0 CCC: $12.00 © 1996 American Chemical Society

8762 J. Am. Chem. Soc., Vol. 118, No. 36, 1996
Scheme 2. Synthesis of faAVIA
Communications to the Editor
Table 1. Inhibitors of PFTase
compound
IC50a (µM)
â-aminoalanine
farnesylamine
faA
>1000
70
51
14
faA-VIA
S-farnesylcysteine
S-farnesyl-CVIA
> 200
> 200
a IC50 values were determined in the presence of 2 µM FPP and 2.5
µM dansyl-GCVIA.
of condensation of FPP with dansyl-GCVIA. IC50s for the
compounds are listed in Table 1. â-Aminoalanine was not an
inhibitor (IC50 > 1 mM). Farnesylamine had an IC50 of 70
µM. This observation is consistent with a previous report that
the amine inhibited farnesylation of proteins in Vitro in a cell-
free extract and in ViVo in a cell culture assay.27 Addition of a
â-alanyl moiety to the amino group in farnesylamine slightly
improved the potency of the inhibitor, while addition of the
full tetrapeptide unit reduced the IC50 5-fold. In contrast,
S-farnesylcysteine and S-farnesyl-CVIA were not inhibitors at
concentrations up to the limits of their solubility (ca. 200 µM).
Inorganic pyrophosphate (PPi) was a poor inhibitor of yeast
PFTase (Ki ) 2.4 mM). However, when faAVIA was incubated
with yeast PFTase in buffer containing 1 mM PPi, the IC50 for
the ammonium analog decreased 35-fold to 370 nM. The
synergism seen between faAVIA and PPi was also observed
for other ammonium-based transition state analog inhibitors of
enzymes in the isoprenoid biosynthetic pathway, including
squalene synthase28,29 and trichodiene synthase.30 In these cases,
the synergistic effects for the ammonium derivatives in the
presence of PPi were larger than observed for yeast PFTase,
although the analogs themselves were less potent than faAVIA.
The relative magnitude of the synergism may reflect the extent
of charge separation between the developing carbocation and
the PPi leaving group in the transition state structures for the
four different enzyme-catalyzed reactions. For the three
enzymes showing potent synergism, the nucleophilic partner is
a carbon-carbon double bond, and one might anticipate a high
degree of cationic character in the farnesyl moiety at the
transition state. In contrast, the thiol, or perhaps thiolate,
nucleophile for protein prenylation might react by an enforced
mechanism through a less highly developed electrophilic
transition state. In summary, the faA analogs represent an
important new class of PFTase inhibitors whose biological
activity supports the hypothesis that prenylation of proteins
occurs by an electrophilic mechanism.
solid phase procedures using the oxime resin developed by
DeGrado and Kaiser.21 Dipeptide fragment Val-Ile, linked to
the resin through the isoleucine carboxyl as a hydroxamate ester,
was coupled to faA by treatment of the dipeptide with the
symmetric anhydride22 of Cbz2-faA. The resulting polymer-
bound tripeptide was cleaved from the solid support with
concomitant formation of the final amide linkage by treatment
with the benzyl ester of alanine to give Cbz2-faAVIA-Bz.23 The
blocked tetrapeptide was purified on silica gel (Rf ) 0.57, 7:3
EtOAc/hexanes), and the protecting groups were removed with
sodium in liquid ammonia to give faAVIA.24
Acknowledgment. This work was supported by Grant GM 21328
from the National Institutes of Health.
JA961214C
(24) HRMS (CI) calcd for C32H57N5O5: 592.4406. Found: 592.4438.
1H NMR (300 MHz, DMSO-d6 and CD3COOD) δ 8.45 (d, 1H, J ) 8.5
Hz, NH), 8.07 (d, 1H, J ) 6.6 Hz, NH), 7.98 (1H, J ) 8.7 Hz, NH), 5.25
(t,1H, J ) 6.8 Hz, H at C2), 5.16-5.02 (m, 2H, H at C6, C10), 3.76 (dd,
1H, J ) 7.8, 5.8 Hz, faA-R), 3.42 (d, 2H, J ) 6.8 Hz, H at C1), 3.00 (dd,
J ) 12.5, 5.8 Hz, faA-â), 2.82 (dd, 1H, J ) 12.5, 7.8 Hz, faA-â), 2.13-
1.83 (m, 9H, val-â, H at C4, C5, C8, C9), 1.82-1.69 (m, 1H, ile-â), 1.68-
1.61 (m, 6H, CH3 at C3, C7), 1.59-1.51 (m, 6H, CH3 at C10, H at C12),
1.25 (d, 3H, ala-â), 1.15-1.03 (m, 1H, ile-γ), 0.97-0.75 (m, 12H, val-γ,
ile-γ, δ); 13C NMR (75MHZ, DMSO-d6 and CD3COOD) δ 174.1, 170.5,
170.3, 141.1, 134.8, 130.7, 124.1, 123.6, 118.0, 57.8, 56.6, 51.2, 49.0, 47.8,
45.1, 39.2, 36.7, 30.6, 26.2, 25.9, 25.4, 24.2, 19.2, 17.8, 17.5, 16.3, 15.7,
15.2, 10.9.
Farnesylamine, â-aminoalanine, transition state analogs faA
and faAVIA, and product analogs S-farnesylcysteine and
S-farnesyl-CVIA were all examined as inhibitors of recombinant
yeast PFTase25 using a fluorescence assay26 to measure the rate
(21) Degrado, W. F.; Kaiser, E. T. J. Org. Chem. 1982, 47, 3258.
(22) Hagenmaier, J.; Frank, J. Hoppe-Seyler’s Z. Physiol. Chem. 1972,
353, 1973.
(23) HRMS (CI) calcd for C55H76N5O9: 950.5643. Found: 950.5619.
1H NMR (300 MHz, CDCl3) δ 7.45-7.22(m, 15H, aromatic Hs), 7.21-
7.10 (m, 2H, NH), 6.96 (d, 1H, J ) 8.7 Hz, NH), 5.25-5.04 (m, 9H, benzyl
Hs, H at C2, C6, C10), 4.62 (apparent t, 1H, J ) 7.3, 7.2 Hz, ala-R), 4.49-
4.37 (m, 2H, R-Hs), 4.35-4.26 (m, 1H, R-H), 3.85 (d, 1H, J ) 5.8 Hz, H
at C1), 3.70 (dd, 1H, J ) 14.8, 8.5 Hz, faA-â), 3.50 (d, J ) 14.8 Hz,
faA-â), 2.24-2.09 (bs, 1H, val-â), 2.10-1.69 (m, 9H, ile-â, H at C4, C5,
C8, C9), 1.65 (s, 3H, CH3 at C3), 1.60-1.40 (m, 10H, ile-γ, CH3 at C7,
C10, H at C12), 1.36 (d, 3H, J ) 7.2 Hz, ala-â), 1.20-0.98 (bs, 1H, ile-γ),
0.95-0.72 (m, 12H, val-γ, ile-γ, δ); 13C NMR (75MHZ, CDCl3) δ 172.4,
170.7, 170.5, 170.2, 157.9, 156.7, 140.1, 136.2, 137.1, 135.3, 135.2, 131.2,
128.4, 128.4, 128.2, 128.1, 128.0, 127.9, 127.7, 124.2, 123.6, 118.9, 67.6,
67.0, 67.0, 58.9, 57.7, 56.6, 48.6, 48.0, 45.6, 39.7, 39.7, 36.5, 30.0, 26.7,
26.3, 25.7, 24.8, 19.2, 18.0, 17.7, 16.2, 16.0, 15.4, 11.4.
(25) Mayer, M. P.; Prestwich, G. D.; Dolence, J. M.; Bond, P. D.; Wu,
H. Y. Poulter, C. D. Gene 1993, 132, 41.
(26) Cassidy, P. B.; Dolence, J. M.; Poulter, C. D. Methods Enzymol.
1995, 250, 30.
(27) Kothapalli, R.; Gutherie, N.; Chambers, A. F.; Carroll, K. K. Lipids
1993, 969.
(28) Poulter, C. D.; Capson, T. L.; Thompson, M. D.; Bard, R. S. J.
Am. Chem. Soc. 1989, 111, 3734.
(29) Steiger, A.; Pyun, H. J.; Coates, R. M. J. Org. Chem. 1992, 57,
3444.
(30) Cane, D. E.; Yang, G.; Coates, R. M. Pyun H. J. J. Org. Chem.
1992, 57, 3454.