Peptidomimetic inhibitors of protein farnesyltransferase show potent antimalarial activity

Article abstract of DOI:10.1016/S0960-894X(01)00055-5

Malaria continues to represent a very serious health problem in the tropics. The current methods of clinical treatment are showing deficiencies due to the increased incidence of resistance in the parasite. In the present paper we report the design, synthe

Full text of DOI:10.1016/S0960-894X(01)00055-5

Bioorganic & Medicinal Chemistry Letters 11 (2001) 761±764
Peptidomimetic Inhibitors of Protein Farnesyltransferase Show
Potent Antimalarial Activity
Junko Ohkanda,a Je€rey W. Lockman,a Kohei Yokoyama,b Michael H. Gelb,b,*
Simon L. Croft,c Howard Kendrick,c Maria Isabel Harrell,d Jean E. Feagin,d,e
Michelle A. Blaskovich,f Said M. Sebtif,* and Andrew D. Hamiltona,*
aDepartment of Chemistry, Yale University, PO Box 208107, New Haven, CT 06520, USA
bDepartments of Chemistry and Biochemistry, University of Washington, Box 351700, Seattle, WA 98195-1700, USA
cDepartment of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street,
London WC1E 7HT, UK
dSeattle Biomedical Research Institute, 4 Nickerson St., Seattle, WA 98109-1651, USA
eDepartment of Pathobiology, School of Public Health and Community Medicine, University of Washington,
Seattle, WA 98195, USA
fDrug Discovery Program, H. Lee Mott Cancer Center and Research Institute, Department of Biochemistry and
Molecular Biology, University of South Florida, Tampa, FL 33612, USA
Received 11 September 2000; accepted 15 January 2001
AbstractÐMalaria continues to represent a very serious health problem in the tropics. The current methods of clinical treatment are
showing de®ciencies due to the increased incidence of resistance in the parasite. In the present paper we report the design, synthesis,
and evaluation of potential antimalarial agents against a novel target, protein farnesyltransferase. We show that the most potent
compounds are active against Plasmodium falciparum in vitro at submicromolar concentrations. # 2001 Elsevier Science Ltd. All
rights reserved.
Malaria is one of the most widespread infectious dis-
eases in the tropics. Nearly 300 million new cases occur
each year, resulting in more than one million deaths
annually. The fatal cases are generally caused by the
mostvirulenthuman malaria parasiet, Plasmodium fal-
ciparum. Current clinical treatment involves the use of
inexpensive antimalarial drugs such as chloroquine.
However resistance has rendered many of these drugs
ine€ective. Other malaria control e€orts have focused
on mosquito eradication programs, the development of
a vaccine against the parasites, and new drug targets
such as protease inhibitors or isoprenoid biosynthetic
pathway inhibitors.1 However, the slow development of
a vaccine and the pressing problem of chloroquine
resistance have created an urgent need for identifying
new drug targets in Plasmodium falciparum and for the
development of e€ective new antimalarial agents. In this
paper we report that a family of inhibitors of the
enzyme protein farnesyltransferase (FTase) shows
potent antimalarial activity in whole cell assays.
Mammalian FTase is a heterodimeric zinc metalloenzyme
composed of a 48 kDa a-subunitand a 46 kDa b-sub-
unit that catalyzes the transfer of a farnesyl group (C15)
from farnesyl pyrophosphate (FPP) to the thiol of a Cys
residue near the carboxyl terminus of target proteins.
The key C-terminal sequence that is recognized by
FTase is CaaX, where C is cysteine, aa is an aliphatic
dipeptide, X is often methionine, glutamine or serine. In
contrast, the related prenyltransferase, protein ger-
anylgeranyltransferase-I (GGTase-I), recognizes and
geranylgeranylates (C20) proteins that terminate in
CaaX, where X is leucine or phenylalanine. In recent
years, FTase has been a major target in the search for
novel anticancer agents. This e€ort was initially based
on the observation that the Ras family of G-proteins
normally cycle between their GTP (active) and GDP
(inactive) bound forms, to regulate intracellular signal
transduction. However, in the mutated form of Ras that
is found in over 30% of human cancers, the protein is
locked in the GTP bound state, and this results in
*Corresponding authors. Tel.: +1-203-432-5570; fax: +1-203-432-
3221; e-mail: andrew.hamilton@yale.edu. Tel.: +1-813-979-6734; fax:
+1-813-979-6748; e-mail: sebti@mott.usf.edu. Tel.: +1-206-543-
7142; fax: +1-206-685-8665; e-mail: gelb@chem.washington.edu.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00055-5

762
J. Ohkanda et al. / Bioorg. Med. Chem. Lett. 11 (2001) 761±764
uncontrolled cell growth. The requirement that Ras be
farnesylated and located in the plasma membrane for
oncogenesis prompted many investigators to target
FTase. In particular, we and others have reported pep-
tidomimetics of the CaaX sequence2À7 that are sub-
nanomolar inhibitors of FTase and suppress tumor
growth in nude mice without apparent gross toxicity.
the basic biphenyl sca€old, 2-phenyl-4-nitrobenzoic
acid. For compounds 2±6, the sca€old was coupled
with methionine methyl ester, and was reduced to the
aniline derivative. Reductive amination with the corre-
sponding 1-substituted 4- or 5-formylimidazole fol-
lowed by hydrolysis gave the free carboxylic acids. For
compounds 7±11, the sca€old was converted to the
t-butyl ester, and was hydrogenated to the amine. After
reductive amination with 1-(4-cyano)benzyl-5-formyl-
imidazole2 under treatment with TiCl4, followed by
deprotection under acidic conditions, the resulting ben-
zoic acid derivative was coupled with an alkylamine
using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride (EDCI)/1-hydroxybenzotriazole (HOBt)
to give the corresponding amides.
Protein prenylation occurs in protozoan parasites. We
have shown that protein farnesylation occurs in the
trypanosomatids Trypanosoma brucei, Trypanosoma
cruzi, and Leishmania mexicana amazonensis, and have
puri®ed, cloned, and expressed FTase from T. brucei.8,9
Recently, the genes coding for the subunits of P. falci-
parum FTase have been identi®ed in genome databases,
and Chakrabarti et al. reported that an FTase inhibitor
showed inhibition activity against the growth of P. fal-
ciparum in human red blood cells (IC50=43 mM).10
These ®ndings suggested that protein farnesylation in P.
falciparum could be a new target for antimalarial agents.
In this paper we report that a new series of tetrapeptide
CaaX mimetics show signi®cant inhibition against P.
falciparum in cell culture.
The whole cell inhibition assay of P. falciparum growth
in human red blood cells was carried outin vitro in a 48
h [3H]-hypoxanthine incorporation assay.12 The ability
of the peptidomimetic FTase inhibitors to slow the
growth of P. falciparum in human red blood cells is
reported in Table 1 as ED50 values and % inhibition at
20 mM. These two experiments were carried out inde-
pendently on the same strain (3D7). Comparison is also
made in Table 1 to the IC50 values for inhibition of
mammalian FTase and of H-Ras processing in NIH
3T3 cells.
Our strategy for the design of potent FTase inhibitors
involved designing mimics of the CaaX tetrapeptide in
which the central aliphatic dipeptide is replaced by a
hydrophobic and rigid spacer, such as a substituted 4-
aminobenzoyl group. This led to a family of highly
potent inhibitors of FTase, such as FTI-276 (1), which
has an IC50 againstht e mammalian enzyme of 0.5 nM,
blocked protein farnesylation in mammalian cells and
slowed the growth of human tumors in nude mouse
models.7 The discovery of FTase in parasites and the
likely critical role of farnesylated G-proteins in the
parasitic life cycle prompted us to investigate the e€ect
of our peptidomimetics as antiparasitic agents. We
recently showed that FTI-276 is also an e€ective inhi-
bitor of T. brucei FTase (IC50=1.7 nM) and blocks
parasite growth in the 10±50 nM range.8 On the basis of
these ®ndings with trypanosomatids we decided to
investigate the potential application of CaaX peptido-
mimetics against P. falciparum. Modi®cations to the
structure of the peptidomimetics were based on the
recent crystal structure of mammalian FTase bound to a
CaaX substrate and an unreactive farnesyl diphosphate
analogue. This showed that the tetrapeptide binds in an
extended conformation with the Cys thiol coordinating
to a zinc in the active site.11 In order to overcome the
problem of metabolic instablility of the thiol group in a
parasitic cell growth experiment, we replaced the
cysteine residue in FTI-276 with imidazole as an alter-
native metal binding functional group.
Compound 1 (FTI-276) did notshow any apparent
inhibition of parasite growth, whereas all of the imida-
zole-containing derivatives (with the exception of 4 and
12) inhibited parasite growth at concentrations below 30
Furthermore, an imidazole group readily allows the
introduction of various substituents to probe the pre-
sence of hydrophobic and/or polar domains near the
active site. The compounds investigated in this study are
listed in Chart 1. The basic synthetic approach used for
the preparation of the CaaX peptidomimetics is shown
in Scheme 1 for the synthesis of 2±11. Suzuki aryl
coupling of 2-bromo-4-nitrotoluene with phenylboronic
acid followed by oxidation of the methyl group a€orded
Chart 1.

J. Ohkanda et al. / Bioorg. Med. Chem. Lett. 11 (2001) 761±764
763
.
Scheme 1. (i) PhB(OH)2, Pd(OAc)2, K2CO3, 89%; (ii) KMnO4, 100%; (iii) HCl H-Met-OMe, EDCI, HOBt, NEt3, 89%; (iv) SnCl2, AcOEt, 90%;
(v) 1-substituted 4- or 5-formylimidazole, NaBCNH3, 54±91%; (vi) LiOH, THF, 72±92%; (vii) SOCl2, ButOK, 72%; (viii) H2/10% Pd±C, THF,
100%; (ix) 1-(4-cyano)benzyl-5-formylimidazole, TiCl4, NaBCNH3, 73%; (x) 50% TFA in CH2Cl2, 100%; (xi) R2NH2, EDCI, HOBt, NEt3,
77±86%. (xii, for 7) LiOH, THF, 90%.
Table 1. ED50 values for P. falciparum 3D7 growth inhibition and IC50
values for inhibition of mammalian FTase and of H-Ras processing
substituents at the C-terminus of the peptidomimetic
increase inhibitory activity (e.g., ED50=3.6 mg/mL for
10; ED50=9.91 mg/mL for 11). Compound 12 (FTI-
2148), one of the most potent mammalian FTase inhi-
bitors (IC50=0.001 mM), did notinhibitparaseit
growth, presumably because of poor membrane perme-
ability from the free methionine carboxylate terminus.
In contrast, the methyl ester form of 12, compound 13
(FTI-2153), showed high inhibition activity against
growth of P. falciparum (ED50=2 mg/mL).
Compds
P. falciparum P. falciparum
Mammalian H-Ras
infected RBC infected RBC processing IC50, mM
ED50 (mg/mL) (%) inhibitiona (IC50 for FTase, mM)
1 (FTI-276)
>30
10
12
>30
5
10
13
3
3
4
10
>30
2
nd
19.4
nd
nd
89.1
72
86.3
nd
nd
nd
nd
0
99
1 (0.0005)
>100 (3)
>100 (6)
nd (0.78)
>100 (0.26)
nd (0.041)
5 (0.0072)
<100 (0.38)
>100 (3.1)
nd (0.64)
2 (FTI-2218)
3 (FTI-2217)
4 (FTI-2237)
5 (FTI-2220)
6 (FTI-2238)
7 (FTI-2289)
8 (FTI-2277)
9 (FTI-2278)
10 (FTI-2279)
11 (FTI-2291)
12 (FTI-2148)
13 (FTI-2153)
The results shown here suggest that the active site
recognition properties of malarial FTase should be dif-
ferent from those of the mammalian enzyme, so that
selective inhibition of parasitic growth over mammalian
FTase inhibition should be possible.
20 (0.22)
0.2 (0.001)
0.02 (34)
aInhibition at 20 mM, RBC=red blood cell.
In summary, we have developed a series of non-thiol
FTase inhibitors that are potent inhibitors of the
growth of P. falciparum. We are continuing to investi-
gate P. falciparum FTase as a novel drug target and are
evaluating the antiparasitic activity of these and other
FTase inhibitors in animal models of malaria.
mg/mL. Compounds 5 and 6, in which the 5-position of
the imidazole ring is linked to the hydrophobic 4-ami-
nobenzoate spacer, showed about 2-fold higher activity
than corresponding 4-substituted isomers, 3 and 4. A
similar dependence on the substitution positions of the
imidazole was seen in the inhibition of mammalian
FTase and re¯ects a well-matched interaction of inhibi-
tors 5 and 6 with the enzyme active site. Interestingly,
compound 5 was more than 20 times more active at
inhibiting the growth of P. falciparum than in blocking
protein farnesylation in mammalian cells, re¯ecting
possible di€erences in FTase inhibition activity and
membrane permeation between the two organisms.
Acknowledgements
This work was supported by grants from the National
Institutes of Health (CA67771 to SMS and ADH, and
CA53874 to MHG) and UNDP/World Bank/WHO
Special Programme for Research and Training in
Tropical Diseases to SLC.
The simpler series of non-methionine-containing com-
pounds 8±11 also showed potency against P. falciparum
in the low mg/mL range. These are unlikely to inhibit
malarial FTase, and thus probably act by a mechanism
other than inhibition of protein prenylation. However,
con®rmation of this conclusion must await inhibition
studies on malarial FTase. Comparison among com-
pounds 8±10 and 11, suggests that larger hydrophobic
References
1. (a) Olliaro, P. L.; Yuthavong, Y. Pharmacol. Ther. 1999,
81, 91. (b) Witter, D. J.; Poulter, C. D. Biochemistry 1996, 35,
925. (c) Francis, S. E.; Gluzman, I. Y.; Oksman, A.; Banerjee,

764
J. Ohkanda et al. / Bioorg. Med. Chem. Lett. 11 (2001) 761±764
D.; Goldberg, D. E. Mol. Biochem. Parasitol. 1996, 83, 189.
(d) Rosenthal, P. J. Emerg. Infect. Diseases 1998, 4, 49. (e)
Jomaa, H.; Weisner, J.; Sanderbrand, S.; Altincicek, B.; Wei-
denmeyer, C.; Hintz, M.; Turbachova, I.; Eberl, M.; Zeidler,
J.; Lichtenthaler, H. K.; Soldati, S.; Beck, E. Science 2000,
285, 1573.
2. Williams, T. M.; Bergman, J. M.; Brashear, K.; Breslin,
M. J.; Dinsmore, C. J.; Hutchinson, J. H.; MacTough, S. C.;
Stump, C. A.; Wei, D. D.; Zartman, B.; Bogusky, M. J.; Cul-
berson, J. C.; Buser-Doepner, C.; Davide, J.; Greenberg, I. B.;
Hamilton, K. A.; Koblan, K. S.; Kohl, N. E.; Liu, D.; Lobell,
R. B.; Mosser, S. D.; O'Neill, T. J.; Rands, E.; Schaber, M. D.;
Wilson, F.; Senderak, E.; Motzel, S. L.; Gibbs, J. B.; Graham,
S. L.; Heimbrook, D. C.; Hartman, G. D.; Oli€, A. I.; Hu€,
J. R. J. Med. Chem. 1999, 42, 3779.
Rosario, J.; Doll, R. J.; Alvarez, C.; Lalwani, T.; Mallams,
A. K.; Rossman, R. R.; Afonso, A.; Girijavallabhan, V. M.;
Ganguly, A. K.; Pramanik, B.; Heimark, L.; Bishop, W. R.;
Wang, L.; Kirschmeier, P.; James, L.; Carr, D.; Patton, R.; Bry-
ant , M. S.; Nomeir, A.; Liu, M.J. Med. Chem. 1999, 42, 2651.
5. Qian, Y.; Marugan, J. J.; Fossum, R. D.; Vogt, A.; Sebti,
S. M.; Hamilton, A. D. Bioorg. Med. Chem. 1999, 7, 3011.
6. Qian, Y.; Vogt, A.; Sebti, S. M.; Hamilton, A. D. J. Med.
Chem. 1996, 39, 217.
7. Sebti, S. M.; Hamilton, A. D. Pharmacol. Ther. 1997, 71, 1.
8. Yokoyama, K.; Trobridge, P.; Buckner, F. S.; Van Voor-
his, W. C.; Stuart, K. D.; Gelb, M. H. J. Biol. Chem. 1998,
273, 26497.
9. Buckner, F.; Yokoyama, K.; Nguyen, L.; Grewal, A.; Erd-
jumen-Bromage, H.; Tempst, P.; Strickland, C. L.; Xiao, L.;
Van Voorhis, W. C.; Gelb, M. H. J. Biol. Chem. 2000, in press.
10. Chakarabarti, D.; Azam, T.; DelVecchio, C.; Qiu, L.;
Park, Y.; Allen, C. M. Mol. Biochem. Parasit. 1998, 1998, 175.
11. Strickland, C. L.; Windsor, W. T.; Syto, R.; Wang, L.;
Bond, R.; Wu, Z.; Schwartz, J.; Le, H. V.; Beese, L.; Weber,
P. C. Biochemistry 1998, 37, 16601.
3. Williams, T. M.; Ciccarone, T. M.; MacTough, S. C.; Bock,
R. L.; Conner, M. W.; Davide, J. P.; Hamilton, K.; Koblan,
K. S.; Kohl, N. E.; Kral, A. M.; Mosser, S. D.; Omer, C. A.;
Pompliano, D. L.; Rands, E.; Schaber, M. D.; Shah, D.; Wil-
son, F. R.; Gibbs, J. B.; Graham, S. L.; Hartman, G. D.; Oli€,
A. I.; Smith, R. L. J. Med. Chem. 1996, 39, 1345.
4. Taveras, A. G.; Deskus, J.; Chao, J.; Vaccaro, C. J.; Njor-
oge, F. G.; Vibulbhan, B.; Pinto, P.; Remiszewski, S.; del
12. Desjardins, R. E.; Can®eld, C. J.; Haynes, J. D. Anti-
microb. Agents Chemother. 1979, 16, 710.