Structurally simple farnesyltransferase inhibitors arrest the growth of malaria parasites

Article abstract of DOI:10.1002/anie.200500674

(Chemical Equation Presented) Antimalarial compounds: Structurally simple acyclic inhibitors of protein farnesyltransferase (active-site model shown) from the malaria parasite Plasmodium falciparum may allow third world countries access to an effective and inexpensive antimalarial therapy to counter the estimated half billion infections that occur annually.

Full text of DOI:10.1002/anie.200500674

Angewandte
Chemie
Medicinal Chemistry
DOI: 10.1002/anie.200500674
Structurally Simple Farnesyltransferase Inhibitors
Arrest the Growth of Malaria Parasites**
Matthew P. Glenn, Sung-Youn Chang, Oliver Hucke,
Christophe L. M. J. Verlinde, Kasey Rivas,
Carrie HornØy, Kohei Yokoyama, Frederick S. Buckner,
Prakash R. Pendyala, Debopam Chakrabarti,
Michael Gelb, Wesley C. Van Voorhis, SaïdM. Sebti,
andAndrew D. Hamilton*
The development of antimalarial agents is not only con-
stricted by the parameters common to the development of all
new drugs (efficacy, toxicity, pharmacology), but importantly,
must also recognize the economic reality of the countries
most affected. It is estimated that there are 300–500 million
acute cases of malaria each year, resulting in 1–3 million
deaths, with 90% of the mortality occurring in Africa.[1] The
severe poverty in the regions most affected by malaria and the
staggering number of people infected necessitate very inex-
pensive treatment. In an effort to decrease the cost of
developing new antimalarial drugs, recent attention has been
[*] Dr. M. P. Glenn, Dr. S.-Y. Chang, Prof. A. D. Hamilton
Department of Chemistry, Yale University
225 Prospect St., New Haven, CT 06511 (USA)
Fax: (+1)203-432-3221
E-mail: andrew.hamilton@yale.edu
Dr. O. Hucke, Prof. C. L. M. J. Verlinde
Department of Biochemistry, University of Washington
Seattle, WA 98195 (USA)
K. Rivas, C. HornØy, Prof. F. S. Buckner, Prof. W. C. Van Voorhis
Department of Medicine, University of Washington
Seattle, WA 98195 (USA)
Dr. K. Yokoyama, Prof. M. Gelb
Department of Chemistry and Biochemistry
University of Washington
Seattle, WA 98195 (USA)
Prof. S. M. Sebti
DrugDiscovery Program, H. Lee Moffitt Cancer Center and
Research Institute
Department of Oncology and Department of Biochemistry and
Molecular Biology
University of South Florida
Tampa, FL 33612 (USA)
Dr. P. R. Pendyala, Prof. D. Chakrabarti
Department of Molecular Biology and Microbiology
University of Central Florida
Orlando, Fl 32826 (USA)
[**] This work was supported by the Medicines for Malaria Venture
(MMV) and NIH grants CA67771 and AI054384. S.-Y.C. thanks the
Postdoctoral Fellowship Program of Korea Science & Engineering
Foundation (KOSEF). O.H. gratefully acknowledges a postdoctoral
fellowship of the German Academy of Natural Scientists Leopoldina
(BMBF-LPD 9901/8-77). We thank David Floyd, Lou Lombardo and
David Williams from Bristol-Myers Squibb for helpful suggestions.
Supportinginformation for this article is available on the WWW
under http://www.angewandte.org or from the author.
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directed at investigating parasitic proteins that are validated
The homology model indicates a large, open, and pre-
dominately hydrophobic cavity for the active site ( ꢀ 20”
20”20 Š3), with the phospholipid binding partner (FPP)
extending across the cavity base. The Zn ion coordinates to
three residues (Cys661, Asp659, and His838) with a water
molecule hydrogen bonded between the terminal phosphate
of FPP and Asp659, defining the limit of the Zn binding
domain (Figure 1). The remainder of the active-site cavity
includes two well-defined hydrophobic pockets (Lys149,
Asn317 Ser150, Phe150 and Trp456, Trp452, Tyr837), a
larger hydrophilic domain formed by Arg564, and three water
therapeutic targets in other diseases, thereby accelerating
access to new classes of inhibitors.[2,3]
Recently, prenylated proteins and associated prenyltrans-
ferases have been identified in the malarial protozoa Plasmo-
dium falciparum.[2] Prenylation is a required modification for
the function of a variety of cellular proteins, including small
G proteins,[4] heterotrimeric G-protein g subunits,[5] protein
kinases,[6] and nuclear lamins.[7] Studies of the role of
prenylation in modulating the function of the GTP-binding
proteins Ras and Rho, which have oncogenic forms common
to many human cancers ( ꢀ 30%), have identified the
ubiquitous eukaryotic enzyme protein farnesyltransferase
(PFTase) as a key target for the interception of aberrant
Ras activity.[8] Subsequent efforts to identify small-molecule
inhibitors of PFTase have led to several potential anticancer
agents currently in clinical trials.[9,10] The application of
related PFTase inhibitors to cells infected with P. falciparum
led to a decrease in farnesylated proteins and to the
associated lysis of the parasites.[11] Importantly, the PFTase
inhibitors studied were found to be significantly more toxic
against the parasite than to mammalian cells. In this study, we
developed a new series of PFTase inhibitors as novel
antimalarial agents, emphasizing simple molecular architec-
ture and straightforward chemical synthesis as a prerequisite
for access to low-cost treatment.
molecules participating in
between Ser449 and Gln152.
a hydrogen-bonded network
Flexible ligand-docking studies (GOLD)[18] indicate that
the four pockets of the homology model active site can be
efficiently accessed from a simple aliphatic tether, affording
PFTase inhibitors with an acyclic scaffold that can be
conveniently prepared through a short series of straightfor-
ward chemical transformations (Figure 1). One of the sim-
plest scaffolds conceivable, ethylenediamine, provides an
inexpensive and fourfold substitutable tether of appropriate
size to project four different groups into the active site
PFTase is one of three closely related heterodimeric zinc
metalloenzymes (protein farnesyltransferase and geranylger-
anyltransferases I and II) that catalyze the transfer of prenyl
groups from farnesyl or geranylgeranyl pyrophosphate to the
free thiol group of a cysteine residue within the tetrapeptide
recognition sequence CaaX (a = aliphatic amino acid, X is
often M, S, A, or Q for PFTase) located at the carboxyl
terminus of the substrate protein.[12] A homology model of
malarial PFTase (PfPFT) was generated with MODEL-
LER,[13] by using the crystal structure of rat PFT complexed
with the nonsubstrate tetrapeptide inhibitor CVFM, and
farnesyl diphosphate (FPP) as the template structure (PDB
entry 1JCR). The sequences of the two subunits (a and b) of
PfPFT were obtained from the PlasmoDB database (gene
loci: PFL2050w, a and chr11.glm_528, b)[14,15] and aligned with
the template with the program T-COFFEE.[16] Only regions
with reasonable reliability in the alignment were included.
The model of PfPFT comprises the following sequence
segments (the residue numbers of the corresponding seg-
ments of the rat PFT subunits are given in parentheses): a:
72–164 (87–179), 300–411 (184–283); b: 421–677 (71–315),
806–896 (330–417). PfPFT and rat PFT share 23% identity
and 53% similarity for the a subunit; the respective values for
the b subunit are 37 and 56%. The catalytic zinc ion, six
structurally conserved water molecules, and FPP were
included in the model. The conformation of FPP was
considered flexible during the model calculations. For this
purpose, the force-field parameters for FPP were added to the
MODELLER force field on the basis of the lipid parameters
of the charmm27 force field.[17] The model with the lowest
value of the objective function of MODELLER from twenty
different calculations was used for docking studies.
Figure 1. Active-site conformation of 15 (colored by atom type), as
determined by flexible ligand docking[18] in the homology model of the
active site of PfPFT (red=hydrophobic, blue=hydrophilic residues).
Values in parentheses refer to the correspondingpositions in rat PFT
(PDB entry 1JCR).
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Table 1: Activity of acyclic ethylene diamine compounds against PfPFT and parasite-infected cells.
pockets. The incorporation of imidazole
provides a zinc binding group that has
been consistently demonstrated to confer
activity in other series of inhibitors, whereas
small hydrophobic (phenyl, benzyl) and
simple sulfonamides (N-methylimidazole,
phenyl) are predicted as suitable substitu-
tions to access the remaining hydrophilic
and hydrophobic pockets.
Compound
IC50 [nm][a]
PfPFT
ED50 [nm][b]
Number
X
R1
R2
R3
H
3D7
K1
8
H
215
2.0
0.5
ND[c]
ND[c]
3200
9
1
Br
CH3
CH3
675
349
CN
375
An initial series of PFTase inhibitors
was prepared as outlined in Scheme 1.
Substituted anilines 4 were formed either
by the reductive amination of aniline (4-H,
4-Br), or by nucleophilic substitution of
para-fluorobenzonitrile with monopro-
tected ethylenediamine. Chemoselective
alkylation of aniline 4 through double
deprotonation with LDA and subsequent
alkylation at the anilide anion proceeds
smoothly in THF with no evidence of
alkylation of the carbamate. Some cyclic
urea is observed, presumably generated
through the elimination of tert-butoxide
from the carbamate to provide the corre-
sponding isocyanate and subsequent intra-
molecular cyclization to the anilide. Alky-
lation of carbamate 5, deprotection, and
10
11
CN
CN
CH3
CH3
8.9
55
2500
300
2250
388
12
13
CN
CN
CH3
CH3
4.5
220
850
>1000
2800
>5000
14
15
CN
CN
CH3
CH3
240
0.6
700
93
2500
150
[a] Inhibitor concentration required to decrease PfPFT activity twofold. [b] Inhibitor concentration
required to decrease hypoxanthine incorporation into parasites twofold. [c] Not determined.
inhibit PfPFT at concentrations of 50n m or lower, with four
compounds having IC50 values below 5 nm (1, 9, 12, and 15).
The growth of parasites in whole cells (3D7, K1) treated with
inhibitors was monitored through the incorporation of
tritium-labeled hypoxanthine. Six compounds displayed
ED50 values below 1 mm, with compound 15 showing signifi-
cant whole-cell activity (ED50: 3D7, 93 nm; K1, 150n m). The
significantly improved activity of compounds 1 and 9, relative
to that of 8, reflects docking solutions that suggest the para
substituent of aniline (X) contacts a hydrophilic domain
formed by Ser150, Asn317, and Lys149, whereas methylation
of the zinc binding group complements a small adjacent
hydrophobic pocket. Of the four substitutions investigated for
the R1 hydrophobic pocket, benzyl and cyclohexylmethyl
groups afforded best activity, whereas tert-butylcarbamate
and 4-phenylbenzyl groups are significantly less active. Three
substitutions were assessed for the hydrophilic cavity formed
predominately by Arg564. The small basic 1-methyl-1H-
imidazole-4-sulfonamide conferred the best potency against
both the enzyme and in whole-cell assays, whereas dansyl
sulfonamide afforded significantly lower enzyme inhibition,
but retained good potency (ED50 300 nm) against whole cells.
Importantly, these early examples of a new series of
simple acyclic PfPFT inhibitors demonstrate their reasonable
metabolic stability, and oral bioavailability. Treatment of 15
with rat liver microsomes resulted in a steady transformation
Scheme 1. Conditions: a) 4-fluorobenzonitrile, TEA, DMSO, 1208C;
b) 4-bromoaniline, NaCNBH3, acetic acid, molecular sieves (3 Š),
MeOH; c) LDA, NaH, 5-chloromethyl-1-methyl-1H-imidazole·HCl (R3),
THF, ꢁ788C!RT; d) NaH, alkyl bromide (R1), DMF, 08C; e) TFA;
f) arylsulfonyl chloride (R2), TEA, DMF. Boc=tert-butoxycarbonyl,
DMF=N,N-dimethylformamide, LDA=lithium diisopropylamide,
TEA=triethylamine, TFA=trifluoroacetic acid.
coupling to sulfonyl chlorides furnishes the desired series of
inhibitors (1, 8–15, Table 1).
A focused set of diversity elements, predicted by docking
studies (GOLD)[18] to efficiently occupy the four binding
domains of the active site, were applied in the preparation of
nine compounds, and their inhibition of PfPFT was deter-
mined by using a scintillation proximity assay with partially
purified PfPFT (Table 1).[19] Eight of the nine compounds
1
of the inhibitor over one hour (t = = 20min), with principal
2
metabolites observed by MS corresponding to oxidation of
the inhibitor (+ O), and loss of the aniline imidazole (R3, data
not shown). The promising metabolic stability of this series of
inhibitors is further reflected by the reasonable plasma
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concentration of inhibitor observed after oral dosing. Oral
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1
Figure 2. a) Metabolism of 15 by rat liver microsomes (t = =20 min);
2
b) inhibitor concentration in plasma of 15 in three rats ꢁa1fter oral
!
1
*
*
garage ( =rat 1, =rat 2, =rat 3; dose: 12.5 mgkg , t = ꢀ33 min,
2
c
max =1.05ꢂ0.21 mm, tmax =40ꢂ17 min).
In summary, a new series of simple acyclic PfPFT
inhibitors was developed, and their potency evaluated in
enzyme and whole-cell assays. Compounds based on this
readily accessible scaffold are highly active inhibitors of
PfPFT, with promising oral bioavailability. Expansion of this
inhibitor series is expected to provide potent, orally available
compounds as promising new low-cost drugs for the treatment
of malaria.
Received: February 22, 2005
Published online: July 11, 2005
Keywords: drugdesign · enzymes · inhibitors · malaria ·
.
medicinal chemistry
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Angew. Chem. Int. Ed. 2005, 44, 4903 –4906