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Farnesyltransferase Inhibitors Inhibit the Growth of Malaria Parasites In Vitro and In Vivo.

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Enzyme Inhibitors
Farnesyltransferase Inhibitors Inhibit the Growth
of Malaria Parasites In Vitro and In Vivo**
Jochen Wiesner, Katja Kettler, Jacek Sakowski,
Regina Ortmann, Alejandro M. Katzin,
Emlia A. Kimura, Katrin Silber, Gerhard Klebe,
Hassan Jomaa, and Martin Schlitzer*
Farnesyltransferase catalyzes the posttranslational modification of numerous proteins involved in intracellular signal
transduction by transferring the farnesyl residue of farnesyl
pyrophosphate to the thiol of a cysteine side chain of the
protein substrate. The cysteine residue belongs to a characteristic carboxy-terminal consensus sequence, the so-called
[*] K. Kettler, Dr. J. Sakowski, Dr. R. Ortmann, Prof. Dr. M. Schlitzer
Ludwig-Maximilians-Universit&t M'nchen
Department Pharmazie—Zentrum f'r Pharmaforschung
Butenandtstrasse 5–13, 81377 M'nchen (Germany)
Fax: (+ 49) 89-2180-79992
Dr. J. Wiesner, Dr. H. Jomaa
Biochemisches Institut der Justus-Liebig-Universit&t Giessen
Friedrichstrasse 24, 35392 Giessen (Germany)
Prof. Dr. A. M. Katzin, Dr. E. A. Kimura
Departamento de Parasitologia
Instituto de CiÞncias BiomEdicas
Universidade de S¼o Paulo
Av. Professor Lineu Prestes, 1374
CEP 05508-900, S¼o Paulo (Brasil)
K. Silber, Prof. Dr. G. Klebe
Institut f'r Pharmazeutische Chemie
Philipps-Universit&t Marburg
Marbacher Weg 6, 35032 Marburg (Germany)
[**] This work was supported by the European Commission (INCO-Dev,
5th Framework Programme, contract no. ICA4-CT-2001-10078). We
thank Dajana Henschker for excellent technical assistance.
Angew. Chem. Int. Ed. 2004, 43, 251 –254
DOI: 10.1002/anie.200351169
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
CAAX box (C: cysteine, A: amino acid with aliphatic side
chain, X: serine or methionine).[1–3] Various inhibitors of
farnesyltransferase have been developed as potential anticancer therapeutics. The compounds of several pharmaceutical companies are in advanced stages of clinical testing.[4, 5]
Farnesyltransferases have been identified in other eukaryotic organisms besides humans, including pathogenic protozoa of the genera Plasmodium,[6, 7] Trypanosoma,[8–10] Leishmania,[10] and Toxoplasma.[11] Therefore, inhibition of the
farnesyltransferase has also been suggested as new strategy
for the treatment of parasitic infections.[4] In this context, the
treatment of malaria tropica caused by the infection with
Plasmodium falciparum is of particular relevance. Approximately 40 % of the world population lives in areas with
malaria risk, and 2 to 3 million people die each year from
malaria. As a consequence of the increasing spread of malaria
parasites resistant to chloroquine and other antimalarials,
there is an urgent need for new treatments.[12, 13]
Table 1: In vitro activity (determined according to ref. [21]), in vivo
The development of farnesyltransferase inhibitors as
activity (in mg kg 1 bw), and solubility of compounds 1–4.
antimalarial drugs is hampered by the fact that the heterCmpd.
IC50 [nm]
Solubility [mm]
ologous expression of the farnesyltransferase gene from
P. falciparum has not been achieved so far, and, therefore,
270 30
< 0.04
< 0.06
no recombinant enzyme is available for routine screening.
270 35
> 3.33
Only the native protein has been purified from in vitro
64 11
< 0.04
< 0.06
parasite cultures and used to demonstrate its inhibition by
210 21
different established farnesyltransferase inhibitors.[7] How[a]
ever, for the development of new chemotherapeutics growthrepeated
inhibition data obtained with cultured blood stages of the
of 22 mg kg 1 bw and an ED90 value of 28 mg kg 1 bw were determined.
parasite seem to be more significant. Gelb et al. have tested a
series of farnesyltransferase inhibitors derived from the group
The introduction of the methylpiperazinyl residue had no
of Sebti and Hamilton against such blood cultures and
influence on the in vitro activity in the case of the compound
reported activities in the micromolar range.[14]
with unsubstituted phenyl ring (cf. 1 and 2 in Table 1). In the
During the last years we have developed a new class of
series of a-unsubstituted compounds, significantly increased
farnesyltransferase inhibitors based on a benzophenone
activity was achieved with a para-chloro substituent on the
scaffold.[15] Compounds of this type suppress the growth of
terminal phenyl group (cf. 1 and 3). Although less prothe multiresistant P. falciparum strain Dd2 with IC50 values in
nounced, this effect was also observed with the a-piperazinyl
the nanomolar range and are thus one to two orders of
derivatives (cf. 2 and 4). These structure–activity relationships
magnitude more active than previously described farnesylcould be rationalized by docking studies based on the X-ray
transferase inhibitors.[16–19] To our disappointment, the inhibitors with the highest in vitro potency, such as
compounds 1 and 3, turned out to be inactive in
a murine malaria model, presumably due to
insufficient solubility in water. We have now
tried to achieve this required in vivo activity by
introducing a methylpiperazinyl residue to the
a-position of the phenyl acetyl moiety of the 2amino group of the benzophenone scaffold.
This modification resulted in significantly
improved solubility in water (Table 1).
For the synthesis of these compounds
(Scheme 1) 2-amino-5-nitrobenzophenone was
treated with commercially available a-chlorophenylacetyl chloride and a-bromo-p-chlorophenylacetyl chloride (prepared according to
ref.[20]). Subsequently, the a-halo substituent
was replaced by the heterocycle. After reducScheme 1. Synthesis of compounds 2 and 4. a) R C6H4 CHX COCl, toluene/dioxane,
tion of the nitro group, a second acylation step 2 h, reflux; b) N-methylpiperazine, acetonitrile, 24 h, reflux; c) SnCl , ethyl acetate, 2 h,
with the biarylacrylic acid resulted in the target reflux; d) NO2 C6H4 C4H2O CH=CH COCl, toluene/dioxane, 2 h, reflux. X = Cl, Br;
compounds 2 and 4.
R = H, Cl.
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2004, 43, 251 –254
structure of the rat farnesyltransferase (PDB code: 1QBQ).[22]
Even though the farnesyltransferases from P. falciparum and
rat show low overall sequence identity due to several
additional external loops of the P. falciparum enzyme, the
active-site residues are nearly identical.
Docking runs were performed with the Lamarckian
genetic algorithm included in AutoDock 3.0[23, 24] (standard
parameters) with 50 independent runs per ligand. For the
protein, polar hydrogens were added with the PROTONATE
utility in AMBER 7,[25] AMBER united-atom force-field
charges[26] were assigned, and solvation parameters were
added with the ADDSOL utility from AutoDock 3.0. For the
ligands, Gasteiger partial atomic charges[27] were assigned,
and all bonds except for amide bonds were kept rotatable. In
contrast to the biological testing with the racemates, only S
enantiomers were considered for molecular docking.
Resulting ligand conformations with similar structures
(rms deviation < 1 C) were clustered together and represented by the conformer with the best docking energy. For
both compounds 2 and 4 the largest cluster was obtained on
rank 3 (AutoDock score). Their representative solutions were
chosen for the structure–activity comparison, and the relative
binding affinity was predicted with the knowledge-based
scoring function DrugScore.[28]
The docking results clearly show that both inhibitors
adopt a similar binding mode (Figure 1). Yet, the chlorosubstituted inhibitor 4 has better interactions with the protein
than compound 2; in particular it could form a N H···Cl
interaction with His 149. The increase in activity attributed to
the chloro subtituent is less pronounced for the piperazinyl
derivatives than for the unsubstituted inhibitors because the
bulky piperazinyl group reduces this interaction.
Importantly, both piperazinyl derivatives display significant in vivo activity in mice infected with P. vinckei. The in
vivo testing was carried out according to a modified standard
protocol (Peters' test).[29] On day 0 Balb/c mice were infected
with 5 I 107 parasites from the blood of a donor mouse. On
days 1 to 3 the mice were treated by intraperitoneal (i.p.)
Figure 1. Superposition of selected docking results of the inhibitors 2
(turquoise) and 4 (yellow) in the binding pocket of the farnesyltransferase. Only the amino acids forming the pocket responsible for the difference in affinity are shown. The zinc ion is represented by a blue
ball. The two inhibitors have a comparable binding mode. The slightly
increased activity of compound 4 can be explained by an additional
N H···Cl interaction with His149 (in part hidden by the surface).
Angew. Chem. Int. Ed. 2004, 43, 251 –254
injection once a day of the test substance with dosages of 6, 13,
25, and 100 mg per kg bodyweight (mg kg 1 bw). Each group
consisted of three (50 and 100 mg kg 1 bw) or four (6, 13, and
25 mg kg 1 bw) mice. On day 4 the parasitaemia (percentage
of infected erythrocytes) was counted microscopically on
Giemsa-stained blood smears. The untreated control mice
developed parasitaemias between 50 and 90 %. In contrast,
after application of the test substances either no or a reduced
number of parasites was detectable in the blood of the
animals. As we had observed in the in vitro experiments, the
introduction of the chloro substituent in the para position of
the phenyl moiety led to an increased activity in the mouse
model; with 4 an ED50 value of 21 mg kg 1 bw was determined, in comparison to 30 mg kg 1 bw with the unsubstituted
compound. The corresponding ED90 values were 25 and
40 mg kg 1 bw, respectively. The small ratio of ED90 to ED50,
together with the high efficacy observed already after a single
dose, is particularly promising for the further development as
antimalarials. As a potential limitation, no oral activity was
observed with the presently available compounds. Toxic
effects were observed only with doses above 100 mg kg 1
bw, providing an acceptable therapeutic range for these lead
In order to confirm the mechanism of action an experiment was performed in which the influence of the test
compounds can be monitored directly on the farnesylation
level of parasite proteins in different developmental stages.[30]
A culture of P. falciparum was exposed for 48 h to a subtoxic
concentration of compound 4. After 30 h of treatment,
tritium-labeld farnesyl pyrophosphate was added. At the
same time a control experiment without 4 was conducted.
Under these conditions, the microscopic examination did not
reveal any differences in growth behavior between the two
cultures. Only when the incubation time was extended over
48 h, enabling the parasites to enter a second replication cycle,
did the number of ring stages diminish under the influence of
4. The different parasite stages were separated by densitygradient centrifugation. Subsequently, protein extracts were
prepared, and comparable amounts of protein from the
inhibition experiment and the control experiment were
separated by gel electrophoresis. Farnesylated proteins were
finally detected by autoradiography. In the untreated control
culture a characteristic pattern of farnesylated proteins was
observed in all parasite stages similar to that described
previously by Chakrabarti et al.[7] and Katzin et al.[30] In the
experiment with compound 4 the intensity of the farnesylation was significantly reduced (Figure 2). This effect was
particularly pronounced in the trophozoite and schizont
stages. In a control experiment the effect of compound 4
was compared with the established farnesyltransferase inhibitor FTI-277[7] (Figure 3). Principally, a comparable inhibition
of the protein farnesylation was observed with both compounds; however, the effect of compound 4 at a concentration
of 10 nm was significantly more pronounced than that of FTI277 at 5 mm. Thus, we could provide strong evidence that the
antimalarial activity of these compounds in fact depends on
the inhibition of the farnesyltransferase of the parasite.
In summary, we could demonstrate that the inhibition of
farnesyltransferase is a valid strategy for the development of
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. Inhibition of the protein farnesyltaion by compound 4. In
vitro cultures of P. falciparum were metabolically labeled with
[1 (n)-3H]farnesylpyrophosphate triammonium salt, and the different
development stages of the parasites (R: ring stages, T: trophozoites,
S: schizonts) separated by discontinuous Percoll gradient centrifugation. Farnesylated proteins were detected by autoradiography after electrophoresis over a 12.5-% sodium dodecylsulfate polyacrylamide gel.
Lane 1: untreated control parasites; lane 2: after incubation with
10 nm compound 4; lane 3: control with uninfected erythrocytes.
Molecular mass standards are indicated in kDa.
Figure 3. Comparison of the inhibition of the protein farnesylation with
FTI-277 (lane 1, 5 mm) and compound 4 (lane 2, 10 nm). Lane 3:
untreated control parasites.
new chemotherapeutics for the treatment of malaria. In
comparison to previously described compounds, our farnesyltransferase inhibitors based on the benzophenone scaffold
possess superior activity. To the best of our knowledge, we
have demonstrated for the first time an in vivo activity of
farnesyltransferase inhibitors against plasmodium infections.
The future development of this class of compounds will
primarily be aimed at achieving oral bioavailability and
further increasing the activity.
Received: February 13, 2003
Revised: September 12, 2003 [Z51169]
Keywords: drug design · enzyme inhibitors · farnesyltransferase ·
malaria · medicinal chemistry
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