Exploring in vitro and in vivo Hsp90 inhibitors activity against human protozoan parasites

Article abstract of DOI:10.1016/j.bmcl.2014.12.048

A set of compounds, previously selected as potent Hsp90α inhibitors, has been studied on a panel of human parasites. 5-Aryl-3,4-isoxazolediamide derivatives (1) were active against two protozoa, Trypanosoma brucei rhodesiense and Plasmodium falciparum, with a good tolerability toward cytotoxicity on non-malignant L6 rat myoblast cell line, unlike the 1,5-diaryl,4-carboxamides-1,2,3-triazole derivatives (2) which, while showing a single-digit nM range activity against the same protozoa, were also highly cytotoxic on L6 cells. In a subsequent in vivo study, two isoxazolediamide derivatives, 1a and 1b, were very efficacious on the sleeping sickness-causing agent with a clear parasitaemia during treatment. These data, however, showed that not all protozoa are sensitive to Hsp90 inhibitors, as well as not all Hsp90 inhibitors are equally active on parasites.

Full text of DOI:10.1016/j.bmcl.2014.12.048

Bioorganic & Medicinal Chemistry Letters 25 (2015) 462–465
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Exploring in vitro and in vivo Hsp90 inhibitors activity against
human protozoan parasites
⇑
Giuseppe Giannini , Gianfranco Battistuzzi
R&D Sigma-Tau Industrie Farmaceutiche Riunite S.p.A., Via Pontina Km 30,400, I-00040, Pomezia, Roma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A set of compounds, previously selected as potent Hsp90
a inhibitors, has been studied on a panel of
Received 18 November 2014
Revised 10 December 2014
Accepted 12 December 2014
Available online 20 December 2014
human parasites. 5-Aryl-3,4-isoxazolediamide derivatives (1) were active against two protozoa, Trypano-
soma brucei rhodesiense and Plasmodium falciparum, with a good tolerability toward cytotoxicity on
non-malignant L6 rat myoblast cell line, unlike the 1,5-diaryl,4-carboxamides-1,2,3-triazole derivatives
(2) which, while showing a single-digit nM range activity against the same protozoa, were also highly
cytotoxic on L6 cells. In a subsequent in vivo study, two isoxazolediamide derivatives, 1a and 1b, were very
efficacious on the sleeping sickness-causing agent with a clear parasitaemia during treatment. These
data, however, showed that not all protozoa are sensitive to Hsp90 inhibitors, as well as not all Hsp90
inhibitors are equally active on parasites.
Keywords:
Heat shock protein inhibitors
Hsp90 inhibitors
Trypanosoma brucei rhodesiense
Plasmodium falciparum
1,2,3-Triazole
Ó 2014 Elsevier Ltd. All rights reserved.
3,4-Isoxazolediamide
In pharmacological research it occurs quite often that a target is
first extensively investigated for oncological application, and then
studied in other areas. Hsp90, a cytosolic molecular chaperone
essential for protein homeostasis and activation of many signaling
proteins in the eukaryotic cell, and not only, does not escape from
this logic.
Hsp90 plays also a pivotal role in the life cycle control of many
parasites. A growing number of researchers are trying to unravel
the cellular mechanisms and the role of Hsp90 in these microor-
ganisms.4–9
Here, we focus on the antiprotozoan activity of a few com-
pounds, potent inhibitors of Hsp90a (Fig. 1; Table 1). Over the last
Heat shock proteins (HSPs) are classified in four main families
according to their molecular weight: Hsp90, Hsp70, Hsp60 and
small HSPs (15–30 kDa) that include Hsp27. They are expressed
either constitutively (i.e., Hsp90b) or regulated inductively (i.e.,
years, many reviews on this topic have been reported in literature.
Therefore, interested readers are encouraged to consult them10–13
as well as the patent literature14 to have a broader view of this
area.
Hsp90
a
), and are present in different subcellular compartments.
There are numerous examples from the literature where Hsp90
chaperone was considered a good target for the control of parasitic
infections, for example against Trypanosoma brucei,15 parasite that
causes sleeping sickness in Africa, and Trypanosoma evansi,16,17
responsible for surra disease of all domestic animals and recently
included in priority list B of significant diseases by the World
Organization of Animal Health (OIE). Besides, Hsp90 inhibitors
were investigated against the malarial parasite Plasmodium
falciparum,7,17 Leishmania braziliensis,6 responsible of visceral
leishmaniasis mainly in patients with human immunodeficiency
virus infection, and Leishmania donovani18 responsible for visceral
leishmaniasis. Few years before Echeverria et al. had reported the
study on Hsp90 on Toxoplasma gondii.9,19 These are only a few
examples that link the Hsp90 chaperone to parasites.
High molecular weight are ATP-dependent chaperones, whereas
small act in an ATP-independent fashion. The most studied stress
inducible HSPs are Hsp90, Hsp70 and Hsp27.1
Hsp90, a chaperone for protein folding and gene regulation, is
highly conserved from bacteria to mammals and it is documented
to interact with more than 200 different ‘client’ proteins involved
in signal transduction, protein trafficking, receptor maturation
and innate and adaptive immunity.2
Although no Hsp90 inhibitors have still been approved by the
FDA or other regulatory bodies worldwide, several drugs are cur-
rently investigated in various stages of clinical trials, mainly
against cancer.3
In recent years, our research group has completed an important
discovery project on new Hsp90 inhibitors. We report here the
⇑
Corresponding author. Tel.: +39 06 9139 3640.
E-mail address: giuseppe.giannini@sigma-tau.it (G. Giannini).
http://dx.doi.org/10.1016/j.bmcl.2014.12.048
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

G. Giannini, G. Battistuzzi / Bioorg. Med. Chem. Lett. 25 (2015) 462–465
463
H
O
O
N
N
N
N
N
N
O
O
N
N
N
H
H
O
H
O
N
N
O
O
N
2a
2b
1d
1a
O
1b
1c
HO
HO
O
O
O
H
O
O
N
N
S
O
N
N
1h
OH
OH
O
N
O
2c
2d
2
N
O
1
1e
1g
1f
1i
a (see synthesis in
F
Figure 2. 1,5-Diaryl,4-carboxamide-1,2,3-triazole-based inhibitors of Hsp90
a (see
synthesis in S.I.- Scheme 2).
Figure 1. 5-Aryl-3,4-isoxazolediamide-based inhibitors of Hsp90
S.I.- Scheme 1).
results of the antiprotozoan activity of a few selected Hsp90 inhib-
itors, having different chemical scaffolds.
HO
O
R= NHEt 3b
R= OEt 3a
N
N
Nine compounds with a 5-aryl-3,4-isoxazolediamide scaffold
(Fig. 1), four compounds with 1,5-diaryl,4-carboxamides-1,2,
3-triazole scaffold (Fig. 2), and two compounds with 4,5,6,7-tetrahy-
dro-isoxazolo-[4,5-c]-pyridine scaffold (Fig. 3), were evaluated.
Since chemistry and biological profile have already been pub-
lished,20–23 here we briefly summarized the synthesis (see S.I.)
O
OH
R
O
3
Figure 3. 4,5,6,7-Tetrahydro-isoxazolo-[4,5-c]-pyridine-based inhibitors of Hsp90
(see synthesis in S.I.- Scheme 3).
a
together with the biological data: binding to Hsp90
activity (Table 1).
a and cytotoxic
Hsp90 binding in the 30 nM range, resulted not to be very
efficacious against any protozoa. In this case, however,
membrane permeation could be the limiting factor, explain-
ing disappointing cell activity to the light of strong binding
affinity;
Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Plasmodium
falciparum and Giardia lamblia were the four protozoan species
selected for this study. Cytotoxicity was evaluated toward the
non-malignant L6 rat myoblast cell line. Two compounds, 1a and
2a, were also tested against Leishmania donovani, found inactive
c) compounds with high cytotoxic activity toward a wide range
of tumor cell lines, such as NCI-H460 and A431 [i.e., 1c, 1d,
1f, 1h, 1i, 2a, 2c], were also active against L6 cells, rendering
impossible any application as antiprotozoan;
at 1 lM and toxic at 3 lM (Table 2).
Through a careful analysis of the in vitro results, few consider-
ations may be highlighted:
d) triazole derivatives, with an IC50 in the single digit nanomo-
lar range, were among the most powerful against
Trypanosoma brucei and Plasmodium falciparum. Unfortu-
nately, they were also highly cytotoxic toward L6 cells.
a) in general, Trypanosoma brucei and Plasmodium falciparum
were more sensitive to these Hsp90 inhibitors than
Trypanosoma cruzi and Giardia, active only in a micro/
sub-micromolar range, with few exceptions;
b) the Hsp90 binding affinity were not highly predictive of the
in vitro antiprotozoan activity: 1h, with a very interesting
in vitro profile against Hsp90 and cytotoxicity, showed only
a weak activity on Trypanosoma brucei, unlike other deriva-
tives of the same class (3,4-isoxazolediamides). Also, the
two tetrahydro-isoxazole derivatives, 3a and 3b, with
2b, the only exception from triazole series, active in single nM
range against two protozoa and poor of cytotoxic toward both
A431 and L6, and two isoxazole derivatives, 1a and 1b, were
selected to assess their in vivo activity against Trypanosoma brucei
rhodesiense mouse model.
Table 1
Binding on Hsp90
a (FP Assay) and cytotoxicity on NCI-H460 (human non-small cell lung cancer) and/or A431 (epidermoid carcinoma) of selected compounds
ID
Lab. code
Binding Hsp90
(IC50; nM)
a
Cytotoxicity (IC50; nM)
NCI-H460
Refs.
A431
5-Aryl-3,4-isoxazolediamides
1a
1b
1c
1d
1e
1f
1g
1h
1i
SST0116CL1
210
71
340
24
5
20
40
2
810
20
110
120
17
13
640
22
200
25
22
20,23
20
20
20
20
20
20
20
20
SST0201CL1
SST0107AA1
SST0226AA1
SST0245AA1
SST0267AA1
SST0295AA1
SST0298AA1
SST0366AA1
13
50
14
11
1
1,5-Diaryl,4-carboxamides-1,2,3-triazoles
2a
2b
2c
2d
SST0287CL1
SST0374AA1
SST0440AA1
SST0441AA1
3
15
1
10
140
7
4
21
21
21
21
3
28
23
34
4,5,6,7-Tetrahydro-isoxazolo-[4,5-c]-pyridines
3a
3b
SST0430AA1
SST0431AA1
34
29
870
450
22
22

464
G. Giannini, G. Battistuzzi / Bioorg. Med. Chem. Lett. 25 (2015) 462–465
Table 2
Growth inhibitory effects of selected derivatives on four protozoa parasites in culture versus L6 cells cytotoxicity. A drug commonly used against each parasite was used as
reference compound (see also Table1-S.I.)24
ID
Drug name or Lab. code
Trypanosoma brucei
rhodesiense IC50 (nM)
Trypanosoma cruzi
IC50 (nM)
Plasmodium falciparum
IC50 (nM)
Giardia lamblia
IC50 (nM)
L6 cells Cytotoxicity
IC50 (nM)
Melarsoprol
3
Benznidazole
Chloroquine
409
3
Metronidazole
Podophyllotoxin
580
5
1a
1b
1c
1d
1e
1f
1g
1h
1i
SST0116CL1
SST0201CL1
SST0107AA1
SST0226AA1
SST0245AA1
SST0267AA1
SST0295AA1
SST0298AA1
SST0366AA1
22
18
14
4
126
18
10
763
12
16,700
6590
435
2700
4200
554
8060
1461
1380
37
25
29
12
117
35
36
26
17
983
146
201
7
15
888
20
279
40
25
9740
3193
8110
7570
4057
12,780
6430
2363
2a
2b
2c
2d
SST0287CL1
SST0374AA1
SST0440AA1
SST0441AA1
1
4
1
3
27
17,500
728
1
4
2
1
67
2
264
8
4460
356
400
4965
11
3a
3b
SST0430AA1
SST0431AA1
52,400
971
616
996
7265
4525
2460
4355
563
110
Table 3
In vivo activity against T.b.rhodesiense model: estimation of the parasitaemia25
% Activity versus control 50 mg/kg; ip qd; DMSO/water; 4 days (after 4 days)
% Activity versus control (after 10 days)
1a
1b
2b
Clear parasitaemia (under the detection limit)*
All mice relapsed on day 11
All mice relapsed on day 9
All mice dead on day 2
Clear parasitaemia (under the detection limit)*
Toxic (mice died after the 2nd administration)
*
The detection limit was about 10,000 parasites/ml.
The in vivo study (Table 3) confirmed the high toxicity of the
The authors would like to thank Dr. Gilles Pain for assistance
with the manuscript.
triazole derivative while the two isoxazole derivatives, after intra-
peritoneal administration (50 mg/kg, qd; 4 days) were found effec-
tive, loading to a total disappearance of the parasitemia after 4
days of treatment, checked in a sample of tail blood. Unfortunately,
parasitemia relapsed in about ten days after the end of treatment,
meanwhile a complete cure was obtained with melarsoprol at
4 mg/kg, qd, 4 days, used as reference drug.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.12.
048.
Taken together, these data showed that not all protozoa are sen-
sitive to Hsp90 inhibitors, as well as not all Hsp90 inhibitors are
equally active on sensitive parasites.
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This work was supported by grants from Sigma-tau Research
Switzerland S.A. Sigma-tau Research Switzerland S.A. retains the
property rights of all the compounds described in the Letter.
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