CHEMMEDCHEM
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Cell culture: Epimastigotes of a Dm28c strain (discrete typing unit I)
were maintained at 268C in liver infusion tryptose (LIT) medium
(Life Technologies, Carlsbad, CA, USA) supplemented with 10%
fetal bovine serum (FBS, Life Technologies), 1% hemin (Sigma–Al-
drich, St. Louis, MO, USA), 1% R9 medium (Sigma–Aldrich), and
50 mgmLÀ1 gentamycin (Novafarma, Anꢀpolis, GO, Brazil). Metacy-
clic trypomastigotes of Y strain (discrete typing unit II) were ob-
tained from the supernatant of infected LLC-MK2 cells and main-
tained in RPMI-1640 medium (Sigma–Aldrich) supplemented with
10% FBS and 50 mgmLÀ1 gentamycin at 378C and 5% CO2. Spleno-
cytes were collected from BALB/c mice and cultivated in RPMI-
1640 medium supplemented with 10% FBS and 50 mgmLÀ1 genta-
mycin. Peritoneal exudate macrophages were elicited by intraperi-
toneal injection of sodium thioglycollate in BALB/c mouse.
fact, we found these compounds are antiparasitic agents and
have a high degree of selectivity. Our structure–activity rela-
tionship (SAR) studies revealed structural determinants for anti-
parasitic activity and led to the identification of thiazolidinone
derivatives, which displayed similar potencies to benznidazole.
Specifically, we demonstrated that thiazolidinone 4h has
strong antiparasitic activity, low cytotoxicity toward host cells
and achieves its anti-T. cruzi activity as a parasiticidal agent.
Most thiazolidinones, including active compound 4h, did not
inhibit cruzain activity, but these compounds affected the
Golgi apparatus as well as ER morphology and produced atypi-
cal cytosolic vacuoles, which ultimately is followed by a necrot-
ic parasite cell death. Consistent with in vitro antiparasitic ac-
tivity, thiazolidinone 4h reduced parasitemia in a mice model
of acute infection. Importantly, this compound displayed low
toxicity in mice and exhibited additive antiparasitic activity
when combined with benznidazole.
Cytotoxicity in splenocytes: Splenocytes of BALB/c mice (200 mL)
were placed into 96-well plates at 5ꢄ106 cells/well. Compounds
were added in a serial dilution (1.1, 3.3, 11, 33 and 100 mgmLÀ1) in
triplicate. To each well, an aliquot of compound suspended in di-
methyl sulfoxide (DMSO) was added. Negative (untreated) and pos-
itive (saponin, Sigma–Aldrich) controls were measured in each
plate, which was incubated for 24 h at 378C and 5% CO2. After in-
cubation, [3H]-thymidine (1.0 mCimLÀ1, PerkinElmer, Waltham, MA,
USA) was added to each well, and the plate was returned to the in-
cubator. Cells were then transferred to a filter paper using a cell
Experimental Section
Chemistry
harvester and measured using
a liquid scintillation counter
Synthetic protocols and spectral data for compounds are described
in the Supporting Information.
(WALLAC 1209, Rackbeta Pharmacia, Stockholm, Sweden). [3H]-Thy-
midine incorporation [%] was measured, and the highest noncyto-
toxic concentration was determined using triplicates.
Antiproliferative activity for epimastigotes: Epimastigotes were
counted in a hemocytometer, and 200 mL were dispensed into 96-
well plates at 106 cells/well. Compounds were added in a serial di-
lution (1.1, 3.3, 11, 33 and 100 mgmLÀ1) in triplicate. The plate was
incubated for 5 days at 268C, and aliquots of each well were col-
lected for counting the number of viable parasites using a Neuba-
uer chamber. The percentage of inhibition was calculated in rela-
tion to untreated cultures. IC50 values were calculated using nonlin-
ear regression on Prism 4.0 (GraphPad). Results are from one single
experiment.
Docking
The structures of all compounds were obtained by application of
the RM1 method,[42] available as part of the SPARTAN 08’ pro-
gram,[43] using internal default settings for convergence criteria.
Some of these new molecules were synthesized as racemic mix-
tures; therefore, the molecular modeling treated the two isomers
(R and S) independently, when appropriate, and the docking proce-
dure used both isomers for each compound. Docking calculations
and analysis were carried using the structure of T. cruzi cruzain
(PDBID: 3IUT) as the target, which is composed of a co-crystallized
complex with inhibitor (referred as “KB2”).[13] The active site was
defined as all atoms within a radius of 6.0 ꢃ from the co-crystal-
lized ligand. Residues Gln19, Cys25, Ser61, Leu67, Met68, Asn70,
Asp161, His162, Trp184 and Glu208 were treated as flexible during
the calculations, using a conformation library for each one. The
GOLD 5.1 program[44] was used for docking calculations, followed
by Binana program,[45] which was used to analyze the molecular in-
teractions present in the best docking solutions, using default set-
ting, except for hydrogen bond distance, which was changed to
a maximum of 3.5 ꢃ. Figures were generated with Pymol (version
1.3r1 edu).[46]
Toxicity for trypomastigotes: Trypomastigotes were collected from
the supernatant of LLC-MK2 cells, and 200 mL were aliquoted into
96-well plate in an axenic media at 4ꢄ105 cells/well. Compounds
were added in a serial dilution (1.1, 3.3, 11, 33 and 100 mgmLÀ1) in
triplicate. The plate was incubated for 24 h at 378C and 5% of CO2.
Aliquots of each well were collected, and the number of viable par-
asites, based on parasite motility, was counted in a Neubauer
chamber. The percentage of inhibition was calculated in relation to
untreated cultures. CC50 calculation was also carried out using non-
linear regression with Prism 4.0 (GraphPad). Two independent ex-
periments were performed.
Intracellular parasite development: Macrophages were seeded at 2ꢄ
105 cells/well for 24 h in a 24-well plate with rounded coverslips on
the bottom in RPMI-1640 medium supplemented with 10% FBS.
Macrophages were infected with trypomastigotes at a ratio of 10
parasites per macrophage for 2 h. Unbound trypomastigotes were
removed by successive washes with saline. Each compound was
dissolved in 5% DMSO and saline in a serial dilution, in triplicate.
Compounds remained on the cell culture for 6 h prior to removal
and addition of fresh media. The plate was incubated for 4 days at
378C and 5% CO2. Cells were fixed in MeOH, stained with Giemsa
and manual counting of at least 100 cells per slide was done using
an optical microscope (Model CX41, Olympus, Tokyo, Japan). The
Biology
Animals: Female BALB/c mice, aged 6–8 weeks, were supplied by
the animal house of Centro de Pesquisas GonÅalo Moniz (Fundażo
Oswaldo Cruz, Bahia, Brazil) and Centro de Pesquisas Aggeu Magal-
haes (Fundażo Oswaldo Cruz, Pernambuco, Brazil). Mice were
maintained in sterilized cages under a controlled environment, re-
ceiving a balanced diet for rodents and water ad libitum. All ex-
periments were carried out in accordance with the recommenda-
tions of ethical guidelines and were approved by the local Animal
Ethics Committee.
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