Molecular design, synthesis and biological evaluation of 1,4-dihydro-4-oxoquinoline ribonucleosides as TcGAPDH inhibitors with trypanocidal activity

Article abstract of DOI:10.1016/j.bmcl.2013.06.029

The 1,4-dihydro-4-oxoquinoline ribonucleoside, Neq135, is the first low micromolar trypanosomatidae inhibitor to show good ligand efficiency (0.28 kcal mol^-1 atom^-1) and good ligand lipophilicity efficiency (0.37 kcal mol^-1

Full text of DOI:10.1016/j.bmcl.2013.06.029

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4597–4601
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Molecular design, synthesis and biological evaluation
of 1,4-dihydro-4-oxoquinoline ribonucleosides as
TcGAPDH inhibitors with trypanocidal activity
Fabyana A. Soares a,b, Renata Sesti-Costa c, João Santana da Silva c, Maria Cecília B. V. de Souza d,
Vitor F. Ferreira d, Fernanda da C. Santos d, Patricia A. U. Monteiro a, Andrei Leitão a,
Carlos A. Montanari a,b,
⇑
a Grupo de Química Medicinal do Instituto de Química de São Carlos, da Universidade de São Paulo, Av. Trabalhador Sancarlense, 400, 13566-590 Carlos/SP, Brazil
b Departamento de Química, Universidade Federal de São Carlos, Rodovia Washington Luiz, Km 235, Caixa Postal 676, CEP 13565-905 São Carlos/SP, Brazil
c Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, 14049-900 Ribeirão Preto/SP, Brazil
d Universidade Federal Fluminense, Instituto de Química, Departamento de Química Orgânica, Outeiro de São João Batista sem no., 24020-150 Niterói/RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
The 1,4-dihydro-4-oxoquinoline ribonucleoside, Neq135, is the first low micromolar trypanosomatidae
inhibitor to show good ligand efficiency (0.28 kcal molꢀ1 atomꢀ1) and good ligand lipophilicity efficiency
(0.37 kcal molꢀ1 atomꢀ1) when acting against Trypanosoma cruzi glyceraldehyde 3-phosphate dehydroge-
nase (TcGAPDH). This and other six ribonucleosides were synthesized using our in-house technology, and
assayed against the GAPDH NAD+ site using isothermal titration calorimetry (ITC). Compound Neq135
Received 23 April 2013
Revised 5 June 2013
Accepted 11 June 2013
Available online 24 June 2013
had acceptable in vitro cytotoxicity, inhibited TcGAPDH with a Kai pp value of 16
lM and killed the trypo-
Keywords:
Chagas disease
Trypanosoma cruzi
GAPDH
Isothermal titration calorimetry
mastigote form of Trypanosoma cruzi Tulahuen strain with a concentration similar to that displayed by
the control drug benznidazole. Neq135 is tenfold lower kinetic affinity against hGAPDH and does not kill
Balb-c fibroblast nor spleen mouse cells. These results emphasize the possibility of integrating ligand-
and target-based designs to uncover potent and selective TcGAPDH inhibitors that expands the opportu-
nity for further medicinal chemistry endeavor towards NAD+ TcGAPDH site.
Ó 2013 Elsevier Ltd. All rights reserved.
Trypanosomiasis is the name of several diseases caused by the
haemo-flagellates of the genus Trypanosoma from which Chagas
disease (caused by Trypanosoma cruzi) is one of the serious health
problems now widespreading out from Latina American countries
to USA, Europe and Japan.1 There are only two drugs for the treat-
ment of Chagas disease (nifurtimox and benznidazole), which are
actually effective in only 50% of all cases and display ghastly and
undesirable side effects.2 For instance, in two recent works,3,4 the
unwanted drug-related adverse events (ADRs) were described to
impose severe discomfort to patients in use of nifurtmox and
benznidazole. For patients in the regime of benznidazole the with-
drawal from treatment was observed to occur within 11 days of
administration.4 Hence, due to the low effectiveness of the
pharmacological treatment and their high toxicity, there is an
overwhelming necessity for new and safe drugs to treat chagasic
people that at present can only rely on such drugs to alleviate their
suffering. Efforts, nevertheless, are on the way and the drug
posaconazole5 is already in clinical studies for oral treatment of
asymptomatic chronic Chagas disease.
The glycosomal GAPDH is a T. cruzi target that has being used
for hit discovery6,7 in the aim that such enzyme inhibitors have a
role in the development of novel chemotherapeutic agents for
the treatment of Chagas disease. To date, either inhibitors acting
at the active or the NAD+ cosubstrate sites have been found so
far, but none has ever evolved to development phases. The ratio-
nale behind NAD+ competitive inhibitors relies on the fact that
such inhibitors can selectively bind to TcGAPDH over the human
enzyme. The most potent competitive inhibitor that binds to
NAD+ site is a disubstituted analog that inhibits TcGAPDH at nano-
molar concentration (see compound 2, Fig. 1), and is selective to
TcGAPDH.8 The NAD+ KM measured value differences (at pH 8.0)
for trypanosomal GAPDH (KM = 258
l
M)9 as compared to the hu-
man enzyme (KM = 62
l
M10 or 50
l
M at pH 7.711) maybe quite
appropriate for the search of selective inhibitors. In addition to
this, if human GAPDH (hGAPDH) is inhibited by 85% no effect will
be seen in the host.12 Therefore, these findings have prompted us
to search for new and improved NAD+ competitive inhibitors and
led us to point out some structure–activity relationship (SAR) for
this type of inhibitor and consequently describe certain useful pat-
terns for paving this desired chemical space.13 Hitherto, however,
our best-synthesized compound proved to be a weak TcGAPDH
⇑
Corresponding author. Tel.: +55 16 3373 9986; fax: +55 16 3373 9985.
E-mail address: carlos.montanari@usp.br (C.A. Montanari).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.06.029

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F. A. Soares et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4597–4601
Figure 1. Representative structures of TcGAPDH NAD+ inhibitors. Compound 1: IC50 = 40
lM; compound 2: IC50 = 0.1 lM; compound 3: IC50 = 16 lM.
inhibitor whose pIC50 value was 4.39 (compound 1, Fig. 1).11 Since
we have managed to develop our synthetic capabilities to obtain
the 1,4-dihydro-4-oxoquinoline ribonucleosides from our previ-
ously protected ribonucleosides,14 we additionally investigated
such compounds against TcGAPDH to show that they have better
ligand and ligand lipophilicity efficiencies than the reported mole-
cules.6 Besides that, molecules of this series are selective towards
TcGAPDH as compared to hGAPDH. For instance, it is noteworthy
that the kinetic affinity ratio Kai ppðhGAPDHÞ=KiappðTcGAPDHÞ for
inhibitor Neq135 (see Table 1), is 10. Additionally, compound
Neq135 is shown, for the first time, to kill the highly infective
Tulahuen T. cruzi strain. Hence, oxoquinoline ribonucleosides here-
in described are promising new scaffolds for the development of
new selective TcGAPDH and T. cruzi inhibitors. Moreover, we have
also carried out some preliminary cytotoxicity studies for some of
our inhibitors against mouse fibroblast and mouse spleen cells.
These molecules showed good selectivity for the desired target
over cytotoxicity and the development of this type of scaffold is
promising. In addition, because the TcGAPDH shares to Saccharo-
myces cerevisiae GAPDH 51%, 52%, and 52% amino acid sequence
for TDH1, TDH2 and TDH3 genes, respectively, we examined their
activities upon this yeast and none of the assayed compounds was
Table 1
Representation of 2D structures of the set and experimental ITC NAD+ KM and Kiapp
values
a
NAD+ KM
(lM)
Kai pp
(lM)
Compounds
R6 = H
49.98 8.73
46.94 2.52
46.98 2.25
62.57 2.33
63.35 2.37
48.51 3.18
67.73 3.71
15.98 1.27
17.10 1.00
18.67 0.96
29.53 1.31
32.27 0.99
57.61 3.84
59.49 2.27
R7 = Br
Neq135
R6 = H
R7 = CH3
Neq137
R6 = OCH3
R7 = H
Neq142
R6 = Br
R7 = H
active up to the concentration of 500 lM.
Neq139
R6 = Cl
Our previous model using principal component analysis (PCA)
depicted compound 1, Figure 1, in a similar region of high positive
PC1 values where most potent GAPDH inhibitors (including
nucleosides, like compound 2, Fig. 1) were also found. Neverthe-
less, the main issue for these molecules was the striking undesir-
able ligand and ligand lipophilicity efficiencies against TcGAPDH.
To address this problem, more chemically tractable derivatives
from 1 would arise by simply hydrolyzing the –Bz groups, as exem-
plified by compound 3 in Figure 1.
R7 = H
Neq140
R6 = CH3
R7 = H
Neq141
R6 = H
R7 = OCH3
Neq138
a
Average KM values for NAD+ in the absence of inhibitors: 38.6 1.48; kcat
/
KM = 1.33 ꢁ 106 sꢀ1 Mꢀ1
.
Briefly, the syntheses of 1,4-dihydro-4-oxo-1-(b-D-dihybofur-
anosyl) quinoline-3-carboxylic acids were achieved by treatment
of meta and para-substituted anilines with diethyl ethoxymethyl-
enemalonate to obtain the enamine-type derivatives, which were
then cyclized in refluxing Dowtherm A, Scheme 1. Protected ribo-
We firstly assayed the newly synthesized 1,4-dihydro-4-oxo-
quinoline ribonucleosides against TcGAPDH using Isothermal Titra-
tion Calorimetry (ITC) as previously described.15,16 The resulting
kinetic parameters can be found in Table 1, with very good statisti-
cal parameters shown in Table 2. Compounds competitively inhib-
nucleosides
ethyl
1,4-dihydro-4-oxo-1-(2,3,5-tri-O-benzoyl-
b- -ribofuranosyl)quinoline-3-carboxylates were prepared by
D
ited TcGAPDH against NAD+ in the range of 16–68
lM, with average
previous silylation of oxoquinolines using bis-(trimethyl)triflu-
oracetamide (BSTFA) containing 1% of trimethylchlorosilane
(TMCS), and subsequent condensation reaction with 1-O-acetyl-
ligand efficiency (LE) of 0.27 kcal molꢀ1 atomꢀ1. This is a sharp
improvement when compared to compound 2 (Fig. 1) that has a
LE of 0.15 kcal molꢀ1 atomꢀ1. These data also show that by hydro-
2,3,5-tri-O-benzoyl-b-
60–70 °C, under
D
-ribofuranose in acetonitrile as solvent, at
trimethylsilyltrifluoromethanesulphonate
lyzing the benzoyl moieties of compound
1
we gain
0.15 kcal molꢀ1 atomꢀ1 in ligand efficiency for these new 1,4-dihy-
dro-4-oxoquinoline ribonucleosides. Furthermore, the averaged
(TMSOTf) catalysis. Removal of the protecting benzoyl groups
was achieved by treating these compounds with ethanolic sodium
hydroxide solution (0.5 M) at 50–60 °C, producing the free
ligand lipophilicity efficiency (LLE) is ca. 0.49 kcal molꢀ1 atomꢀ1
,
ribonucleoside 1,4-dihydro-4-oxo-1-(b-D-ribofuranosyl)quinoline-
which is remarkably good to evaluate the druglikeness quality of
these compounds.
Some noteworthy results emerged when changing Cl, Br, CH3,
OCH3 positions from carbon-6 to carbon-7 in the oxoquinoline
3-carboxylic acids, and characterized as previously described.14
See Supplementary data for synthetic details and structure
characterization.

F. A. Soares et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4597–4601
4599
Scheme 1. Synthetic route used for preparing 1,4-dihydro-4-oxo-1-(b-D-ribofuranosyl)quinoline-3-carboxylic acids.
Table 2
Table 3
Fit of ITC experimental data parameters as in Table 1
Statistical parameters for the ITC experimental fit of compounds Neq138, 135 and 142
whose Kai pp values were determined against TcGAPDH G3P site
b
AICca
R2
Sum of squares
Sy.x
p Test
AICc
R2
Sum of
squares
Sy.x
p Test
Kai pp
(lM)
Neq135
Neq137
Neq142
Neq139
Neq140
Neq141
Neq138
ꢀ1010.61
ꢀ1111.48
ꢀ1160.74
ꢀ1348.52
ꢀ1517.80
ꢀ1298.13
ꢀ1506.56
0.92
0.96
0.96
0.97
0.99
0.95
0.98
0.12
0.06
0.04
0.01
0.003
0.01
0.004
2.94 ꢁ 10ꢀ2
2.07 ꢁ 10ꢀ2
2.00 ꢁ 10ꢀ2
9.09 ꢁ 10ꢀ3
5.05 ꢁ 10ꢀ3
1.08 ꢁ 10ꢀ2
5.25 ꢁ 10ꢀ3
<0.001
<0.001
<0.001
<0.001
<0.001
<0.005
<0.001
Neq138 105.10 3.39
Neq135 150.94 3.62
Neq142 305.44 30.41 ꢀ608.767 0.91 0.15
ꢀ591.277 0.93 0.07
ꢀ629.492 0.93 0.12
0.02 <0.001
0.04 <0.001
0.04 <0.001
a
Second-order Akaike’s information criterion.
The standard deviation of errors in regression.
assayed against both the active and cosubstrate sites could prefer-
entially bind to where it is easier to compete for the site. It is also
noteworthy that the enzyme efficiency is higher for G3P than for
NAD+ at both expenses of larger kcat (faster turnover) and smaller
KM value. Moreover, the KM and Vmax values obtained when we var-
ied the G3P concentration are indicative of the noncompetitive
inhibition, since the KM values are the same despite the variation
of Vmax values.
b
ring. For instance, we get an increase of twofold in kinetic affinity
by just changing the bromine atom from position 6 to 7 (com-
pounds Neq139 and Neq135). Variation at the R6 position from
bromine (Neq139) to a chlorine atom (Neq140) takes place with-
out a significant change in the kinetic affinity. Following this trend,
by exchanging the methyl group from R6 (Neq141) to R7 (Neq137)
we get an increase of threefold plus in kinetic affinity. On the other
hand, by replacing a hydrogen with a methoxy group at the posi-
tion R6 to get compound Neq142, we observe a threefold increase
in the kinetic affinity, as compared to substitution at position R7
(Neq138). It maybe therefore speculated that this observation is
due to a stronger pKa perturbation of compound Neq142 that bears
the methoxy group at the C7 position. Moreover, the ITC KM values
for assaying study compounds against the TcGAPDH NAD+ always
The compound with lower Kai pp value towards TcGAPDH acting
on the NAD+ site, Neq135, was also evaluated against the hGAPDH,
and showed a tenfold increase on its inhibition constant value. This
is remarkably interesting considering that the NAD+ KM value for
hGAPDH has dropped to 22.31 0.48 M with an enzyme efficiency
of 31.
All results obtained so far compelled us to further understand
the biological activity of these molecules using in vitro models.
The first screening for cytotoxicity assessment of some chemicals
was performed on Saccharomyces cerevisiae17, once it grows fast,
with easy maintenance and detection methods like the fluorogenic
compound FUN-1Ò (Invitrogen/Molecular Probes).18 FUN-1 accu-
mulates in dead or metabolic compromised cells and they present
diffused green fluorescence at the cytoplasm, while live yeasts
metabolize FUN-1, being concentrated into cylindrical intravacuo-
lar structures (CIVS) with red fluorescence.19 Cell viability is deter-
increased, while Vmax remained constant at 0.24 0.01 l .
M sꢀ1
This means we can ascertain the competitive mechanism for these
inhibitors. Non-linear least-squares regression analysis is thor-
oughly used to calculate the kinetic parameters. Therefore, the
competitive mechanism is directly obtained from ITC measure-
ment curves and any transformation to linearize data, as in the
case of well-known double-reciprocal analysis of Lineweaver–
Burk,14 is not necessary. Nonetheless, when doing so the straight
lines cross the same point at 1/Vmax, which is, as a result, un-
changed. Since KM becomes KM + KM[I]/KI and thus the apparent
ES association gets weaker, KM gets larger, as demonstrated for
the competitive inhibition towards NAD+. In order to further vali-
date this, we have carried out the inhibition assays for compounds
mined as
a red/green ratio referring to live/dead yeasts,
respectively. This fast screening was performed to a subset of com-
pounds at the highest concentration that did not result in solubility
problems (500 l
mol Lꢀ1). Cytotoxicity was observed for Neq138
and Neq139 only (Fig. 2). This screening gave promising results
to undertake further studies of these molecules in other cell-based
assays due to the overall low cytotoxicity.
Neq135, 138 and 142 against the TcGAPDH active site where D-
glyceraldehyde-3-phosphate (G3P) is converted to the product
1,3-biphosphoglicerate (1,3-BPG), Table 3.
Table 4
Kinetic parameters for TcGAPDH G3P and NAD+
Neq138, 135 and 142 have inhibition constant ðKai ppÞ values that
are far higher when assayed against the TcGAPDH G3P site than
when assayed against the TcGAPDH NAD+ site. This provides an-
other evidence in favor of the NAD+ competitive binding mode
for the whole set, which are also noncompetitive in relation to
the G3P substrate. The lower KM value for TcGAPDH G3P over
NAD+ (Table 4) indicates the enzyme needs lower concentration
of G3P than NAD+, which suggests the same set of compounds
Parameters
Vmax
M sꢀ1
KM M)
G3P
NAD+
(l
)
0.63 0.01
34.10 1.23
146 0.61
4.28
0.24 0.01
40.14 3.13
51.37 0.33
1.28
0.917
0.011
(
l
kcat (sꢀ1
)
kcat/KM (ꢁ106 sꢀ1 Mꢀ1
)
R2
0.986
0.011
Sy.x

4600
F. A. Soares et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4597–4601
same range of the reported Balb-c 3T3 cell cytotoxic value of
mol Lꢀ1 retrieved from the literature.21
The most promising TcGAPDH inhibitors from the biochemical
2
l
studies (Neq135 and Neq142) were also tested in vitro using T. cru-
zi and mouse spleen cells. Trypomastigote T. cruzi Tulahuen strain
was cultured and compounds were assayed as previously de-
scribed.22 Spleen cells from C57BL/6 mice were isolated by
mechanical dissociation and screened according to our previous
work.23 Compounds were incubated in 96-well plates with
5.106 cells mLꢀ1 during 24 h at 37 °C with 5% CO2. Cells were har-
vested, following incubation with 10 l
g mLꢀ1 propidium iodide
and data were acquired using a FACSCantoll flow cytometer.
Neq135 was equipotent to benznidazole in these assays (Fig. 4).
In contrast to Neq135, Neq142 was inactive against the parasite,
which led to strikingly different selectivity index (Table 5).
In summary, the study compounds had low cytotoxicity for S.
cerevisiae, mouse fibroblast and spleen cells, which gave a good
safety index for all compounds in these short-term in vitro assays.
Among all, Neq135 was the best compound of the series being ac-
tive against the trypomastigote T. cruzi Tulahuen strain with high
Figure 2. Viability screening of NEQUIMED compounds in S. cerevisiae.
The second assay employed the MTT colorimetric method for
Balb-c 3T3 mouse fibroblast cell lineage.20 Balb-c 3T3 is one of the
most common cell lineages in use to access the cytotoxicity
profile of bioactive compounds. As depicted in Figure 3, five com-
pounds were not cytotoxic (Neq135, Neq137, Neq138, Neq141
and Neq142) while Neq140 exhibited some cytotoxicity at the
highest concentration only, causing 30% of cell death. Neq140 was
Table 5
Trypanocidal activity and in vitro cytotoxicity with selectivity index
Trypomastigotea
mol Lꢀ1
Mouse
spleen
Balb-c 3T3
mol Lꢀ1
Selectivity
index
cytotoxic above the concentration of 100
l
mol Lꢀ1 and Neq139
Compound
(
l
)
(
l
)
was cytotoxic above 10
l
mol Lꢀ1. The highest cytotoxic compound
(l )
mol Lꢀ1
was Neq139. For the sake of comparison, posaconazole (an anti-
Neq135
Neq142
Benznidazole 19
18
>500
>500
>500
>500
ND
>500
ND
>27
1
>26
fungal drug in phase 2 clinical trial for Chagas Disease, http://clini-
caltrials.gov) inhibited cell growth at the rate of 20% at 500 l .
mol L-1
Doxorubicin (an anticancer drug) was used as a control for the col-
orimetric assay. The IC50 obtained (3.24 0.39
a
l
mol Lꢀ1) is in the
Trypanosoma cruzi Tulahuen strain. ND = not determined.
120
110
100
90
80
70
60
50
40
30
110
100
90
80
70
60
50
40
30
20
Neq135
Neq137
Neq138
Neq139
Neq140
Neq141
Neq142
20
10
0
Posaconazole
Doxorubicin
Benznidazole
10
0
log [cpd]
log [cpd]
Figure 3. In vitro cytotoxic assays using Balb-c 3T3 fibroblast cells. Results were normalized according to the untreated cells cultured with 0.5% DMSO.
Figure 4. Biological evaluation of benznidazole (Bz), Neq135 and Neq142 in mouse spleen (a) and trypomastigote strain (b). Average value plus the standard deviation of two
experiments in duplicate are shown for each sample.

F. A. Soares et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4597–4601
4601
13. Leitão, A.; Andricopulo, A. D.; Oliva, G.; Pupo, M. T.; de Marchi, A. A.; Vieira, P.
C.; Silva, M. F. G. G.; Ferreira, V. F.; Souza, M. C. B. V.; Sa, M. M.; Moraes, V. R. S.;
Montanari, C. A. Bioorg. Med. Chem. Lett. 2004, 14, 2199.
selectivity index and potency as good as for the reference drug
benznidazole (Table 5). Neq135 is a fairly potent NAD-driven small
molecule inhibitor of TcGAPDH with molecular weight below
500 Da. Therefore, Neq135 is a promising lead-like compound for
additional development considering it has high ligand and lipo-
philicity efficiencies, act through a novel mode of action compared
to compounds in development and has a comparable selectivity in-
dex towards the parasite in relation to the benznidazole drug.
14. (a) Canuto, C. V. B. S.; Gomes, C. R. B.; Marques, I. P.; Faro, L. V.; Santos, F. C.;
Frugulhetti, I. C. P. P.; Souza, T. M. L.; Cunha, A. C.; Romeiro, G. A.; Ferreira, V. F.;
de Souza, M. C. B. V. Lett. Drug Des. Disc. 2007, 4, 404; (b) Deffin, G. F.; Kendall, J.
D. J. Am. Chem. Soc. 1948, 70, 893; (c) Snyder, H. R.; Freier, H. E.; Kovacic, P.;
Heyningen, E. M. V. J. Am. Chem. Soc. 1947, 69, 371; (d) Lauer, W. M.; Arnold, R.
T.; Tiffany, B.; Tinker, H. J. Am. Chem. Soc. 1946, 68, 12; (e) Ramsey, V. G.;
Cretcher, L. H. J. Am. Chem. Soc. 1947, 69, 1659; (f) Riegel, B.; Lappin, G. R.;
Adelson, B. H.; Jackson, R. I.; Albisetti, C. J., Jr.; Dodson, R. M.; Baker, R. H. J. Am.
Chem. Soc. 1946, 68, 1264; (g) Roberts, R. B. J. Am. Chem. Soc. 1948, 70, 277.
15. Wiggers, H. J.; Cheleski, J.; Zottis, A.; Oliva, G.; Andricopulo, A. D.; Montanari, C.
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Acknowledgments
16. Cheleski, J.; Wiggers, H. J.; Citadini, A. P.; Da Costa Filho, A. J.; Nonato, M. C.;
Montanari, C. A. Anal. Biochem. 2010, 399, 13.
17. S. cerevisiae was acquired from The Brazilian Collection of Environmental and
Industrial Microorganisms (CBMAI code 0194) and cultured in a shaker at 37 °C
with 220 rpm in Luria Broth medium (Fermentas) supplied with 2% glucose
and acetate buffer (10 mmol Lꢀ1, pH 5).
The Brazilian granting agencies CAPES, CNPq and FAPESP
(grants #2011/01893-3 and 2011/07025-3, São Paulo Research
Foundation - FAPESP). Are acknowledged for supporting this re-
search. Fabiana Rosini for helpful aid in laboratory maintenance
and TcGAPDH expression and purification.
18. (a) Essary, B. D.; Marshall, P. A. J. Microbiol. Methods 2009, 78, 208; (b) Van der
Heggen, M.; Martins, S.; Flores, G.; Soares, E. V. Appl. Microbiol. Biotechnol. 2010,
88, 1355; (c) Kwolek-Mirek, M.; Zadrag-Tecza, R.; Bartosz, G. Cell Biol. Toxicol.
2012, 28, 1; (d) Millard, P. J.; Roth, B. L.; Thi, H. P. T.; Yue, S. T.; Haugland, R. P.
Appl. Environ. Microbiol. 1997, 63, 2897.
Supplementary data
19. LIVE/DEADÒ Yeast Viability Kit protocol from Invitrogen/Molecular Probes was
used with some minor modifications, described here. Log phase S. cerevisiae
was centrifuged for 5 min at 9500 rpm and ressuspended with NaHEPES buffer
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.06.
029.
(pH 7.2) to reach 107 cells mLꢀ1. 100
of a black 96-well plate. Upon use, working solutions of 200
l
L of this solution was added to each well
l
mol Lꢀ1 FUN-1 in
NaHEPES were prepared and incubated during 30 min with and without the
inhibitors to stain the yeast. All plates were read at 37 °C every 10 min for 1 h
using Synergy HT Multi-Mode Microplate Reader (BioTec Instruments, Inc.).
FUN-1 was excited at 485 nm, emitting at 530 nm (green) and 620 nm (red)
and a red/green ratio was defined for live/dead yeast. Yeast viability was
determined using control (no treatment) and all compounds with 0.6% DMSO
in each well. DMSO alone did not disturb yeast growth, according to the
absorbance measured at 700 nm and cell counting using Neubauer chamber.
20. Balb-c 3T3 mouse fibroblasts cells were acquired from the Rio de Janeiro Cell
Bank (BCRJ) and grown in a CO2 incubator using 5% CO2 atmosphere at 37 °C,
using DMEM medium (Cultilab, Brazil) supplemented with 10% FBS (Sigma–
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Aldrich) and 1% penicillin/streptomycin solution (Cultilab, Brazil). 100 lL of
cells (105 cells mLꢀ1) were pipetted in 96-wells plates and incubated for 24 h.
Medium was discarded and compounds were added to each well with 24 h
incubation. Following medium removal, 100
was added to the wells. After 3 h incubation, supernatant was discarded and
100 L solubilizing solution (10 g SDS, 100 mL DMSO, and 0.6 mL glacial acetic
l )
L MTT solution (1.25 mg mLꢀ1
l
acid) was pipetted into each well. Plates were read at 570 nm after 1 h
incubation (two experiments in quadruplicate were performed).
ˇ
´
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