2-Octadecynoic acid as a dual life stage inhibitor of Plasmodium infections and plasmodial FAS-II enzymes

Article abstract of DOI:10.1016/j.bmcl.2014.07.050

The malaria parasite Plasmodium goes through two life stages in the human host, a non-symptomatic liver stage (LS) followed by a blood stage with all clinical manifestation of the disease. In this study, we investigated a series of 2-alkynoic fatty acids (2-AFAs) with chain lengths between 14 and 18 carbon atoms for dual in vitro activity against both life stages. 2-Octadecynoic acid (2-ODA) was identified as the best inhibitor of Plasmodium berghei parasites with ten times higher potency (IC₅₀ = 0.34 μg/ml) than the control drug. In target determination studies, the same compound inhibited three Plasmodium falciparum FAS-II (PfFAS-II) elongation enzymes PfFabI, PfFabZ, and PfFabG with the lowest IC₅₀ values (0.28-0.80 μg/ml, respectively). Molecular modeling studies provided insights into the molecular aspects underlying the inhibitory activity of this series of 2-AFAs and a likely explanation for the considerably different inhibition potentials. Blood stages of P. falciparum followed a similar trend where 2-ODA emerged as the most active compound, with 20 times less potency. The general toxicity and hepatotoxicity of 2-AFAs were evaluated by in vitro and in vivo methods in mammalian cell lines and zebrafish models, respectively. This study identifies 2-ODA as the most promising antiparasitic 2-AFA, particularly towards P. berghei parasites.

Full text of DOI:10.1016/j.bmcl.2014.07.050

Bioorganic & Medicinal Chemistry Letters 24 (2014) 4151–4157
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Octadecynoic acid as a dual life stage inhibitor of Plasmodium
infections and plasmodial FAS-II enzymes
b
c
d
Néstor M. Carballeira a, , Angela Gono Bwalya , Maurice Ayamba Itoe , Adriano D. Andricopulo ,
⇑
María Lorena Cordero-Maldonado e, Marcel Kaiser f,g, Maria M. Mota c, Alexander D. Crawford e,
Rafael V. C. Guido d, Deniz Tasdemir b,h,
⇑
a Department of Chemistry, University of Puerto Rico, PO Box 23346, San Juan 00931-3346, Puerto Rico
b Department of Biological and Pharmaceutical Chemistry, University of London, School of Pharmacy, London WC1N 1AX, UK
c Instituto de Medicina Molecular, Universidade de Lisboa, Lisbon 1649-028, Portugal
d Laboratório de Química Medicinal e Computacional, Centro de Pesquisa e Inovação em Biodiversidade e Fármacos, Instituto de Física de São Carlos,
Universidade de São Paulo, São Carlos, SP 13563-120, Brazil
e Chemical Biology Group, Luxembourg Centre for Systems Biomedicine, University of Luxembourg, L-4362 Esch-sur-Alzette, Luxembourg
f Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, CH-4002 Basel, Switzerland
g University of Basel, Petersplatz 1, CH-4003 Basel, Switzerland
h School of Chemistry, National University of Ireland, Galway, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
The malaria parasite Plasmodium goes through two life stages in the human host, a non-symptomatic
liver stage (LS) followed by a blood stage with all clinical manifestation of the disease. In this study,
we investigated a series of 2-alkynoic fatty acids (2-AFAs) with chain lengths between 14 and 18 carbon
atoms for dual in vitro activity against both life stages. 2-Octadecynoic acid (2-ODA) was identified as the
Received 7 July 2014
Revised 15 July 2014
Accepted 17 July 2014
Available online 24 July 2014
best inhibitor of Plasmodium berghei parasites with ten times higher potency (IC50 = 0.34
control drug. In target determination studies, the same compound inhibited three Plasmodium falciparum
FAS-II (PfFAS-II) elongation enzymes PfFabI, PfFabZ, and PfFabG with the lowest IC50 values (0.28–0.80 g/
lg/ml) than the
Keywords:
Acetylenic fatty acids
Blood stage
Liver stage
Malaria
2-Octadecynoic acid
Plasmodium
Type II fatty acid synthase
l
ml, respectively). Molecular modeling studies provided insights into the molecular aspects underlying
the inhibitory activity of this series of 2-AFAs and a likely explanation for the considerably different inhi-
bition potentials. Blood stages of P. falciparum followed a similar trend where 2-ODA emerged as the most
active compound, with 20 times less potency. The general toxicity and hepatotoxicity of 2-AFAs were
evaluated by in vitro and in vivo methods in mammalian cell lines and zebrafish models, respectively.
This study identifies 2-ODA as the most promising antiparasitic 2-AFA, particularly towards P. berghei
parasites.
Ó 2014 Elsevier Ltd. All rights reserved.
With more than one third of the planet’s population at risk of
infection, malaria remains the most devastating parasitic disease.
Past and current research for antimalarial drug discovery has lar-
gely focused on easily cultivable blood stage (BS) parasites for
the treatment of the disease. The major problem in this area is
the emergence of drug resistance. Even against the artemisinins
and their combination therapies there are already reports of signs
of resistance.1,2 Liver stage, the first, clinically silent stage preced-
ing the symptomatic BS in humans, is recently gaining research
interest, due to low parasite load, hence a lower risk of resistance
development. Most importantly, elimination of liver stage (LS)
parasites provides causal malaria prophylaxis through blockage
of the initiation of the BS. Sadly, primaquine, the only FDA
approved drug with LS activity is associated with many drawbacks
and limitations. Therefore, new approaches might be necessary for
both prevention and treatment of malaria. This could include the
identification of new drug candidates with selectivity against one
life stage, as well as dual inhibition of both liver and blood forms.
Among the vast number of acetylenic fatty acids known to date,
the 2-alkynoic fatty acids (2-AFAs) are among the most interesting
for the diversity of biological activities that they display coupled to
the challenging mechanisms of action underlying their toxicity
towards a wide range of pathogenic cells and organisms. In this con-
text, 2-hexadecynoic acid (2-HDA) is among the best-studied 2-
AFAs. 2-HDA displays in vitro antimycobacterial,3 antileishmanial,4
antiplasmodial,5 antibacterial,6 antifungal7 and cytotoxic activity.8
⇑
Corresponding authors.
E-mail addresses: nestor.carballeira1@upr.edu (N.M. Carballeira), deniz.tasdemir@
nuigalway.ie (D. Tasdemir).
http://dx.doi.org/10.1016/j.bmcl.2014.07.050
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

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N. M. Carballeira et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4151–4157
Different mechanisms proposed to date responsible for the bioactiv-
ity of the 2-AFAs include dual inhibition of fatty acid biosynthesis by
two 2-HDA metabolites (3-oxohexadecanoic acid and 3-hexadecy-
noic acid) that block key metabolic enzymes responsible for fatty
acid biosynthesis and degradation,3 inhibition of important proto-
zoal enzymes such as topoisomerases IB and type II fatty acid syn-
thase (FAS II) enzymes,4,5 inhibition of fatty acid elongation and
acylation, in particular triglyceride synthesis in cancer cells,8 as well
as necrosis.9
2-ODA
Previous studies on 2-HDA and other analogs have shown that
the alkyl chain length is the most important determinant for the
biological activity of the 2-AFAs. The important work by Morbidoni
et al. (2006) has identified a relationship between fatty acid chain
length and antimycobacterial activity against Mycobacterium
smegmatis.3 In the latter work Morbidoni established that the
antimycobacterial activity of the 2-AFAs decreased in the order
2-TDA
C
18 > C16 > C19 > C14 > C20 ꢀ C22 ꢀ C12. This trend has remained con-
stant against other biological systems as well, in particular with
respect to their antiprotozoal activities.4 We recently reported on
the slightly better antileishmanial activity of 2-octadecynoic acid
(2-ODA, C18) when compared to 2-HDA (C16) against Leishmania
donovani (EC50 value of 11 lM vs 17.8 lM, respectively). The same
trend also translates into target enzyme inhibition since in the lat-
ter study 2-ODA turned out to be a better inhibitor of the Leish-
mania topoisomerase IB enzyme as compared to either 2-HDA or
2-tetradecynoic acid (2-TDA, C14) with EC50 values of 5
lM vs 28
and 68
l
M, respectively.4 A plausible explanation for this tendency
2-HDA
has not been proposed yet. Sanabria-Ríos et al. (2014) determined
the critical micelle concentration (CMC) of both 2-AFAs and found
that the CMC of 2-HDA (CMC >90
lg/ml) is higher than the CMC of
2-ODA (CMC = 50
l
g/ml).6 However, whether this translates into
their biological activities remains to be investigated.
In a recent study, we reported in vitro antiprotozoal activity of
2-HDA towards BS of Plasmodium falciparum (IC50 = 10.4
lg/ml)
Figure 1. Impairment of P. berghei infection in human hepatoma cells, Huh7, by 2-
AFAs. Human hepatoma cells were infected with luciferase-expressing P. berghei
sporozoites and treated at 2hpi with 2-fold dilutions of test compounds; 2-ODA, 2-
TDA, 2-HDA or DMSO (vehicle), or 15 lM primaquine (internal control). Infection
(expressed as percentage of control) was analyzed at 48 hpi. Red lines indicate cell
and LS forms of Plasmodium yoelii (IC50 = 15.3
l
g/ml).5 Moreover,
we were able to show that 2-HDA was a potent inhibitor of the
P. falciparum type II fatty acid synthase (PfFAS-II), a key system
for de novo production of fatty acids in the late LS development
of the parasite.5 2-HDA blocks the activity of three crucial PfFAS-
II enzymes responsible for enoyl reduction (PfFabI), b-hydroxyacyl
dehydration (PfFabZ), and b-ketoacyl reduction (PfFabG) with IC50
confluency at the time of analysis. AU: arbitrary units.
by fluorescence intensity measurements after incubation with
the active plasma membrane labeling dye Alamar Blue (red line,
Fig. 1). Confocal imaging of parasites immunostained with anti-
PbHsp70 (P. berghei heat shock protein 70, green) antibody reveals
that parasites were greatly impaired in development as shown by
representative images (Fig. 2). As shown in Table 1, 2-ODA was the
most potent P. berghei active compound with an IC50 value
values ranging between 0.38 and 3.50 l
g/ml.5 Based on these pre-
liminary results, we have extended our work to the synthesis and
antimalarial activity assessment of 2-ODA and 2-TDA against both
BS (P. falciparum K1) and LS (Plasmodium berghei) parasites. The
potential activity of these 2-AFAs against the same PfFAS-II
enzymes was also studied by in vitro enzyme inhibition assays
and docking studies. Finally, in vitro and in vivo toxic and hepato-
toxic potential of 2-ODA, 2-TDA, 2-HDA, as well as palmitic acid
(PA) was investigated on cell lines and zebrafish larvae to allow
the identification of the most promising acetylenic fatty acid of
the series.
The synthesis of 2-TDA, 2-HDA, and 2-ODA was previously
reported by us and others.4,10 These compounds are synthesized
from the reaction of the corresponding 1-alkyne with n-BuLi in
THF at À70 °C followed by quenching with CO2 followed by proton-
ation with NH4Cl. The purity of all the final AFAs was >97%, as
determined by capillary GC–MS and 13C NMR.
We first studied the growth inhibitory effects of 2-ODA and
2-TDA against P. falciparum K1 and P. berghei parasites, and com-
pared these to those of 2-HDA and PA. To achieve this, hepatoma
Huh7 cells were infected with rodent malaria parasite, luciferase-
expressing P. berghei sporozoites and treated with compounds for
48 h, as described previously.11 Treatment with 2-ODA and
2-TDA greatly impaired infection (Fig. 1) with no apparent effects
on cell viability of host human hepatic cell (Huh7) as determined
(0.34
primaquine. This potency is even superior to that of 2-HDA
(IC50 = 0. 48 g/ml) on P. berghei (Table 1). Interestingly, we previ-
ously determined a lower anti-LS activity of 2-HDA against another
rodent model, P. yoelii, with IC50 values of 15.3 g/ml (assessed by
flow cytometry) and 4.88 g/ml (assessed by immunofluorescence
activity).5 The potency of 2-TDA on the hepatic stage P. berghei par-
asites was much poorer (IC50 2.87 g/ml), whereas PA was devoid
of any LS activity at the highest test concentration (25 g/ml).
lg/ml) that was ten times lower than the control compound,
l
l
l
l
l
In our effort to find a plausible mechanism of action of 2-ODA
towards the Plasmodium parasites, as well as to compare these
results to those previously reported by us for 2-HDA, we assessed
the inhibitory potency of the three 2-AFAs towards PfFAS-II
enzymes, namely PfFabI, PfFabZ, and PfFabG. All of these enzymes
were recombinantly prepared and evaluated following a previously
published procedure.12 Again 2-ODA appeared as the most effec-
tive inhibitor of the key elongation enzymes in the PfFAS-II path-
way of Plasmodium, following the order PfFabI ꢀ PfFabZ > PfFabG
(Table 1). Notably, the enzyme inhibitory potential of 2-ODA

N. M. Carballeira et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4151–4157
4153
DMSO
2-TDA
2-HDA
2-ODA
Figure 2. P. berghei liver stage development is impaired in hepatoma cells by 2-AFAs. Human hepatoma cells, Huh7, were infected with GFP-expressing P. berghei sporozoites
and treated with 2-AFAs; 2-ODA (53 M), 2-TDA (33.4 M), 2-HDA (19.7 M) or DMSO (vehicle, at 0.4%) at 2 hpi for 46 h. Cells were fixed with 4% PFA and P. berghei exo-
erythrocytic forms were immuno-labeled with anti-PbHsp70 antibody followed by Alexa-488 secondary antibody (green). Nuclei are stained with Hoechst. Scale bar: 10 m.
l
l
l
l
(IC50’s 0.28 to 0.8
hei antiparasitic activity (IC50 0.34
the relative enzyme inhibition potential of 2-AFAs was in the order
of 2-ODA > 2-HDA > 2-TDA. In addition, it is evident that 2-TDA,
with the shortest alkyl chain, was less competitive with regards
to inhibiting these enzymes. As reported before,5 PA was inactive
l
g/ml) was identical or comparable to its P. berg-
g/ml). As depicted in Table 1,
protocols were carried out with GOLD 4.1 (Cambridge Crystallo-
l
graphic Data Centre, Cambridge, UK).16 Default parameters were
used to investigate the possible binding conformations of the
ligands within the PfFabG and PfFabZ binding pockets. The X-ray
crystallographic data for PfFabG determined at 1.5 Å (PDB ID
2C07)17 and PfFabZ determined at 2.09 Å (PDB ID 1Z6B)18 used in
the docking simulations were retrieved from the Protein Data Bank
(PDB). The binding site of PfFabG was defined as all the amino acid
residues encompassed within a 20 Å radius sphere centered on the
three dimensional coordinates of the side chain CG of Asn147.
Similarly, the binding site of PfFabZ was defined as all the amino
acid residues encompassed within a 20 Å radius sphere centered
on the three dimensional coordinates of the side chain NE2 of
His98 (Chain B). The docking procedures were repeated 10 times
for each inhibitor. The GOLDScore scoring function and visual
inspection were employed to select the representative conforma-
tion for each inhibitor.
against all three enzymes tested (IC50 >100 lg/ml). Therefore, the
presence of a triple bond at C-2, coupled to the right alkyl chain
length (longer) seem to be essential for the inhibition of both LS
parasites and target enzyme activity.
On the basis of the information obtained from the enzymatic
assays, molecular modeling studies were performed as a crucial
step towards understanding the mode of the preferential interac-
tion of 2-ODA over 2-TDA and 2-HDA with these enzymes. To
achieve this 2-ODA, 2-TDA, and 2-HDA were docked into the active
site of both PfFabZ and PfFabG, (Figs. 3 and 4, respectively). The 3D
structures of the fatty acid inhibitors were constructed using stan-
dard geometric parameters of the molecular modeling software
package SYBYL 8.0. Each single optimized conformation of each
molecule in the data set was energetically minimized employing
the Tripos force field13 and the Powell conjugate gradient
algorithm14 with a convergence criterion of 0.05 kcal/mol Å and
Gasteiger–Hückel charges.15 Molecular docking and scoring
According to the model, the alkyl tails of the 2-ODA, 2-TDA, and
2-HDA inhibitors adopt similar extended conformations into the
PfFabZ binding site (Fig. 3). In these conformations, non-polar
interactions play a key role in orientating the inhibitors within
the binding cavity. Specifically, the alkyl tails are in favorable ori-
entation to make extensive van der Waals contacts to the side

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N. M. Carballeira et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4151–4157
Figure 3. Predicted binding mode of the inhibitors 2-ODA (dark gray), 2-TDA (light gray) and 2-HDA (wheat) into the binding site of PfFabZ. Upper panel: inhibitors and
protein residues involved in the ligand-receptor binding are indicated as ball-and-stick and stick models, respectively. Chain A and Chain B residues are indicated as green and
cyan, respectively, and hydrogen bonds are illustrated as yellow dashed lines. Lower panel: view of the active site showing the solvent accessible surface of PfFabZ and the
spatial complementarity of the inhibitors.
chain of Ala150, Gly154, Cys157, Leu158, Phe171, Phe198, Ala205,
Phe226 and Ala227. In addition, favorable polar interactions assist
the stabilization of the inhibitors. However, the hydrophilic heads
bind to different regions of the binding site. In the case of 2-TDA,
the carboxylic acid substituent accepts two hydrogen bonds from
the main chain NHs of Gly142 and Val143. With respect to
2-ODA and 2-HDA, the carboxylic acid substituents are in favorable
orientation to accept three hydrogen bonds, one from the side
chain NH2 of Gln 145, while the other two from the main chain
NHs of Lys181 and Val183. Additionally, the triple bond of
2-ODA and 2-HDA orients the methylene groups towards a hydro-
phobic cavity formed by the side chains of Leu170, Trp179, Pro182
and Ile189, which favors additional hydrophobic contacts related
to the inhibitor 2-TDA. This finding is in good agreement with
the enzymatic inhibition data and might explain the differences
in the inhibitory activities, that is, 2-ODA > 2-HDA > 2-TDA.
The proposed binding modes of 2-ODA, 2-TDA and 2-HDA to
PfFabG indicate that the inhibitors occupy a conserved positively
charged patch on the surface of the enzyme (Fig. 4). Previous muta-
genesis studies on 3-oxoacyl-acyl-carrier (OAR), and a homologue

N. M. Carballeira et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4151–4157
4155
Figure 4. Predicted binding mode of the inhibitors 2-ODA (dark gray), 2-TDA (light gray) and 2-HDA (wheat) into the binding site of PfFabG. Upper panel: inhibitors and
protein residues involved in the ligand-receptor binding are indicated as ball-and-stick and stick models, respectively. Hydrogen bonds are illustrated as yellow dashed lines.
Lower panel: view of the active site showing the solvent accessible surface of PfFabG and the spatial complementarity of the inhibitors.
protein of Escherichia coli, suggested that two arginine residues
(e.g., Arg129 and Arg172) play a key role in the binding of a natural
substrate.17 These residues are conserved in PfFabG (e.g., Arg190
and Arg233) and form part of the positively charged region.
According to the model, the negatively charged carboxylic acid
substituents of 2-ODA, 2-TDA and 2-HDA are coordinated by ionic
interactions with the side chain of the conserved Arg233 residue
and accepts a hydrogen bond from the side chain of Asn188
(Fig. 4). Furthermore, extensive van der Waals contacts are made
between the alkyl tail of the inhibitors and the side chains of
Ile187, Arg185, Arg184, Ile182, Leu135 and Ile131. The enhanced
inhibitory activity of 2-ODA related to the other 2-AFAs can be
explained by the additional hydrophobic interactions establish by
the extra carbon atoms of 2-ODA with a hydrophobic pocket
flanked by Pro181, Phe176, Tyr177, Gln180, and Ser128 (Fig. 4).
Next we assessed the in vitro blood stage antiplasmodial
activity of 2-ODA and 2-TDA against the multidrug-resistant
P. falciparum K1 strain, using the established 3H-hypoxanthine

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N. M. Carballeira et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4151–4157
Table 1
Inhibitory activity of the 2-AFAs and palmitic acid (PA) against PfFAS-II enzymes, liver stage (LS) P. berghei and blood stage (BS) P. falciparum K1 parasites
Compound
LS P. berghei
PfFabI
PfFabZ
PfFabG
BS P. falciparum
L6 cell toxicity⁄
2-ODA
2-TDA
2-HDA
PA
0.34
2.87
0.48
>25
3.4a
0.30
1.2
0.28
3.5
0.80
5.5
6.0
5.6⁄
27.3
9.2⁄
0.38⁄⁄
>100⁄⁄
0.015b
0.58⁄⁄
>100⁄⁄
0.03c
3.5⁄⁄
>100⁄⁄
0.2c
10.4⁄⁄
3.8⁄⁄
0.117d
85.9⁄⁄
52.7⁄⁄
0.02e,⁄
Standard
IC50 values (lg/ml) represent the average of at least two independent assays performed in duplicates. For the berghei assays, human hepatoma cells were infected with
luciferase-expressing P. berghei sporozoites and treated at 2 h after infection with various concentration of each fatty acid or with DMSO. Parasite load was determined at
44 hpi and IC50 values were determined using Graphpad Prism software. Standard compounds aPrimaquine, bTriclosan, c(À)-Epigallocatechin gallate, dChloroquine, ePodo-
⁄
⁄⁄
and 3
phyllotoxin, Symbols and show data from our previous work2
respectively.
method as described.5 As shown in Table 1. and 2-ODA emerged as
the most active, hence, the best 2-AFA with dual activity against
Following previously described guidelines,25 we first evaluated
the general toxicity of all four compounds in zebrafish larvae at
3 days post-fertilisation (3 dpf). The maximum-tolerated concen-
trations (MTCs) were determined during the course of the treat-
ment (3–6 dpf) according to the following parameters: body
shape, touch response, pericardial oedema, cardiovascular defects,
and death. PA revealed no signs of general toxicity up to the high-
both Plasmodium species (IC50’s of 6.0 and 0.34
l
g/ml, respec-
tively), followed by 2-HDA (IC50’s of 10.4 and 0.48
lg/ml, respec-
tively). As expected, the least effective of the series was 2-TDA
with IC50 values of 27.3 and 2.87 g/ml, respectively. Notably, all
l
2-AFAs exhibited 10–20 times less BS activity. As reported previ-
ously, PA was the C16 saturated fatty acid with highest BS inhibi-
est concentration tested (179.2
itation in a concentration-dependent manner was observed with
this compound. For the 2-AFAs, the MTCs at 6dpf were: 0.20 g/
ml (=0.8 M) for 2-HDA, 0.27 g/ml (=1.2 M) for 2-TDA and
0.28 g/ml (=1.0 M) for 2-ODA.
Next, the hepatotoxic effects of PA, 2-HDA, 2-TDA and 2-ODA
were assessed in 3 dpf larvae with a range of concentrations up to
their respective MTCs. At 6 dpf (3 days post-treatment) the livers
were scored according to five phenotypes: normal liver, reduced
fluorescence, reduced size, enlarged liver, and absent liver. As
shown in Figure 5, when compared to the untreated control (vehicle
only, 1% DMSO) no hepatotoxic effects were induced by PA at the
lg/ml = 700 lM), however, precip-
tory effect (IC50 3.8 l
g/ml).5
Finally we turned to the assessment of toxic potential of 2-AFAs
in comparison to PA. As displayed in Table 1, all four fatty acids
bear some general, in vitro toxicity against L6 cells, a primary cell
line derived from rat skeletal myoblasts. This followed the order of
2-ODA > 2-TDA > PA > 2-HDA, with 2-ODA being the most toxic.
However, the compounds did not exhibit significant toxicity to
the human hepatoma Huh7cell line at the highest test concentra-
tions. In order to gain more insights into that aspect, we tested
all three 2-AFAs, as well as PA, in zebrafish larvae for the in vivo
assessment of general toxicity and hepatotoxicity. Beyond their
many genetic, physiological and pharmacological similarities to
mammals,19,20 zebrafish (Danio rerio) have proven to be an attrac-
tive, versatile vertebrate animal model for drug discovery. Embry-
onic and larval zebrafish can be readily adapted to diverse low- to
high-throughput drug-induced toxicity screens, including in vivo
assays for geno-, cardio-, neuro- and hepatotoxicity.21–24
l
l
l
l
l
l
highest concentration tested in our assay (700
In the same manner, compounds 2-HDA and 2-ODA were also
non-hepatotoxic at their MTCs (0.8 and 1 M, respectively). How-
ever, treatment with 2-TDA induced liver toxicity at the MTC
(1.2 M = 0.27 g/ml), which was revealed by livers with slightly
reduced size and rounded shape, and with reduced fluorescence.
lM = 179.2 lg/ml).
l
l
l
Figure 5. In vivo hepatotoxic effects of PA and 2-AFAs in zebrafish larvae. All larvae, displaying DsRed fluorescent livers, are 6 days post-fertilization (6 dpf), photographed
(Nikon SMZ25 stereomicroscope) after 3 days of exposure to compounds. (A) Control larvae treated with the vehicle only (1% DMSO); (B) positive control larvae treated with
paracetamol 300
l
M showing smaller-sized liver; (C) compound PA 700
l
M showing normal liver; (D) compound 2-HDA 0.8
M showing normal liver.
lM showing normal liver; (E) compound 2-TDA
1.2 M showing round-shaped liver with reduced fluorescence; (F) compound 2-ODA 1
l
l

N. M. Carballeira et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4151–4157
4157
Similar hepatotoxic effects were observed in larvae exposed to par-
acetamol (300 M), drug used in our assay as a positive control due
to its drug-induced liver injury effects.24,26 A slightly lower concen-
tration of 2-TDA (1.0 M = 0.22 g/ml), however, did not induce
any apparent signs of liver toxicity.
Portugal—Grant EXCL/IMI-MIC/0056/2012). M.A.I. was sponsored
by FP7- Marie Curie Actions Initial Training Networks-Interventions
Strategies against malaria-InterMal Training fellowship:
PITN-GA-2008-215281.
l
l
l
Although a limited number of compounds have been evaluated,
this study sheds additional light into structural requirements for
antiplasmodial activity of 2-AFAs in both life stages of the malaria
parasite in the human host, as well as their general toxicity and
hepatotoxicity profiles. From the data discussed above, it is obvi-
ous that a C-2 acetylenic bond and a longer (C18) alkyl chain are
crucial for parasite growth and target enzyme inhibition. In that
respect, the potency always followed the trend 2-ODA >
2-HDA > 2-TDA. A similar, but not identical tendency was also
observed with in vitro general toxicity towards mammalian pri-
mary cells, but not on human hepatic cells. Notably, the 2-AFAs
(2-ODA, 2-HDA, 2-TDA) did not exhibit any specific in vivo hepato-
toxicity in zebrafish larvae despite testing them at their MTCs (1,
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Acknowledgments
The project described was supported by Award Number
SC1GM084708 from the National Institutes of General Medical
Sciences of the NIH and the UPR-Rio Piedras RISE program. We grate-
fully acknowledge financial support from FAPESP (The State of Sao
Paulo Research Foundation, grant #2013/07600-3) and CNPq (The
National Council for Scientific and Technological Development),
Brazil. We thank M. Cal and S. Keller-Maerki for parasite assay
results. A.G.B. thanks the Commonwealth Scholarship Commission
for financial support. M.M.M. has received funding from the
European Community’s Seventh Framework Programme (FP7/
2007-2013) under grant agreement N° 242095. M.M.M. is also
funded by the European Research Council (Grant ERC-2012-
StG_20111109) and Fundação para a Ciência e Tecnologia (FCT,
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