2-Alkynoic fatty acids inhibit topoisomerase IB from Leishmania donovani

Article abstract of DOI:10.1016/j.bmcl.2012.08.019

2-Alkynoic fatty acids display antimycobacterial, antifungal, and pesticidal activities but their antiprotozoal activity has received little attention. In this work we synthesized the 2-octadecynoic acid (2-ODA), 2-hexadecynoic acid (2-HDA), and 2-tetradecynoic acid (2-TDA) and show that 2-ODA is the best inhibitor of the Leishmania donovani DNA topoisomerase IB enzyme (LdTopIB) with an EC₅₀ = 5.3 ± 0.7 μM. The potency of LdTopIB inhibition follows the trend 2-ODA > 2-HDA > 2-TDA, indicating that the effectiveness of inhibition depends on the fatty acid carbon chain length. All of the studied 2-alkynoic fatty acids were less potent inhibitors of the human topoisomerase IB enzyme (hTopIB) as compared to LdTopIB. 2-ODA also displayed in vitro activity against Leishmania donovani (IC₅₀ = 11.0 μM), but it was less effective against other protozoa, Trypanosoma cruzi (IC₅₀ = 48.1 μM) and Trypanosoma brucei rhodesiense (IC ₅₀ = 64.5 μM). The antiprotozoal activity of the 2-alkynoic fatty acids, in general, followed the trend 2-ODA > 2-HDA > 2-TDA. The experimental information gathered so far indicates that 2-ODA is a promising antileishmanial compound.

Full text of DOI:10.1016/j.bmcl.2012.08.019

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6185–6189
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Bioorganic & Medicinal Chemistry Letters
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2-Alkynoic fatty acids inhibit topoisomerase IB from Leishmania donovani
Néstor M. Carballeira a, , Michelle Cartagena a, David Sanabria b, Deniz Tasdemir c, Christopher F. Prada d,
⇑
Rosa M. Reguera d, Rafael Balaña-Fouce d
a Department of Chemistry, University of Puerto Rico, PO Box 23346, San Juan 00931-3346, Puerto Rico
b Department of Natural Sciences, Interamerican University, Metropolitan Campus, San Juan 00919, Puerto Rico
c School of Chemistry, National University of Ireland Galway, Galway, Ireland
d Department of Biomedical Sciences, University of León, Campus de Vegazana s/n, 24071 León, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
2-Alkynoic fatty acids display antimycobacterial, antifungal, and pesticidal activities but their antiproto-
zoal activity has received little attention. In this work we synthesized the 2-octadecynoic acid (2-ODA), 2-
hexadecynoic acid (2-HDA), and 2-tetradecynoic acid (2-TDA) and show that 2-ODA is the best inhibitor
Received 22 June 2012
Revised 20 July 2012
Accepted 1 August 2012
Available online 10 August 2012
of the Leishmania donovani DNA topoisomerase IB enzyme (LdTopIB) with an EC50 = 5.3 0.7 lM. The
potency of LdTopIB inhibition follows the trend 2-ODA > 2-HDA > 2-TDA, indicating that the effectiveness
of inhibition depends on the fatty acid carbon chain length. All of the studied 2-alkynoic fatty acids were
less potent inhibitors of the human topoisomerase IB enzyme (hTopIB) as compared to LdTopIB. 2-ODA
Keywords:
Acetylenic fatty acids
Leishmania donovani
2-Octadecynoic acid
Topoisomerase IB
also displayed in vitro activity against Leishmania donovani (IC50 = 11.0
against other protozoa, Trypanosoma cruzi (IC50 = 48.1 M) and Trypanosoma brucei rhodesiense
(IC50 = 64.5 M). The antiprotozoal activity of the 2-alkynoic fatty acids, in general, followed the trend
lM), but it was less effective
l
l
2-ODA > 2-HDA > 2-TDA. The experimental information gathered so far indicates that 2-ODA is a prom-
ising antileishmanial compound.
Ó 2012 Elsevier Ltd. All rights reserved.
Acetylenic fatty acids have entertained both medicinal chemists
and natural products chemists for the diversity of biological activ-
ities that they have displayed.1,2 Among these fatty acids the octad-
ecynoic acids are worth mentioning. Earlier studies identified the
5-octadecynoic acid (5-ODA) as a natural product from the root
of the plant Ximena americana with pesticidal activity as demon-
strated by the inhibition of the hatching of the pod-sucking bug
Clavigralla tomentosicollis eggs.3 On the other hand, the roots of
Pentagonia gigantifolia yielded the acid 6-octadecynoic acid (6-
ODA), which displayed potent antifungal activity against a series
of fluconazole resistant Candida albicans strains with IC50 values
tion of key enzymes in Leishmania parasites by these acids has been
reported.
Several other 2-alkynoic fatty acids show antiprotozoal activity
and inhibit protozoal enzymes. For example, our team studied the
antiprotozoal activity of 2-hexadecynoic acid (2-HDA) and found
that it effectively inhibited plasmodial FAS-II enzymes (IC50’s be-
tween 1.5 and 13.9 lM) and arrests erythrocytic and liver stage
Plasmodium infections.7 In addition, we showed that 2-HDA dis-
plays antiprotozoal activity against Leishmania donovani amastig-
otes (IC50 = 17.8 lM) but no studies on key L. donovani enzymes
amenable for therapeutic intervention were performed. These ini-
tial results motivated us to study 2-TDA, 2-HDA and 2-ODA as anti-
protozoal compounds so as to determine the antiprotozoal activity
of the acids as a function of carbon chain length. Therefore, in this
work we synthesized 2-TDA, 2-HDA, and 2-ODA and determined
their growth inhibitory activities against L. donovani, T. cruzi, and
Trypanosoma brucei rhodesiense in vitro. The best antiprotozoal re-
sults were obtained against L. donovani and therefore, we studied
the inhibition of the LdTopIB enzyme by 2-TDA, 2-HDA, and 2-
ODA and compared it to hTopIB.
DNA topoisomerases have been the target of many studies since
early successes with the camptothecins and similar structural ana-
logs.8 In particular, type I DNA topoisomerases are a well-recog-
nized target for cancer therapy. The trypanosomal and leishmania
type IB DNA topoisomerases differ significantly from the homolo-
gous mammalian structures since they are phylogenetically
between 0.45–0.65 l
g/mL.4 Among a series of 2-alkynoic fatty
acids the 2-octadecynoic acid (2-ODA) and its metabolites dis-
played the best antimycobacterial activity against Mycobacterium
smegmatis and Mycobacterium bovis BCG by inhibiting fatty acid
biosynthetic pathways of considerable importance for mycobacte-
ria.5 Interesting to mention is that by placing the unsaturation at
the other extreme of the acyl chain, the acid 17-octadecynoic acid
(17-ODA) is obtained, which is a well-known suicide inhibitor of
the leukotriene B4
inhibitor of the renal CYP450
x
-oxidase (IC50 < 5
lM) as well as a selective
x
-hydroxylase.6 All of these findings
attest to the biological potential of the octadecynoic acids as anti-
infective agents and enzyme inhibitors, but no data on the inhibi-
⇑
Corresponding author.
E-mail address: nestor.carballeira1@upr.edu (N.M. Carballeira).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.019

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N. M. Carballeira et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6185–6189
unique and possess an anomalous dimeric structure.9,10 These
enzymes have been recognized as excellent targets for the
development of antiparasitic drugs. However, no clear structure
activity relationships (SAR) exist for the interaction of drugs, in
particular fatty acids, against these type IB topoisomerases. Our
series of 2-alkynoic fatty acids give us an excellent tool to gain bet-
ter insights into the structural requirements needed for effective
LdTopIB inhibition by fatty acids, in particular if these activities
can be correlated to the toxicity against L. donovani. The results of
this study indicate that C18 is an effective carbon chain length
among the 2-alkynoic acids for inhibiting LdTopIB, which happens
to correlate with the toxicities observed against L. donovani axenic
amastigotes.
The synthesis of 2-TDA, 2-HDA, and 2-ODA followed an already
published procedure7 wherein commercially available 1-tridecyne,
1-pentadecyne, or 1-heptadecyne was reacted with n-BuLi in THF
at -70 °C followed by quenching with CO2 and final protonation
with NH4Cl (Fig. 1). The yields ranged from 45–79%. The purity of
the synthesized compounds was determined to be >95% by capil-
lary GC–MS and 13C NMR. The spectral data of the synthesized 2-
alkynoic fatty acids were in agreement with those previously
reported.5,7
main mechanism by which the 2-alkynoic acids kill the Leishmania
promastigotes.
Other mechanisms of toxicity for 2-TDA, 2-HDA, and 2-ODA in L.
donovani were examined by concentrating on the inhibition of the
Leishmania donovani topoisomerase IB enzyme (LdTopIB) (Table 2,
Fig. 3). One of the most recent contributions against protozoan-
caused infectious diseases takes advantage of the structural differ-
ences between the protozoan and the host TopIB, since the unusual
heterodimeric TopIB of kinetoplastid parasites, such as LdTopIB,
can be used for the development of new compounds targeting only
the parasite TopIB without interfering with the monomeric TopIB
of the human host.13 For this reason, the inhibition of LdTopIB by
the 2-alkynoic fatty acids was examined and compared to hTopIB.
As expected, 2-ODA was the most efficient inhibitor with an
EC50 = 5.3 0.7
lM followed by 2-HDA with an EC50 = 28.7
1.3 M (Fig. 3). The effectiveness of inhibition followed the order
l
2-ODA > 2-HDA > 2-TDA. This trend correlates quite well with the
toxicity displayed by the 2-alkynoic acids towards the L. donovani
amastigotes.
In the latter experiment the inhibition of 2-ODA towards hTopIB
was compared to LdTopIB and the results are also shown in Table 2
and Figure 3. While 2-ODA was able to inhibit LdTopIB at 5.3
was less effective against hTopIB (EC50 = 51.9 M). In fact, neither
2-TDA nor 2-HDA was inhibitory towards hTopIB at 100 M. These
lM, it
The antiprotozoal activities of the synthesized 2-alkynoic fatty
acids were studied against Leishmania donovani (axenic amastig-
otes), Trypanosoma brucei rhodesiense (bloodstream forms), and
Trypanosoma cruzi (intracellular amastigotes in L6 rat skeletal
myoblasts) as previously described.7 As shown in Table 1, among
the 2-alkynoic fatty acids 2-ODA displayed the best antiprotozoal
activity against all the studied protozoa with IC50 values ranging
l
l
results clearly reveal that it will be possible to preferentially inter-
fere with LdTopIB without inhibiting the human enzyme, a finding
that could have therapeutic value. It is evident that LdTopIB is
more sensitive to fatty acid inhibition than hTopIB.
The next study contemplated if the 2-alkynoic fatty acids inhib-
ited hTopIB and LdTopIB with a similar or different mechanism as
camptothecin (CPT), a well-known topoisomerase I inhibitor.8
Although, 2-HDA is a bit less inhibitory than 2-ODA towards
LdTopIB, the availability of compound prompted us to choose
2-HDA as the model fatty acid to study the fatty acid-CPT mecha-
nism. From Figure 4 it is evident that 2-HDA inhibits the catalytic
between 11.0 and 64.5
17.8 and 83.6 M) and finally 2-TDA (IC50’s between 24.7 and
255.4 M). Therefore, the 2-acetylenic fatty acids were effective
lM, followed by 2-HDA (IC50’s between
l
l
in killing the protozoa by following the order 2-ODA > 2-
HDA > 2-TDA. Leishmania donovani amastigotes were the most sus-
ceptible to the 2-alkynoic fatty acids (IC50’s between 11.0 and
24.7
cruzi (IC50’s between 62.4 and 80.0
(IC50’s between 64.5 and 255.4 M). Overall, 2-ODA showed the
broadest spectrum of antiprotozoal activity, but the most effective
effect was observed against L. donovani.
Aimed at exploring a possible mechanism responsible for the
antileishmanial activity displayed by the 2-alkynoic fatty acids
apoptosis was probed in Leishmania infantum (2-HDA displayed
l
M), but the test compounds were not as effective against T.
activity of LdTopIB at 100 lM concentration (DNA is supercoiled in
lM) and T. brucei rhodesiense
the presence of the acid) but there is no inhibition of hTopIB since
the DNA substrate is totally relaxed at the same 2-HDA concentra-
tion. However, from Figure 4a (lane 5 under human) it seems that
2-HDA enhances the stabilization of cleavable complexes between
CPT and hTopIB and prevents the formation of the corresponding
cleavable complex made between CPT and LdTopIB (Fig. 4a, lane 5
under Leishmania). In order to further explore the mechanism of
2-HDA inhibition two parallel experiments were then performed.
In a control experiment LdTopIB was first reacted with the super-
coiled plasmid DNA and CPT in DMSO for 15 min at 4 °C and in an-
other experiment LdTopIB was pre-incubated (15 min) first with
l
an IC50 of 14.9 lM against L. infantum promastigotes) by detecting
the translocation of phosphatidylserine (PS) to the cell surface with
the Annexin-FITC reagent. To test for apoptosis in Leishmania is
quite reasonable since it has been reported that Leishmania
amastigotes can fake its own death by exposing PS on its surface
and gain access to macrophages.11 This PS translocation results in
the inhibition of NO production and the induction of TGF-b secre-
tion and IL-10 synthesis.11 In our experiment (Fig. 2) L. infantum
promastigotes were treated with 2-TDA, 2-HDA, and 2-ODA at con-
2-HDA (100 lM) followed by the addition of 100 lM CPT and the
experiment was run for the same amount of time as the control.
The left panel in Figure 4b shows that CPT inhibited LdTopIB in a
reversible way when it was pre-incubated with DMSO (control),
and after 4 min the supercoiled band was less intense and the re-
laxed DNA band became stronger. In addition, the nicked DNA band
in Figure 4b (left) grew in intensity due to the stabilization of the ter-
nary cleavage complexes (poisoning effect of CPT). The right panel of
Figure 4b shows an assay where a pre-incubation of 2-HDA and
LdTopIB was performed before the addition of DNA and CPT. Two
interesting findings were obtained: i) there was no relaxation of
the DNA at any extent, since only the supercoiled band was present
when the gel was run, and ii) the nicked band did not grow in inten-
sity with respect to the control lane, so CPT was unable to stabilize
the cleavable complexes. If the enzyme were to cut the DNA sub-
strate, CPT could interact with the DNA-protein complexes and thus
stabilize them. Therefore, we can hypothesize that 2-HDA inhibits
the LdTopIB-mediated DNA relaxation by a complete different
mechanism as CPT, and prevents the formation of cleavable
centrations ranging from 100 to 400
lM. At the highest concentra-
tion of 400 M (Fig. 2) no significant concentration of apoptotic
l
cells were observed for neither of the acetylenic fatty acids tested,
but a positive test for hydrogen peroxide as the control was ob-
served.12 This experiment demonstrates that apoptosis is not the
Figure 1. Synthesis of 2-TDA, 2-HDA, and 2-ODA with the corresponding yields.

N. M. Carballeira et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6185–6189
6187
Table 1
Antiprotozoal activities of the 2-alkynoic fatty acids. The IC50 values are in
lM.
Compounds
Trypanosoma brucei rhodesiense
Trypanosoma cruzi
Leishmania donovani
L6 cell cytotoxicity
2-TDA
255.4
83.6
62.4
80.0
48.1
1.47b
24.7
17.8
11.0
0.49c
40.9
2-HDA
2-ODA
Standard
340.3
20.0
64.5
0.0075a
0.02d
The values represent the average of at least two independent assays performed in duplicates. Reference compounds are: aMelarsoprol, bBenznidazole, cMiltefosine,
dPodophyllotoxin.
Figure 2. Apoptosis analysis by staining with Annexin-V-FITC and propidium iodide. Untreated and H2O2 treated cell were used as negative and positive controls,
respectively. Top right quadrant shows the percent of late apoptotic cells. Apoptosis was detected by translocation of phosphatidyl serine (PS) to the cell surface with the
annexin V-FITC reagent (BD Pharmigen). Fraction of Annexin V-positive cells was measured with CellQuest software (BD Biosciences, San Jose, CA). To estimate the number of
apoptotic cells L. infantum promastigotes was treated with 400
l
M compounds for 24 h, harvest, wash twice with PBS, and resuspended in 1 Â 106 cells/ml. Cells were
fluorescently labeled by addition of 20 l of binding buffer, 5 ll of Annexin V-FITC and 5 ll of propidium iodide. After incubation at room temperature in the dark for 15 min,
l
cells were applied to flow cytometry analysis. A minimum of 10,000 cells in the gated region was analyzed using a BD FACS Calibur Flow Cytometer. Results were interpreted
by the percentage of total cells appearing in each quadrant. The experiment is representative of three independent trials.
Table 2
normally observed with CPT, which strongly reduces the topoiso-
merase IB religation rate.14 Taken as a whole, the most plausible
Inhibition of the relaxation activities of the LdTopIB and hTopIB enzymes by the
studied fatty acids and their toxicities against murine macrophages (lM)
explanation in our system is for the 2-alkynoic fatty acids to be
interacting with LdTopIB by binding in a region close to the topoiso-
merase active site and either inhibiting the enzyme binding to DNA
or blocking the cleavage reaction step.
Toxicity of the 2-alkynoic fatty acids was determined against L6
cells, a primary cell line from rat skeletal myoblasts. As shown in
Table 1 the 2-ODA was the most toxic of the three 2-alkynoic fatty
Compounds LdTopIB
hTopIB
EC50
Murine macrophages BALB/c
IC50
EC50
2-TDA
2-HDA
2-ODA
CPT
67.8 1.1
28.7 1.3
5.3 0.7
>100
>100
51.9 1.0
2.0 1.0
>100
>100
>100
0.62 0.13
0.67 0.08
acids towards L6 cells (IC50 = 20
lM). Surprisingly, 2-HDA dis-
played negligible toxicity towards L6 cells (IC50 = 340.3
lM), which
complexes avoiding the ability of CPT to stabilize enzyme-DNA com-
plexes. This is consistent with the findings of Castelli and co-workers
where they analyzed the effect of conjugated eicosapentaenoic acid
(cEPA) on the homologous human enzyme catalytic cycle.14 In par-
ticular, pre-incubation of cEPA with hTopIB before addition of DNA
did not permit the stabilization of the cleavable complex by CPT
and, in addition, cleavage inhibition was enhanced.14 Additional
experiments with a suicide substrate, where the enzyme is not able
to carry out the religation step, revealed an identical religation rate
with or without the presence of cEPA. This is in contrast to what is
makes it a more therapeutically valuable compound. While 2-HDA
seems to be an outlier in Table 1 we also determined that palmitic
acid displays an IC50 of 209 lM against these L6 cells. This marked
difference between the toxicity of fatty acids towards L6 cells does
not seem to be novel. For example, oleate and palmitate display
different effects with respect to mitochondrial function, apoptosis,
and insulin signaling in L6 skeletal muscle cells.15 Selective toxicity
of the 2-alkynoic acids was also studied against BALB/c murine
macrophages (Table 2). In this system, none of the 2-alkynoic fatty
acids displayed significant toxicity, that is, in all three examples

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N. M. Carballeira et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6185–6189
Figure 3. Comparison of the inhibition of the relaxation activity of human TopIB (left) and recombinant LdTopIB (right) by 2-ODA. One unit of recombinant LdTopIB was
assayed in a plasmid DNA relaxation assay for 30 min at 37 °C in the presence of 3.125–100 M 2-ODA. Reaction products were resolved in agarose gel and subsequently
visualized by ethidium bromide staining. The relative position of the negatively supercoiled DNA substrate is indicated by Sc, N is the nicked DNA, whereas the ladder of
l
relaxed DNA topoisomer bands is shown in between. Reactions were stopped with a mixture of 1% SDS and 6.1
and lane 2 DMSO.
lg of proteinase K. Lane 1 contains 0.5 lg of pSK plasmid DNA
Figure 4. Supercoiled plasmid DNA TopIB-mediated cleavage assay comparing the inhibition and poisoning of recombinant hTopIB (A left) and LdTopIB (A right) by 2-HDA
and CPT. Both enzymes were assayed in the presence of DMSO (drug vehicle), 100 M CPT (positive control for cleavage stabilization), 100 M 2-HDA and a mix of 100 M 2-
HDA/CPT. Samples were incubated for 4 min at 25 °C, stopped with 1% SDS and digested for one extra hour at 37 °C in the presence of 1 mg/ml proteinase K. DNA was
extracted with one volume of phenol-chloroform and samples were run on a 1% agarose gel containing ethidium bromide to a final concentration of 40 g/ml in order to
l
l
l
l
separate supercoiled and relaxed DNA. The bottom figure (B left panel) shows that a pre-incubation (15 min at 4 °C) with DMSO had no influence on the CPT inhibiting/
poisoning mechanism, as the nicked DNA band increased its relative intensity with respect to the control, at different time intervals. When LdTopIB was pre-incubated with a
2-HDA excess (100 lM) previous to the addition of 0.5 lg of pSK DNA the enzymatic activity was fully inhibited by 2-HDA and CPT poisoning effect did not take place (B right
panel). After the time-course assay samples were extracted and run as previously described in 4A. The nicked band corresponds to the stabilized cleavage complexes. The
results are representative of three independent trials.
the IC50 value was >100
l
M. This result indicates that the 2-alky-
2-TDA we can only speculate at this point since there is no com-
plete crystal structure for LdTopIB available, only partial structures
of regions similar to hTopIB or computational simulations of the
whole enzyme. However, the increased affinity of 2-ODA could
be simply due to an increase number of van der Waals interactions
with LdTopIB by the extra methylene groups in the molecule. More
research is evidently needed in order to elucidate the subtle differ-
ences in the interaction of fatty acids with LdTopIB vs. hTopIB.
However, as mentioned above, one possible mechanism of inhibi-
tion is for the 2-alkynoic fatty acids to interact with LdTopIB by
noic fatty acids display different toxicity towards different cell
lines. The difference in toxicity between BALB/c and L6 cells for
2-ODA could also lie in the fact that we used a primary cell line
for the latter.
The results presented herein indicate that among the studied 2-
alkynoic fatty acids 2-ODA is the most toxic against L. donovani
amastigotes with a mechanism of action that could involve,
although not exclusively, the inhibition of the LdTopIB enzyme.
As to the reasons why 2-ODA is a better inhibitor than 2-HDA or

N. M. Carballeira et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6185–6189
6189
2. Li, X.-C.; Jacob, M. R.; Kahn, S. I.; Ashfaq, M. K.; Babu, K. S.; Agarwal, A. K.;
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binding in a region close to the topoisomerase active site and just
blocking the cleavage reaction, as it was demonstrated in the inter-
action of cEPA with hTopIB.14 In any instance, 2-ODA or 2-HDA
stand out as useful fatty acids that could be used in drug formula-
tions to treat leishmaniasis.
5. Morbidoni, H. R.; Vilchèze, C.; Kremer, L.; Bittman, R.; Sacchettini, J. C.; Jacobs,
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Acknowledgments
7. Tasdemir, D.; Sanabria, D.; Lauinger, I. L.; Tarun, A.; Herman, R.; Perozzo, R.;
Zloh, M.; Kappe, S. H.; Brun, R.; Carballeira, N. M. Bioorg. Med. Chem. 2010, 18,
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8. Pommier, Y. Nat. Rev. Cancer 2006, 6, 789.
9. Das, A.; Mandal, C.; Dasgupta, A.; Sengupta, T.; Majumder, H. K. Nucleic Acid Res.
2002, 30, 794.
The project described was supported by Award Number
SC1GM084708 from the National Institutes of General Medical Sci-
ences of the NIH. M. Cartagena thanks the UPR RISE program for a
graduate fellowship. We thank Gabriel Cintrón for technical assis-
tance. This research was also supported in part by Ministerio de
Ciencia e Innovation (Grant AGL2010-16078/GAN), by Junta de
Castilla y León (Grant Gr-238) and Instituto de Salud Carlos III
(Grant PI09/0448 and the Tropical Diseases Network RICET) from
Ministerio de Salud y Consumo of the Spanish Kingdom. Prada CF
is a pre-doctoral fellow granted by Junta de Castilla y León (ESF;
European Social Founding).
10. Bodley, A. L.; Chakraborty, A. K.; Xie, S.; Burri, C.; Shapiro, T. A. Proc. Natl. Acad.
Sci. U.S.A. 2003, 100, 794.
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