AAC Accepted Manuscript Posted Online 12 January 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.03972-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
SQ109: A New Drug Lead for Chagas Disease
2 3
Phercyles Veiga-Santos1,2, Kai Li3, Lilianne Lameira1, Tecia Maria Ulisses de Carvalho1,2,
4
Guozhong Huang4, Melina Galizzi4, Na Shang5, Qian Li5, Dolores Gonzalez-Pacanowska6,
5
Vanessa Hernandez-Rodriguez7, Gustavo Benaim7, Rey-Ting Guo5, Julio A. Urbina8, Roberto
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Docampo4, Wanderley de Souza1,2,9and Eric Oldfield3,*
7 8
1
9
Filho, Universidade Federal do Rio de Janeiro, Bloco G, Ilha do Fundão, Rio de Janeiro. CEP
Laboratório de Ultraestrutura Celular Hertha Meyer, CCS, Instituto de Biofísica Carlos Chagas
2
10
21941-902, Brazil;
11
Bioimagens, Universidade Federal do Rio de Janeiro, Cidade Universitária, Ilha do Fundão, Rio
12
de Janeiro, Brazil; 3Department of Chemistry, University of Illinois at Urbana-Champaign, 600
13
South Mathews Avenue, Urbana, Illinois 61801, USA; 4Center for Tropical and Emerging
14
Global Diseases and Department of Cellular Biology, University of Georgia, Athens, Georgia
15
30602, USA;
16
Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China;
17
Parasitología y Biomedicina "López-Neyra", Consejo Superior de Investigaciones Cientificas,
18
Granada, Spain; 7Laboratorio de Señalización Celular y Bioquímica de Parásitos, Instituto de
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Estudios Avanzados (IDEA), Caracas, Venezuela; 8Instituto Venezolano de Investigaciones
20
Cientificas, Caracas, Venezuela; 9Instituto Nacional de Metrologia, Qualidade e Tecnologia -
21
Inmetro, Duque de Caxias, Rio de Janeiro, Brazil
5
Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e
Industrial Enzymes National Engineering Laboratory, Tianjin Institute of
22 23
1
6
Instituto de
24 25 26
Data deposition: Crystallography, atomic coordinates, and structure factors have been deposited
27
in the Protein Data Bank, www.pdb.org (PDB ID codes 3WSB, 3WSA).
28 29
*To whom correspondence may be addressed. E-mail:
[email protected]
30 31
ABSTRACT
32
We tested the anti-tuberculosis drug SQ109, currently in advanced clinical trials for the treatment
33
of drug-susceptible and drug-resistant tuberculosis, for its in vitro activity against the
34
trypanosomatid parasite Trypanosoma cruzi, the causative agent of Chagas disease. SQ109 was
35
a potent inhibitor of the trypomastigote form of the parasite with an IC50 for cell killing of 50 ± 8
36
nM but had little effect (EC50~80 μM) in a red blood cell hemolysis assay. It also inhibited
37
extracellular epimastigotes (IC50 = 4.6 ± 1 μM) and the clinically relevant, intracellular
38
amastigotes (IC50~0.5-1 μM) with a selectivity index of ~10-20. SQ109 caused major ultra-
39
structural changes in all three life-cycle forms, as observed by light microscopy, SEM and TEM.
40
It rapidly collapsed the inner mitochondrial membrane potential (∆\m) in succinate-energized
41
mitochondria, acting in the same manner as the uncoupler FCCP, and caused alkalinization of
42
internal acidic compartments, effects that are likely to make major contributions to its
43
mechanism of action. The compound also had activity against squalene synthase, binding to its
44
active site; it inhibited sterol side-chain reduction and, in the amastigote assay, acted
45
synergistically with the anti-fungal drug posaconazole with a fractional inhibitory concentration
46
index (FICI) of 0.48, but these effects are unlikely to account for the rapid effects seen on cell
2
47
morphology and cell killing. SQ109 thus most likely acts, at least in part, by collapsing ∆\/∆pH,
48
one of the major mechanisms demonstrated previously for its action against Mycobacterium
49
tuberculosis. Overall, the results suggest that SQ109, currently in advanced clinical trials for the
50
treatment of drug-susceptible and drug-resistant tuberculosis, may also have potential as a drug
51
lead against Chagas disease.
52 53 54
INTRODUCTION
55
Chagas disease is caused by the trypanosomatid parasite Trypanosoma cruzi and affects ~8-10
56
million individuals, mostly in Latin America (1) with the United States Centers for Disease
57
Control and Prevention estimating that ~300,000 individuals are also infected in the United
58
States (2) . The drugs benznidazole (1) and nifurtimox (2) (Fig. 1) are used to treat the acute and
59
early chronic forms of the disease, but their efficacy against established chronic infections, the
60
most common presentation of the disease, is lower and variable. Chagas disease is calculated to
61
have a $7B annual global cost and represents the largest parasitic disease burden of the American
62
continent(3). There is thus interest in the development of new therapeutic options. Among these
63
are inhibitors of ergosterol (3) biosynthesis. Ergosterol is an essential membrane sterol in yeasts,
64
fungi and many protozoa and is the functional equivalent of cholesterol (4) in humans, and anti-
65
fungal sterol biosynthesis inhibitors (such as posaconazole (5) are of interet as new T. cruzi
66
drugs, as we discuss in more detail below.
67 68 69
In previous work we noticed reports (4-6) that the anti-arrhythmic drug amiodarone 6 (used to
3
70
treat arrhythmias in Chagas disease patients), also had activity against the yeast Saccharomyces
71
cerevisiae, and that amiodarone potentiated the effects of azole drugs. This suggested that
72
amiodarone might also inhibit ergosterol 3 biosynthesis in T. cruzi because at least in yeast, it
73
acted synergistically with azoles (which inhibit lanosterol 14D-demethylase). This was found to
74
be the case (7) with amiodarone inhibiting the enzyme oxidosqualene cyclase (lanosterol
75
synthase) in T. cruzi (7), thereby decreasing ergosterol levels. In addition, it acted synergistically
76
with posaconazole against T. cruzi in vitro and was active in vivo in a mouse model of infection
77
(7). Similar results were later found with Leishmania spp. (8, 9) and amiodarone has now been
78
used clinically for the etiological treatment of chronic Chagas disease (10), as well as
79
disseminated cutaneous leishmaniasis (11), as discussed in a recent review (12). Similar results
80
have also been obtained with the newer (and perhaps less toxic) analog of amiodarone,
81
dronedarone (13) (7, Fig. 1). What is interesting about amiodarone and dronedarone is that they
82
also release Ca2+ from intracellular stores in both T. cruzi and L. amazonensis, an activity
83
probably related to the fact that these compounds are uncouplers (14-16), lipophilic bases that
84
can transport H+.
85 86
In this work, we investigated another lipophilic base, SQ109 (17-21), an ethylene diamine
87
containing N-geranyl and N-adamantyl groups (8, Fig. 1). SQ109 is currently in Phase II clinical
88
trials for the treatment of drug-sensitive and drug-resistant tuberculosis (18-20). One major
89
mode of action for SQ109 in Mycobacterium tuberculosis has been proposed (21) to be
90
inhibition of MmpL3 (Mycobacterial membrane protein Large 3), a trehalose monomycolate
91
transporter that is used in cell wall biosynthesis in M. tuberculosis. Our interest in SQ109 arose
92
since, in addition to inhibiting M. tuberculosis cell growth, it also inhibits the growth of other
4
93
bacteria such as Helicobacter pylori (22), Clostridium difficile (18), Entercocci spp. (18),
94
Haemophilus influenza (18), Neisseria gonorrhoeae (18) and Streptococcus pneumoniae (18), as
95
well as the fungi Candida albicans (23), Aspergillus fumigatus (18) and Cryptococcus
96
neoformans (18), and the malaria parasite, Plasmodium falciparum (24). Since none of these
97
bacteria, fungi or the malaria parasite contain bioinformatically identifiable mmpL3 orthologs,
98
there must be an alternative site (or sites) of action in these organisms and in recent work (24) we
99
found that SQ109 can inhibit enzymes involved in quinone biosynthesis (MenA and MenG). In
100
addition, it acts as an uncoupler, collapsing pH gradients (∆pH) and membrane potentials (∆\) in
101
bacterial systems (24), thereby reducing ATP synthesis. In unrelated work, we also reported (25)
102
that SQ109 was an inhibitor of dehydrosqualene synthase (from Staphylococcus aureus), a
103
protein whose three-dimensional structure (26) and mechanism of action (27) is very similar to
104
that of squalene synthase (SQS) (28), which catalyzes the first committed step of sterol
105
biosynthesis in eukaryotes, such as T. cruzi, suggesting that SQ109 might also inhibit SQS.
106 107
Here, we report the antiproliferative and ultrastructural effects of SQ109 against the three life
108
cycle stages of T. cruzi: trypomastigotes, epimastigotes and amastigotes; its synergistic
109
interaction with posaconazole; its uncoupling activity on T. cruzi mitochondria; its alkalinizing
110
effects on acidic compartments; its effects on sterol biosynthesis, as well as the X-ray structures
111
of SQ109 bound to T. cruzi and human squalene synthase.
112 113
MATERIALS AND METHODS
114 115
Parasites and host cell culture.
5
116
Assays were performed in most cases using T. cruzi epimastigotes, trypomastigotes or
117
intracellular amastigotes of the Y strain (TcII) (29). T. cruzi trypomastigotes were obtained from
118
the supernatants of previously infected LLC-MK2 cells (ATCC CCL-7; American Type Culture
119
Collection, Rockville, MD) cultured in RPMI 1640 medium with garamycin (GIBCO, Grand
120
Island, NY) and 10% fetal bovine serum (FBS) (Cultilab, São Paulo, Brazil) at 37 °C in a 5%
121
CO2 atmosphere. Sub-confluent cultures of LLC-MK2 cells were infected with 5 × 106
122
trypomastigotes. Extracellular parasites were removed after 2 h, the cells were washed and the
123
cultures maintained in RPMI 1640 medium containing 10% FBS until trypomastigotes emerged
124
from the infected cells (usually after 120 h). Epimastigotes were cultivated in liver infusion
125
tryptose (LIT) medium supplemented with 10% FBS (30) and were collected by centrifugation at
126
350 × g after 96 h of cultivation.
127 128
Drug solutions.
129
Stock solutions of SQ109 and analogs (0.01 mM) were prepared in dimethyl sulfoxide (DMSO)
130
(Merck, Darmstadt, Germany), with the final concentration of DMSO in the experiments never
131
exceeding 0.05%.
132 133
Effects of SQ109 and analogs on LLC-MK2 cells.
134
LLC-MK2 cells were treated with SQ-109 (2.5 - 20 μM) and incubated for 96 hours at 37 ºC.
135
Fresh RPMI-1640 medium containing only 10% FBS was added to untreated samples (control).
136
To determine toxicity, the MTS/PMS (3-(4,5-dimethyl-2-thiazolyl)-5-(3-carboxymethoxy-
137
phenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt/5-methylphenazinium methyl sulfate) assay
138
was performed as described by Henriques et al. (31). The selectivity index of SQ109 was
6
139
determined based on its activity against the trypomastigote and intracellular amastigote forms of
140
T. cruzi as the ratio: (CC50) mammalian cells/ (IC50 or LC50) T. cruzi. All experiments were
141
performed in duplicate. The means were determined based on at least 3 experiments.
142 143
Antitrypanosomal activity.
144
Trypomastigotes were obtained from the supernatant of a previously infected cell line (LLC-
145
MK2). Between 5 to 7 days after infection, protozoa were collected from the supernatants of the
146
infected LLC-MK2 cells and were then incubated with fresh RPMI-1640 medium supplemented
147
with 0.5% FBS, with or without SQ109 (0.05 to 5 μM), for 24 hours at 37 ºC under a 5% CO2
148
atmosphere. The concentration of SQ109 at which 50% of the parasites were lysed (LC50) was
149
calculated by counting the cells in a Neubauer chamber. The experiment was performed in
150
duplicate for each of the three different experiments.
151 152
For the antiproliferative assay involving epimastigotes, 106 parasites/mL were cultivated in LIT
153
medium supplemented with 10% FBS. After 24 hours of epimastigote growth, concentrations of
154
0.5 to 10 μM SQ109 (or analogues) were added to the culture and incubated for 120 hours at 28
155
ºC. Cells were collected every 24 hours, for counting in a Neubauer chamber. Two controls were
156
used and consisted of LIT (liver infusion broth/tryptose) supplemented with 10% FBS, and LIT +
157
0.05% DMSO. Cells were allowed to adhere to the coverslips with 0.1 mg/mL poly-L-lysine,
158
fixed with Bouin’s solution and stained with Giemsa for morphological analysis using a Zeiss
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Axioplan 2 light microscope (Oberkochen, Germany) equipped with a Color View XS digital
160
video camera.
161
7
162
To investigate the effect of SQ109 on amastigotes, LLC-MK2 cells were incubated with T. cruzi
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trypomastigotes at a ratio of 10 parasites to 1 cell, for 2 hours. Non-internalized parasites were
164
removed by washing, and the host cells were incubated for 24 h at 37 qC to allow full
165
internalization and differentiation of trypomastigotes to amastigotes. Fresh 10% FBS–RPMI-
166
1640 medium alone (control) or containing the inhibitors (0.5-6 PM) was added to infected cells,
167
which were then incubated for 96 h at 37 qC. Infected cultures were fixed in Bouin’s solution and
168
stained with Giemsa. Parasite infection was quantified using a Zeiss Axioplan (Jena, Germany)
169
light microscope equipped with a 100u lens. The antiproliferative assay was performed as
170
described by Veiga-Santos et al. (32).
171 172
Infection indices (i.e., the percentage of infected host cells multiplied by the average number of
173
intracellular amastigotes per infected host cell) were determined by counting a total of 500 host
174
cells.
175 176
Inhibitor activity was calculated using the SigmaPlot® (version 10) program (Systat Software
177
Inc., San Jose, California, USA). The results are expressed as the mean of three independent
178
experiments.
179 180
We also carried out an additional set of amastigote assays using the CL strain overexpressing a
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tdTomato red fluorescent protein, basically as described earlier (33).
182 183
For evaluation of antiproliferative synergism against intracellular amastigotes, fractional
184
inhibitory concentration indices (FICIs) were calculated as described by Hallander et al. (34). An
8
185
isobologram was constructed by plotting the concentrations of the drugs that alone or in
186
combination induced an IC50 of intracellular amastigotes, as described above.
187 188
To compare control and treated groups among all assays, paired t-tests were applied using a 95%
189
confidence interval (GraphPad Prism v.5.00 for Windows; GraphPad Software Inc., San Diego,
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CA).
191 192
Scanning electron microscopy (SEM).
193
Trypanosoma cruzi epimastigotes were treated with 4.6 μM SQ109 and incubated for 24 and 48
194
hours at 28 °C. Trypomastigotes were treated with 75 nM SQ109 and incubated for 6 and 12
195
hours at 37 °C. Samples were subsequently fixed in 2.5% glutaraldehyde in 0.1 M phosphate
196
buffer (pH 7.2) for 1 h, washed and adhered to a specimen support coated with poly-L-lysine.
197
Cells were then post-fixed in 1% OsO4 and 0.8% potassium ferrocyanide in 0.1 M sodium
198
cacodylate buffer (pH 7.2) at room temperature for 20 min, washed, dehydrated in ethanol and
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critical-point dried in CO2. In addition, samples were ion-sputtered with a chrome layer using a
200
Leica EM SCD500 (Leica Microsystems, Nussloch, Germany) high vacuum sputter coater and
201
observed under a Quanta X50 Scanning Electron Microscope. Images were obtained and
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processed using XTm version 4.1.1.1935 (FEI Company).
203 204
Transmission electron microscopy (TEM).
205
For TEM, T. cruzi epimastigotes were treated with SQ109 (4.6 μM) and were incubated for 24
206
and 48 hours at 28 °C. Trypomastigotes were incubated with 75 nM of the drug for 24 hours.
207
LLC-MK2 host cells infected with T. cruzi trypomastigotes were treated with 1.5 μM SQ109 and
9
208
incubated for 96 h at 37 °C. After the experimental procedure, samples were processed as
209
described by de Macedo-Silva et al. (9). Ultrathin sections were stained with uranyl acetate and
210
lead citrate and were observed with a JEOL JEM-1200EX (JEOL, Akishima, Japan) transmission
211
electron microscope operating at 80 kV.
212 213 214
Haemolytic assay.
215
The haemolytic activity of SQ109 was evaluated as described by Veiga-Santos et al. (35). A 4%
216
suspension of fresh de-fibrinated human blood was prepared in a sterile 5% glucose solution and
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treated with SQ109 (10-80 μM) for 24 hours at 37 ºC. Then, the cells were centrifuged at 250 xg
218
for 5 min. The supernatant absorbance was determined at 540 nm to determine percent
219
haemolysis. Amphotericin B (Cristalia, São Paulo, Brasil) was used as the haemolytic reference
220
drug, and Triton X-100 (Vetec, Rio de Janeiro, Brasil) was used as the positive control. Each
221
experiment was conducted in duplicate and repeated at least 3 times.
222 223
Analysis of mitochondrial membrane potential.
224
We monitored the mitochondrial membrane potential spectrofluorometrically using safranine as
225
the probe (36, 37). T. cruzi epimastigotes (108 cells) were added to buffer (2.4 mL) containing 2
226
mM succinate and 5 μM safranine, and the reaction initiated with or without 60 μM digitonin,
227
with or without the additives shown in Fig. 9. Incubations were at 28 oC. Fluorescence changes
228
were monitored using a Hitachi 4500 spectrofluorometer (excitation wavelength = 496 nm;
229
emission wavelength = 586 nm).
230
10
231
Sterol biosynthesis inhibition.
232
T. cruzi epimastigotes (Y strain) were grown for 96 hours at 28 oC in LIT medium supplemented
233
with 10% heat inactivated fetal bovine serum in the absence or presence of 6 μM SQ109. Cells
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from cultures were washed with Buffer A with glucose (116 mM NaCl, 5.4 mM KCl, 0.8 mM
235
MgSO4, 5.5 mM D-glucose, and 50 mM HEPES at pH 7.0) then pelleted and
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chloroform/methanol (2:1 v/v) added (~30 mL of solvent per gram of wet cells) followed by
237
vigorous stirring and overnight incubation at 4oC. The extract was then filtered using Whatman
238
#2 paper and the filtrate (a single phase) dried under nitrogen. The extract was re-extracted using
239
chloroform/methanol (9/1; v/v) and dried under nitrogen. The extracted lipids were saponified
240
with 5 mL of ethanolic-potassium hydroxide (5 g KOH, 7 mL water, 14 mL ethanol) for 1 hour at
241
80 oC. After cooling, 2.5 mL of water and 1.5 mL hexane were added and the phases allowed to
242
separate. The aqueous phase was then re-extracted twice with hexane. The hexane extracts were
243
dried under nitrogen, dissolved in chloroform and converted to the trimethylsilyl derivatives by
244
using bis-trimethylsilyl acetamide. Both standards and samples (1 μL) were injected in split
245
mode (10:1) on a GC/MS system, which consisted of an Agilent 6890 (Agilent Inc, Palo Alto,
246
CA, USA) gas chromatograph, an Agilent 5973 mass selective detector and an HP 7683B auto-
247
sampler. Samples were analyzed on a 30 m DB5 column with 0.32 mm I.D. and 0.25 μm film
248
thickness with an injection port temperature of 300 oC, the interface was set to 300 oC, the ion
249
source adjusted to 230 oC, MS Quadrupole 150 oC. The helium carrier gas was set at a constant
250
flow rate of 2.7 mL min-1. The temperature program was: 2 min at 250 oC, followed by an oven
251
temperature increase of 25 oC min-1 to 320 oC, for 10 min. The mass spectrometer was operated
252
in positive electron impact mode (EI) at a 69.9 eV ionization energy in the m/z 50-800 scan
253
range. The spectra of all chromatogram peaks were analyzed by using the HP Chemstation
11
254
(Agilent, Palo Alto, CA, USA) program. Identifications and quantifications were performed
255
using mass spectra obtained from authentic standards and with NIST08 and W8N08 libraries
256
(John Wiley & Sons, Inc., USA).
257 258
Synthetic Aspects
259
SQ109 and its analogs were from the batches whose syntheses and characterization were
260
described previously (24).
261 262
Expression and purification of T. cruzi squalene synthase TcSQS.
263
TcSQS expression was induced with 0.8 mM isopropyl E-thiogalactopyranoside (IPTG) at 25 °C
264
for 18 hours, and human SQS (HsSQS) with 1.0 mM IPTG at 37 °C for 6 hours. Cell pastes were
265
harvested by centrifugation at 4,000 xg for 15 min and re-suspended in lysis buffer containing 25
266
mM Tris-HCl, pH 7.5, 20 mM imidazole, and 150 mM NaCl. Cell lysate was prepared with a
267
French® pressure cell press (AIM-AMINCO Spectronic Instruments), then centrifuged at 17,000
268
xg for 30 min to remove cell debris. The cell-free extract was loaded onto a lysis buffer-
269
equilibrated Ni-NTA column, followed by washing with 20 mM imidazole-containing buffer.
270
The His-tagged enzyme was then eluted with an imidazole gradient (from 20-250 mM), dialyzed
271
twice against 5 L of 25 mM Tris-HCl, pH 7.5, and loaded onto a 20 mL DEAE Sepharose Fast
272
Flowcolumn (GE Healthcare Life Sciences). The buffer and gradient were 25 mM Tris, pH 7.5,
273
and 0-500 mM NaCl. Purity of the recombinant proteins (> 95%) was checked by SDS-PAGE
274
analysis.
275 276
Crystallization, data collection and structure determination of TcSQS and HsSQS.
12
277
We used the hanging-drop vapour-diffusion method at room temperature for crystallization. In
278
general, 2 PL TcSQS protein solution (8 mg/ml TcSQS, 25 mM Tris-HCl, pH 7.5, 2.5 mM FSPP
279
was mixed with 2 PL of reservoir solution. Single crystals were obtained in 0.1 M Tris, pH 8.5,
280
21% PEG 3350. Crystals of TcSQS could only be obtained in the presence of FSPP. To obtain
281
SQ109-bound crystals we soaked TcSQS-FSPP crystals with cryoprotectant (0.15 M Tris, pH
282
8.5, 25% PEG3350 and 8% glycerol) that contained 10 mM SQ109, for 3 hrs.
283 284
For HsSQS, 2 PL protein solution (5 mg/mL HsSQS, 25 mM Tris-HCl, pH 7.5) was mixed with
285
2 PL of reservoir solution. Single crystals were obtained in 0.2 M sodium citrate, pH 8.2, 28%
286
PEG3350. Crystals of HsSQS in complex with SQ109 were obtained by co-crystallization (0.2
287
M sodium citrate, pH 8.2, 24-28% PEG 3350, 5 mM SQ109). Prior to data collection at 100 K,
288
the crystals were mounted in a cryoloop and flash-frozen in liquid nitrogen with 0.3 M sodium
289
citrate, pH 8.2, 30% PEG3350 and 2% glycerol, as a cryoprotectant. Diffraction data were
290
collected at beam line BL13A1 of the National Synchrotron Radiation Research Center
291
(NSRRC, Hsinchu, Taiwan), and processed using the HKL2000 program. Prior to structural
292
refinements, 5% randomly selected reflections were set aside for calculating Rfree. The crystal
293
structures of TcSQS and HsSQS were solved by using the molecular replacement method with
294
the CNS program (38). The previously determined TcSQS-FSPP (PDB code 3WCA) and
295
HsSQS-FSPP structures (PDB code 3WC9) were used as the search models. The TcSQS crystal
296
belongs to the P212121 space group and contains four protein molecules in an asymmetric unit.
297
The HsSQS crystal belongs to the P21 space group and contains six protein molecules in an
298
asymmetric unit. Subsequent incorporation of FSPP, SQ109 and water molecules were at the 1.0
299
V map level. All structural refinements were carried out using the CNS (38) and Coot (39)
13
300
programs(38). Data collection and structure refinement statistics are summarized in Table S1. All
301
of the structural diagrams were drawn using PyMol software (40).
302 303
RESULTS AND DISCUSSION
304 305
SQ109 rapidly kills trypomastigotes but does not lyse red blood cells.
306
We first investigated whether SQ109 had any effect on survival of the infective, non-proliferative
307
trypomastigote stage of the T. cruzi life cycle, of potential interest in the context of the
308
development of blood-sterilizing agents in areas with a high prevalence of Chagas disease, as
309
well as for the treatment of both acute and chronic T. cruzi infections.
310
trypomastigotes from the first burst release of T. cruzi-infected LLC-MK2 cells and incubated
311
them with SQ109 for 24 hours at 37˚C, Fig. 2. As can be seen in Fig. 2A, parasite survival was
312
greatly decreased in the presence of SQ109 with an IC50 of 50 ± 8 nM. For comparison, crystal
313
violet (previously used in some areas as a blood sterilizing agent) has an IC50 of ~12 μM in this
314
assay, while benznidazole (the drug used to treat acute infections) is essentially inactive under
315
the same conditions (IC50> 400 μM). SQ109 had little effect on red blood cell hemolysis (Fig.
316
2B) where the EC50 was ~80 μM, leading to a selectivity index (EC50 red cells/IC50 T. cruzi
317
trypomastigotes) of ~1600.
We obtained
318 319
Scanning electron microscopy (SEM) of drug-treated trypomastigotes revealed the formation and
320
release to the extracellular medium of vesicles, Fig. 3. Only a few vesicles were seen after a 6
321
hour incubation, and most were associated with the parasite’s cell surface (Fig. 3A, 3B).
322
However, after a 12 hour treatment, the trypomastigotes displayed a very large number of
14
323
vesicles having diameters of ~0.5 nm (Fig. 3C, D), indicating that incubation with SQ109 leads
324
to cell lysis.
325 326
We also studied drug-treated trypomastigotes by using transmission electron microscopy (TEM),
327
Fig. 4. Untreated parasites exhibited a characteristic ultrastructure with normal flagellum and
328
kinetoplast organization (Fig. 4A). Incubation with 75 nM SQ109 for 12 hours caused an intense
329
alteration of the kinetoplast organization (Fig. 4B, C), as well as release to the extracellular
330
medium of membranous material (Fig. 4C, D), resembling a membrane-shedding process. Such
331
fast and potent activity suggests that the primary mechanism of action against this stage of the
332
parasite may not be due to inhibition of sterol biosynthesis because in these non-proliferating
333
cells, the sterol turnover rate is expected to be extremely slow (several days). Likewise, it seems
334
unlikely that inhibition of quinone biosynthesis is involved (again because of the times needed to
335
deplete quinone pools), suggesting – as with bacteria (24), that one mode of SQ109 action in
336
trypomastigotes may be as an uncoupler, collapsing the inner mitochondrial membrane potential
337
(and ∆pH), as reported elsewhere for dronedarone and amiodarone (12, 13) against T. cruzi and
338
L. amazonesis. We examined this possibility and reported the results later in the Text.
339 340
Effects of SQ109 on epimastigotes.
341
We next investigated the effects of SQ109 and the three analogs 9-11 (24) shown in Fig. 5 on the
342
growth of T. cruzi epimastigotes (equivalent to the proliferative stages present in the hindgut of
343
the insect vectors), grown axenically in LIT medium.
344 345
A representative set of cell proliferation curves as a function of time in the presence of varying
15
346
concentrations of SQ109 (500 nM to 10 μM) is shown in Fig. 6A which yields a growth
347
inhibition IC50 of 4.6 ± 1 μM. IC50 values for the three other compounds tested were similar (9
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= 6.9 μM; 10 = 9.3 μM; 11 = 6.1 μM; data not shown). We investigated these three compounds
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since they have been reported to have activity against both M. tuberculosis as well as the malaria
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parasite, P. falciparum, with 10 being 5x more active than was SQ109 against M. tuberculosis,
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but with T. cruzi, all three compounds were slightly less active than was SQ109. As can be seen
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in the photomicrographs (obtained by using a light microscope) in Fig. 6B-D, SQ109 treatment
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resulted in the rounding of many parasites, just as observed with amiodarone treatment of T.
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cruzi (32). This can be seen even more clearly in the scanning electron microscopy (SEM) results
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shown in Fig. 6E-H, in which rounding of the cell body in the treated epimastigotes, as well as
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the appearance of large depressions (Fig. 6F-H), are apparent.
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Thin sections of treated epimastigotes observed by transmission electron microscopy (TEM) also
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showed drastic changes in the Golgi complex cisternae, the appearance of mitochondrial
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swelling, the formation of myelin-like figures as well as cytoplasmic vesiculation (Supporting
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Information, Fig. S1and S2).
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Effects of SQ109 on intracellular amastigotes, and synergy with posaconazole.
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We next tested SQ109 (and the three analogs) against the proliferation of T. cruzi (Y strain)
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intracellular amastigotes in cultured murine peritoneal macrophages in a 96-hour assay. Typical
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dose-response results (for SQ109) are shown in Fig. 7A from which we deduce an IC50 = 1.2 ±
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0.4 μM. For the analogs, the IC50 values were 9 = 1.6 μM; 10=1.9 μM and 11 = 1.7 μM (data
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not shown). In a second series of experiments with SQ109, against the CL strain, we found IC50
16
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520 ± 70 nM under conditions where the IC50 for benznidazole was 1.3 ± 0.5 μM ( n = 3). As can
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be seen from the isobologram shown in Fig. 7B, SQ109 acts synergistically with posaconazole
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against amastigotes with a FICI (fractional inhibitory concentration index) = 0.48. By way of
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comparison, the FICI for the amiodarone-posaconazole combination reported previously is
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slightly better, FICI = 0.42 (7), but any FICI
