AAC Accepts, published online ahead of print on 3 February 2014 Antimicrob. Agents Chemother. doi:10.1128/AAC.01240-13 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
Dronedarone, an Amiodarone Analog with an Improved Anti-
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Leishmania mexicana Efficacy. Gustavo Benaima,b, Paola Casanovaa,b, Vanessa HernandezRodrigueza, Sheira Mujica-Gonzaleza, Nereida Parra-Gimeneza, Lourdes PlazaRojasa, Juan Luis Concepcionc, Yi-Liang Liud, Eric Oldfieldd,e , Alberto PanizMondolfif and Alirica I. Suarezg. a
Instituto de Estudios Avanzados (IDEA). Caracas, Venezuela. Instituto de Biología Experimental. Facultad de Ciencias. Universidad Central de Venezuela (UCV). Caracas. Venezuela. c Laborartorio de Enzimologia de Parásitos. Facultad de Ciencias, Universidad de Los Andes, Mérida, Venezuela. d Center for Biophysics & Computational Biology, University of Illinois at UrbanaChampaign, Urbana, IL, 61801, USA e Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA; f Instituto de Biomedicina (MPPSPS/UCV)/ Instituto Venezolano de los Seguros Sociales (IVSS). Caracas, Venezuela. g Facultad de Farmacia, Universidad Central de Venezuela. b
Abstract
22
Dronedarone and amiodarone are cationic, lipophilic benzofurans used to treat
23
cardiac arrythmias. They also have activity against the parasitic protozoan
24
Trypanosoma cruzi, the causative agent of Chagas disease. They function by
25
disrupting parasite intracellular Ca2+ homeostasis, as well as by inhibiting
26
membrane sterol (ergosterol) biosynthesis. Amiodarone also has activity
27
against Leishmania mexicana, suggesting that dronedarone might likewise be
28
active against this organism. This could be of therapeutic interest since
29
dronedarone is thought to have fewer side-effects in humans than does
30
amiodarone. We show here that dronedarone effectively inhibits the growth of
31
L. mexicana promastigotes in culture and more importantly, has excellent
32
activity against amastigotes inside infected macrophages (the clinically relevant
33
form) without affecting the host-cell, with the IC50 values against amastigotes
34
being three orders of magnitude lower than those obtained previously with T.
35
cruzi amastigotes (0.65 nM vs 0.75 μM). As with amiodarone, dronedarone
36
affects intracellular Ca2+ homeostasis in the parasite, inducing an elevation of
37
intracellular Ca2+. This is achieved by rapidly collapsing the mitochondrial
38
membrane potential and inducing an alkalinization of acidocalcisomes at a rate
39
that is faster than that observed with amiodarone. We also show that
40
dronedarone inhibits parasite oxidosqualene cyclase, a key enzyme in
41
ergosterol biosynthesis, known to be vital for survival. Overall, the results
42
suggest the possibility of a re-purposing dronedarone as a treatment for
43
cutaneous and perhaps other leishmaniases.
44 45
Introduction
46
Leishmania mexicana is a trypanosomatid parasite responsible for cutaneous
47
and muco-cutaneous leishmaniasis. Drugs used to treat these diseases, such
48
as glucantime, amphotericin B and miltefosine, frequently exhibit adverse side-
49
effects (1-2) so there is a growing interest in new drug targets, and leads. We
50
have demonstrated that amiodarone (1, Figure 1), a commonly used anti-
51
arrhythmic, has promising activity against both Leishmania mexicana (3) as well
52
as Trypanosoma cruzi parasites (4-5). Moreover, the combination of
53
amiodarone and miltefosine resulted in the complete parasitological cure of
54
mice infected with L. mexicana (6). It has been proposed that disruption of
55
parasite intracellular Ca2+ homeostasis is the target of action of several drugs,
56
including miltefosine and amiodarone (7). Thus, miltefosine opens a Ca2+
57
channel in the plasma membrane of these parasites (6), probably related to the
58
recently described sphingosine-induced L-type voltage-dependent Ca2+ channel
59
in L.mexicana (8), causing a large increase in the intracellular Ca2+
60
concentration ([Ca2+]i) of the parasites, while amiodarone also increases the
61
[Ca2+]i in L. mexicana promastigotes, by collapsing the mitochondrial
62
electrochemical potential and alkalinizing acidocalcisomes. However, in spite of
63
its frequent use in humans, the use of amiodarone may lead to a number of side
64
effects, due at least in part to the presence of two iodine atoms in its structure,
65
which may contribute, for example, to its thyroid toxicity. For this reason, an
66
analog of amiodarone was developed that does not contain iodine: dronedarone
67
(2, Figure 1). Recently, we found that dronedarone shows even greater potency
68
than does amiodarone against T. cruzi epimastigotes, as well as against
69
amastigotes present in infected VERO cells, which constitute the clinically
70
relevant form of the parasite (9). This opens up the possibility of using
71
dronedarone, already FDA approved for treating arrythmias, against Chagas'
72
disease (4) and perhaps, leishmaniasis. Here, we report the effects of
73
dronedarone on L. mexicana finding that it has potent activity against both
74
promastigotes as well as amastigotes inside macrophages, whose growth is not
75
affected by the drug. As with amiodarone, dronedarone functions by disrupting
76
Ca2+ homeostasis as well as by inhibiting parasite oxidosqualene cyclase
77
(OSC), a key enzyme in ergosterol synthesis. This multi-site targeting is likely to
78
contribute to the potent activity found.
79 80
Chemicals.
81
Amiodarone (1, Figure 1), digitonin, EGTA, carbonylcyanide p-(trifluoromethoxy)
82
phenylhydrazone (FCCP), and nigericin were purchased from Sigma (St. Louis,
83
MO). Fura 2-acetoxymethyl ester (FURA 2-AM), acridine orange, Rhodamine
84
123 and Rhod 2-AM were from Molecular Probes (Eugene, OR).
85 86
Dronedarone extraction
87
At room temperature, two commercial tablets of dronedarone (MULTAQ®
88
Sanofi-Aventi, 400 mg each) were added to 20 ml of methanol. 10 ml of H2O
89
were added and the mixture stirred for 15 minutes. The mixture was extracted
90
with 10 ml of chloroform, four times, the combined chloroform extracts dried
91
over anhydrous magnesium sulphate, filtered and evaporated under reduced
92
pressure to yield colorless crystals. The product was further purified using
93
column chromatography on silica gel (eluent chloroform:hexane 9:1) and
94
fractions containing pure dronedarone were evaporated to obtain pure
95
dronedarone as colorless crystals (mp 65 °C).
96 97
Culture of L. mexicana epimastigotes and growth inhibition by
98
dronedarone.
99
L. mexicana (Bel 21 strain) promastigotes were cultured in LIT (Liver Infusion-
100
Tryptose) medium supplemented with 10% fetal-bovine serum using continuous
101
agitation (100 rpm) at 29 °C, as reported previously (8). A growth curve was
102
performed to evaluate susceptibility of parasites to either dronedarone or
103
amiodarone at different concentrations; live parasites were counted daily using
104
a Neubauer chamber. The initial parasite concentration was 106 parasites/ml
105
and either the drug or the vehicle (DMSO) was added after 24 h. At least 3
106
independent experiments were performed for each drug and dose, and the IC50
107
values were determined using Prism GraphPad 5.0.
108 109
Amastigote growth inhibition.
110
L. mexicana amastigotes were incubated with murine macrophages (J774)
111
maintained in RPMI-1640 (GIBCO), supplemented with 10% fetal-bovine serum
112
and incubated at 37 °C in humidified 95% air/ 5% CO2 (6). 500 macrophages
113
were seeded per cover-slip in a 24-well plate and infected with promasitigotes
114
using 10 parasites per cell, for 12 h. After infection, cells were washed three
115
times to remove the external, non-adherent parasites, and fresh culture medium
116
was added, with or without dronedarone at different concentrations. The
117
infected cells were incubated for 96 h as previously described (6). After 96 h,
118
macrophages were fixed with methanol and stained with Giemsa to determine
119
the percentage of infected cells.
120 121
Determination of the intracellular Ca2+ concentration.
122
L. mexicana promastigotes were loaded with the fluorescent ratiometric
123
indicator Fura 2 as reported previously (8) in order to evaluate the effect of
124
dronedarone on [Ca2+]i. Briefly, 1 x108 parasites were collected by
125
centrifugation at 600g for 2 min and washed twice with a loading buffer (137
126
mM NaCl, 4mM KCl, 1.5 mM KH2PO4, 8.5 mM Na2HPO4, 11 mM glucose, 1 mM
127
CaCl2 0.8 mM, MgSO4 and 20 mM HEPES-NaOH, pH 7.4). The pellet was then
128
re-suspended and loaded with Fura 2-AM (1 μM), probenecid (2.4 mM) and
129
Pluronic acid (0.05 %) in loading buffer. Parasites were incubated at 29 °C in
130
the dark with continuous agitation for 1 h. Fura 2-loaded parasites were washed
131
twice with the same buffer, either in the presence or absence of Ca2+.
132
Fluorescence measurements were carried out on stirred cells at 29 oC using a
133
Perkin Elmer LS 55 spectrofluorimeter using Ex 340 nm/380 nm and Em 510
134
nm.
135
The [Ca2+]i was calculated as described by Grynkiewicz et al. (1985) (10),
136
using the equation:
137
[Ca2+]i = Kd x (R-Rmin/Rmax-R) x Fmin (380)/Fmax(380),
138
where Kd = dissociation constant (244 nM), R = ratio of measured fluorescence
139
intensities (at 340 nm and at 380 nm), Rmin = lowest R value at 0 Ca2+
140
concentration, Rmax = highest R value at saturating concentrations of Ca2+, Fmax
141
= lowest value of measured fluorescence at 380 nm, at 0 Ca2+ concentration,
142
and Fmin = highest value of measured fluorescence at 380 nm, at saturating
143
concentrations of Ca2+.
144
Maximum and minimum values were obtained after the addition of 30 μM
145
digitonin, which allows the flow of Ca2+ to the interior of the cell. Then, 8 mM
146
EGTA was added to chelate the remaining Ca2+.
147 148
Mitochondrial membrane potential.
149
The effect of dronedarone on the mitochondrial membrane potential of L.
150
mexicana promastigotes was evaluated using the fluorescent dye Rhodamine
151
123 as reported previously (9). Briefly, 108 parasites were collected by
152
centrifugation at 600 x g for 2 min and washed in PBS buffer plus 1% glucose.
153
The pellet was re-suspended in the same buffer and loaded with 20 μM of
154
Rhodamine 123 for 45 min. at 29 °C with mild agitation, in the dark.
155
Subsequently, parasites were washed twice and re-suspended in the same
156
buffer and transferred into a magnetically stirred cuvette. Measurements (Ȝext
157
488 nm, Ȝem 530 nm) were made in a HITACHI 7000 spectrofluorimeter at 29
158
°C. FCCP (1 μM) was used as positive control.
159 160
Acidocalcisomes alkalinization.
161
Evaluation of the effect of dronedarone on the alkalinization of acidocalcisomes
162
was performed using acridine orange (9). Promastigotes (108 cells/ml) were
163
collected and washed, then incubated with 2 ȝM acridine orange in PBS buffer,
164
for 5 min at 29 ºC with constant stirring. Measurements were performed at Ȝext
165
488 nm and Ȝem 530 nm at 29 °C in a HITACHI 7000 spectrofluorimeter under
166
magnetic stirring. Nigericin (1 μM) was used as positive control.
167 168
Subcellular fractionation.
169
L. mexicana promastigotes in exponential phase (2 L of culture) were
170
homogenized and fractionated following established procedures (11). Briefly,
171
cells were collected at 3000 x g for 10 min and washed twice in buffer A (25 mM
172
Tris–HCl, pH 7.4, 1 mM EDTA, 250 mM sucrose) and broken by abrasion in a
173
1:1 (wet wt:wt) mixture of cells with silicon carbide (200 mesh) in a chilled
174
mortar to produce an homogenate that was diluted to 100 ml of buffer B (25 mM
175
Tris–HCl, pH 7.2, 250 mM sucrose, 25 mM NaCl, 2 mM EDTANa2, 5 mM
176
dithiothreitol) in the presence of a cocktail of protease inhibitors (10 μM
177
leupeptin, 50 μg ml-1 trypsin inhibitor, 1 mM benzamidine, 50 μM PMSF, 100 μM
178
TLCK, 0.2 μM antipain, 1 μM pepstatin, 1 μM E-64, 1 μM chemostatin, 1 μM
179
bestatin). The homogenate was centrifuged at 1000 x g for 10 min to remove
180
unbroken cells and nuclei and then at 5000 x g for 10 min, which produced a
181
large granule fraction (containing mitochondria and large cellular debris); a
182
small granule pellet was obtained at 33,000 x g for 15 min. The post-small
183
granule supernatant was subjected to 105,000 x g for 90 min to produce a
184
microsomal pellet and a soluble (cytosolic) fraction. The microsomal fraction
185
pellet was re-suspended in 6 ml of 50 mM MOPS/NaOH (pH 7.4), 1 mM EDTA,
186
0.1% Triton X-100 and 0.1% Tween 80, homogenized with a Potter-Elvehjem
187
homogenizer, then centrifuged at 105,000 x g for 90 min. The resulting
188
supernatant was collected and used for the enzyme inhibition assays.
189 190
Determination of protein concentration
191
Proteins were precipitated with 10% (w/v) trichloroacetic acid at 47 ºC. The
192
protein precipitate was washed with cold acetone, re-suspended in distilled
193
water, and finally assayed using a modification (12) of Folin phenol in the
194
presence of 0.1% sodium dodecyl sulfate; the procedure was designed to
195
eliminate the interference from substances present in the fractionation and
196
assay media, including EDTA and sucrose. Bovine serum albumin was used as
197
a standard.
198
199
Enzyme inhibition assay
200
Oxidosqualene cyclase (OSC) was assayed using the radioactive method
201
described by Milla et al. 2002 (13). The microsomal extract (0.7 mg of
202
protein/ml) was incubated with 3S-2,3-[14C]oxidosqualene (35 μM) in the
203
presence of Triton X-100 (0.1%) and Tween 80 (0.1%) in 50 mM MOPS/NaOH
204
(pH 7.4) and 1 mM EDTA for 30 min at 28 ºC under vigorous shaking on a
205
water bath. The reaction was stopped by adding of 20% KOH/ 50%EtOH, and
206
lipid was saponified at 80 ºC for 30 min. The non-saponifiable lipids were
207
extracted twice with 1 ml of petroleum ether and separated on TLC plates
208
(Merck)
209
Radioactivity in 2,3-oxidosqualene and lanosterol were quantified by using an
210
imaging plate on a System 2000 Imaging Scanner (Packard). The amount of
211
lanosterol product formed was used for the calculation of enzyme activity.
212
Chromatographic
213
squalene, 2,3-oxidosqualene and ergosterol. The standards were visualized on
214
the TLC plate with iodine vapor. Enzyme inhibition was carried out in basically
215
the same way, but in the presence of varying concentrations of dronedarone.
216
Lineweaver-Burke plots were utilized to derive apparent Ki values (average of
217
triplicates ± standard deviation).
using
toluene-diethyl
standards
ether
were
(9:1)
as
the
developing
2,3,22,23-dioxidosqualene,
solvent.
lanosterol,
218 219
Computational aspect
220
Docking of dronedarone to a Phyre2 (14) model of LmOSC was carried out by
221
using the Glide program (15) from Schrödinger. The common feature
222
hypothesis was produced using 3-7, known OSC inhibitors (5) using the MOE
223
program (16). All the structural figures were made by using Pymol (17).
224 225
Data analysis
226
Analyses of kinetic data and IC50 results were carried out using non-linear
227
regression methods implemented in the SIGMA-PLOT software packages.
228 229
Results and Discussion
230
We first studied the effect of dronedarone at different concentrations on the
231
growth of L. mexicana promastigotes. Figure 2A shows that dronedarone at
232
concentrations above 0.25 μM has a profound effect on the growth of
233
promastigotes in culture. The calculated IC50 value was of 115 nM (as shown in
234
the insert). This concentration is much smaller than that we previously obtained
235
in L. mexicana with amiodarone, IC50 ~900 nM (6). We also analysed the
236
percentage of growth inhibition induced by dronedarone in promastigotes in
237
Figure 2B, where a clear dose-dependent response was obtained. We then
238
evaluated the effect of dronedarone on the growth of intracellular amastigotes in
239
infected macrophages. As shown in Figure 3, dronedarone has a marked effect
240
on the parasites, with a calculated IC50 value of 0.65 nM; again, significantly
241
less than the IC50 of 8 nM observed using amiodarone under the same
242
conditions (6). Interestingly, this concentration is also dramatically lower than
243
that obtained using dronedarone on T. cruzi amastigotes in infected cells where
244
in previous work (9) we reported an IC50 of 0.75 μM (i.e. a ~1000-fold higher
245
IC50 value). Since amastigotes inside macrophages represent the clinically
246
relevant form of L. mexicana, this result points to the much higher effectiveness
247
of dronedarone in L. mexicana. Additionally, it can be seen (Figure 3) that
248
dronedarone at the concentrations employed does not have any discernible
249
effect on macrophages: over the entire dose range investigated. At therapeutic
250
doses (in humans, to treat arrhythmias) the plasma level of dronedarone is ~0.2
251
μM but in liver, levels are ~10-20x higher (18).
252
The 0.65 nM IC50 value in amastigotes is ~300x less than the 0.2 μM plasma
253
level using normal therapeutic dosing and suggests a potentially good
254
therapeutic index.
255
Since we found in previous work that amiodarone induced an increase in the
256
intracellular, cytoplasmic levels of calcium, [Ca2+]i, we next investigated whether
257
dronedarone had a similar effect on [Ca2+]i using FURA 2-loaded promastigotes.
258
As shown in Figure 4A, the addition of dronedarone (2.5 μM) induces a marked
259
increase in [Ca2+]i which reaches a plateau after a few minutes. To determine
260
whether this increase was due to entry of extra-cellular Ca2+ or as a
261
consequence of its release from intracellular organelles, the same experiment
262
was performed in the absence of external Ca2+ (i.e. a Ca2+-free buffer as well as
263
in the presence of EGTA). As can be seen in Figure 4B, in the absence of
264
external calcium, dronedarone causes the same increase in [Ca2+]i as that
265
obtained in the presence of external calcium, meaning that it was released from
266
intracellular stores. It should be noticed that under both conditions, a biphasic
267
response can be observed that we attribute to the action of dronedarone on at
268
least two intracellular compartments, namely acidocalcisomes and the
269
mitochondrion, given that these are the intracellular organelles known to be
270
involved in Ca2+ accumulation (7;19-20). We calculated the initial slopes of this
271
biphasic response finding that dronedarone acts about twice as fast as does
272
amiodarone (Figure 4D). However, when the experiments were performed using
273
the IC50, instead of 2.5 μM dronedarone (Figure 4C), while there was still a
274
marked increase in [Ca2+]i , the biphasic response was not discernible and the
275
initial slope of the curve with dronedarone was about one third of that obtained
276
in the presence of 2.5 μM of the drug (Figure 4D).
277
In order to test the effect of dronedarone on acidocalcisomes, several
278
experiments were performed using promastigotes loaded with acridine orange,
279
a compound that accumulates in acidic compartments, enabling the
280
visualization of any alkalinization. Figure 4A shows that dronedarone (2.5 μM)
281
induces a rapid alkalinization of acidocalcisomes. In the same experiment,
282
amiodarone, also known to lower the acidity in these organelles (6), was added
283
at 10 μM in order to compare its effect with dronedarone. As can be seen in
284
Figure 5A, dronedarone’s effect was significantly faster than that seen with
285
amiodarone. The subsequent addition of nigericin induced the further
286
alkalinization in these organelles (Figure 5A). Nigericin promotes the
287
electroneutral exchange of H+ and K+, and loading with acridine orange followed
288
by exposure to nigericin leads to the rapid release of the fluorescent dye from
289
the acidocalcisomes with a concomitant increase in fluorescence (9).
290
Performing the same experiment but inverting the order of addition (i.e., adding
291
nigericin first and then dronedarone or amiodarone) shows once again a large
292
rise in acidocalcisome alkalinization (Figure 5B). Subsequent addition of
293
dronedarone or amiodarone induced a further increase in the release of acridine
294
orange. Once again, the effect of dronedarone was more rapid than that seen
295
with amiodarone (Figure 5B), suggesting perhaps better cell membrane
296
permeability. Since nigericin completely disrupts acidocalcisome function, this
297
further effect of the drugs suggests that another separate compartment may be
298
involved in their action. Analysis of the initial slopes in Figure 5A,B indicates
299
(Figure 5C) that the rate of alkalinization of dronedarone is similar to that
300
obtained with nigericin, while that of amiodarone is significantly slower. This
301
could help explain the lower IC50 obtained with dronedarone when compared to
302
amiodarone (Figure 5C). We also observed that dronedarone when added at
303
the IC50, (instead of at 2.5 μM), was again able to induce alkalinization of the
304
acidocalcisomes, albeit with a lower velocity (Figure 5D).
305
The large, unique mitochondrion in L. mexicana is also thought to be involved in
306
[Ca2+]i regulation (21-22), as well as in the leishmanicidal effects of amiodarone
307
(6). We thus next investigated the effects of dronedarone on the mitochondrial
308
membrane potential using Rhodamine 123, which is known to accumulate in
309
energized mitochondria (4,9), resulting in self-quenching of the dye’s
310
fluorescence. An increase in Rhodamine 123 fluorescence corresponds to de-
311
energization. As shown in Figure 6A, dronedarone (2.5 μM) induces a rapid
312
increase in fluorescence, due to a collapse of the mitochondrial membrane
313
potential. Again, the effect is significantly faster with dronedarone than with
314
amiodarone (at 10 μM, Figure 6A). Furthermore, addition of the uncoupler
315
FCCP after adding either amiodarone or dronedarone induces a small
316
additional release of Rhodamine 123, as deduced from the increased
317
fluorescence. When FCCP is added first (Figure. 6B), it induces a rapid but
318
small increase in fluorescence, since FCCP dissipates the mitochondrial
319
membrane potential by permeabilizing the inner mitochondrial membrane to
320
protons. In the same figure it is also evident that the subsequent addition of
321
dronedarone (2.5 μM) causes a rapid release of Rhodamine 123, faster again
322
than that obtained with amiodarone (at 10 μM, Figure. 6B). The calculated initial
323
slope of these curves indicate that the effect of dronedarone is faster than that
324
of amiodarone (about 5-fold), and twice as that obtained with FCCP (Figure
325
6C). We also performed experiments on the mitochondrial electrochemical
326
potential using the IC50 of dronedarone. As can be seen in Figure 6D,
327
dronedarone is able to induce a robust release of Rhodamine 123, even when
328
added after FCCP. This strongly suggests that the action of dronedarone on
329
mitochondria could be part of the improved performance of dronedarone as
330
compared to amiodarone.
331
From these results it is clear that dronedarone induces a rapid collapse of the
332
parasite’s mitochondrial membrane potential, although its specific target is
333
unknown. The effect of dronedarone or amiodarone after FCCP is not easy to
334
explain since it might be expected that the uncoupler would release all the
335
fluorescent dye. In a previous study with epimastigotes of T. cruzi (9) we also
336
observed this behaviour with dronadarone (and amiodarone) after FCCP
337
addition. It is conceivable that FCCP is not properly acting under these
338
experimental conditions. Another possible explanation is that part of the action
339
of dronedarone (and amiodarone) is by affecting also another unknown
340
compartment.
341
We also performed experiments using the IC50 of dronedarone on the
342
electrochemical mitochondrial potential. It can be seen in Figure 6D that this
343
drug is also able to induce a robust release of Rhodamine 123, even when
344
added after FCCP. This strongly suggests that the action of dronedarone on
345
mitochondria could be part of the better performance of dronedarone when
346
compared with amiodarone on these parasites.
347
With amiodarone inhibition of T. cruzi growth (6), we reported previously that
348
not only did amiodarone affect Ca2+i and the mitochondrial membrane potential,
349
it also blocked formation of the essential membrane sterol ergosterol by
350
inhibiting the enzyme oxidosqualene cyclase (OSC). We thus investigated
351
whether dronedarone inhibited the activity of L. mexicana OSC (LmOSC) in a
352
similar fashion.
353
Figure 7A shows that the enzyme exhibits classic Michaelis–Menten kinetics
354
with respect to substrates with Km,app [(3S)-2,3-oxidosqualene] = 36 μM, Vmax,app
355
= 0.82 μM . min-1, and Vmax/Km = 0.023 min-1. Dronedarone behaves as a potent
356
non-competitive inhibitor with respect to [(3S)-2,3-oxidosqualene], with Ki = 0.7
357
μM. The dose-response curves for the activity of dronedarone against LmOSC
358
(Figure 7B) are consistent with noncompetitive inhibition with Ki § IC50; these
359
Ki values are 1 to 2 orders of magnitude lower than the Km of the substrates.
360
Similar experiments were performed with amiodarone, in order to compare it
361
with dronedarone (not shown). The Ki value obtained was of 9.0 μM, much
362
weaker inhibition than with amiodarone.
363
To investigate how dronedarone (2) might bind to LmOSC, we used
364
computational docking with the Glide program (15). The top scoring pose is
365
shown in Figure 8A,B, superimposed on the structure of the OSC inhibitor Ro-
366
48-8071 (3, Figure 1) bound to human OSC (PDB ID code 1W6J). Clearly, it
367
seems very likely that dronedarone binds to the LmOSC substrate binding site
368
with its cationic (trialkylammonium) side-chain located close to the essential
369
D718, corresponding to D455 in Homo sapiens OSC (HsOSC) that catalyzes
370
the protonation-initiated electrocyclization of epoxysqualene. The binding of
371
amiodarone (1) is also similar to that of Ro-48-8071 (3, Figure 7C) and a
372
comparison between the amiodarone (1) and dronedarone (2) docked LmOSC
373
structures is shown in Figure 7D. A common feature pharmacophore obtained
374
using the potent known HsOSC inhibitors (3-7, Figure 1) is shown in Figure 8E
375
superimposed on the structure of dronedarone (2). The key features of this
376
pharmacophore that are also present in dronedarone are the presence of the
377
cationic feature (that presumably mimics the carbo-cation obtained on
378
protonation of epoxysqualene) that is likely to interact with the conserved D718,
379
together with 3 hydrophobic features. In conclusion, the results indicate that
380
dronedarone binds to the same catalytic/hydrophobic pocket as does
381
amiodarone (in T. cruzi OSC), as does the HsOSC inhibitor Ro-48-8071-whose
382
structure is known (PDB ID code 1W6J) and which has a similar
383
pharmacophore. The results also show that the cationic moiety in dronedarone
384
docks very close to the very highly conserved D718 which in human OSC is a
385
key catalytic residue (D455) involved in initial squalene epoxide protonation.
386
Given that amastigotes are inhibited with a very low IC50 (0.65 nM) it seems
387
unlikely that there will be significant OSC inhibition at the sub-micromolar levels
388
that might be anticipated to have therapeutic utility. Nevertheless, the
389
development of more potent analogs that target LmOSC is a potentially
390
important goal since multiple-site targeting would be expected to increase
391
efficacy.
392 393 394
In conclusion, the results we have shown above are of interest for several
395
reasons. First, we show that the anti-arrhythmia drug dronedarone has activity
396
against L. mexicana promastigotes. Second, that it has potent activity against
397
intracellular amastigotes, in macrophages, with an IC50 of 0.65 nM while host
398
cell proliferation is not inhibited. These effects are similar to those observed
399
previously with amiodarone but are more rapid as well as far more pronounced.
400
Third, we find that dronedarone increases intracellular Ca2+ and that this
401
increase in [Ca2+]i arises from Ca2+ release from acidocalcisomes and the
402
mitochondrion. Finally, we find that dronedarone inhibits L. mexicana
403
oxidosqualene cyclase, potentially opening up a future route to blocking
404
formation of the essential membrane sterol, ergosterol. Overall, these results
405
are of considerable interest since not only is dronedarone more potent than is
406
amiodarone against L. mexicana, but also it is known to have less adverse side-
407
effects (in humans, when used as an anti-arrhythmic) since it lacks the iodine-
408
substituents that make amiodarone resemble thyroxine/tri-iodothyronine.
409
From a clinical perspective these findings are of importance because
410
mixed infections involving both Trypanosoma cruzi and different Leishmania
411
species is becoming a common clinical scenario in many countries in Central
412
and South America (23). Thus, having a single drug that can target both
413
parasites is of therapeutic interest. Moreover, Leishmania mexicana is a major
414
causative agent of the difficult-to-treat diffuse cutaneous leishmaniasis (24),
415
where again new drugs are needed.
416
It remains to be seen whether dronedarone is more efficacious than is
417
amiodarone in humans since at least part of the efficacy of amiodarone against
418
cutaneous leishmaniasis is likely due to its unusual, dermal elimination from the
419
body. That is, concentration in the skin may be much higher than with
420
dronedarone, and further work on determining these levels with dronedarone is
421
needed, as are studies aimed at other Leishmania parasites. However, the
422
considerably increased efficacy of dronedarone may offset the decrease in skin
423
elimination.
424
The repositioning of old drugs for their use in other diseases has been a
425
matter of recent interest, since the trials in humans have been already
426
successfully achieved, and most of them are not expensive since their
427
respective patents have expired (25). We postulate that since both dronedarone
428
and
429
clinical trials against both leishmaniasis as well as Chagas’ disease, both drugs
430
could be repurposed in the near future for the treatment of these human
431
infections. It seems likely that the more potent as well as less toxic dronedarone
432
may be more efficacious in humans.
amiodarone are inexpensive and amiodarone has been used in some
433
Because the role of dronedarone in patients with severe heart failure is
434
still controversial, we believe that its use should be avoided in severe cardiac
435
disease. However, its potential use in otherwise healthy patients with cutaneous
436
leishmaniasis and/or mild-to-moderate heart failure or arrhythmias remains a
437
feasible therapeutic option, even more considering its potentially high efficacy.
438 439
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20. Docampo R, Ulrich P, Moreno SNJ. 2010. Evolution of acidocalcisomes
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21. Benaim G, Bermudez R, Urbina JA. 1990. Ca2+ transport in isolated
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Pharmacol Sci. 34: 267-272.
521 522
Acknowledgments: This work was supported by grants from Fondo Nacional
523
de Ciencia, Tecnología e Investigación, Venezuela (FONACIT) (2011000884),
524
and the Consejo de Desarrollo Científico y Humanístico (CDCH-UCV) grant
525
PG-03-8728-2013. Universidad Central de Venezuela to G.B. and in part by
526
NIH grant GM065307 to E.O.
527 528
FIGURES LEGEND
529
Figure 1. Structures of molecules of interest.
530 531
Figure 2. Inhibition of L. mexicana promastigotes growth by dronedarone.
532
A. Susceptibility of promastigotes to dronedarone. Each point represents the
533
mean ± SD of at least three independent experiments. Insert: Dose-response
534
curve from A; IC50 = 115 nM. B. Percentage of growth inhibition induced by
535
dronedarone in promastigotes. All the values were taken from the data of Figure
536
2A using the values obtained at 10 days after addition of the corresponding
537
drug concentration. Each point represents the mean ± SD.
538 539
Figure 3. Effect of dronedarone against intracellular amastigotes.
540
Macrophages (J774 cells) infected with L. mexicana amastigotes were exposed
541
to different concentrations of dronedarone. The percentage of infected
542
macrophages (filled squares) and the effect on non-infected macrophages (filled
543
circles) were determined at 72 hours after the addition of the drug. Around 100
544
cells were counted in every experiment. Each point represents the mean ± SD
545
of at least three independent experiments.
546 547
Figure 4: Effect of dronedarone on the [Ca2+]i of L. mexicana
548
promastigotes. Promastigotes were loaded as described under “Materials and
549
Methods”. A. Effect of 2.5 μM dronedarone (arrow) on the parasite cytoplasmic
550
Ca2+ concentration in the presence of 2 mM external Ca2+. B. Effect of 2.5 μM
551
dronedarone (arrow) on Fura 2-loaded promastigotes in the absence of external
552
Ca2+ (EGTA). C. Effect of 0.65 nM dronedarone (arrow) on the parasite
553
cytoplasmic Ca2+ concentration in the presence of 2 mM external Ca2+. The
554
figures shown are representative of at least three independent experiments. D.
555
Initial slopes (300 sec) of the curves obtained in the presence of 2 mM external
556
Ca2+. The first and second slopes were taken from at least three independent
557
experiments similar to that shown in Figure 4A. The slope named IC50 was
558
taken from at least three independent experiments similar to that shown in
559
Figure 4C. Each point represents the mean ± SD. Asterisks indicate significant
560
differences (measured using a Student’s t test, P
Dronedarone, an Amiodarone Analog with an Improved Anti-
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Leishmania mexicana Efficacy. Gustavo Benaima,b, Paola Casanovaa,b, Vanessa HernandezRodrigueza, Sheira Mujica-Gonzaleza, Nereida Parra-Gimeneza, Lourdes PlazaRojasa, Juan Luis Concepcionc, Yi-Liang Liud, Eric Oldfieldd,e , Alberto PanizMondolfif and Alirica I. Suarezg. a
Instituto de Estudios Avanzados (IDEA). Caracas, Venezuela. Instituto de Biología Experimental. Facultad de Ciencias. Universidad Central de Venezuela (UCV). Caracas. Venezuela. c Laborartorio de Enzimologia de Parásitos. Facultad de Ciencias, Universidad de Los Andes, Mérida, Venezuela. d Center for Biophysics & Computational Biology, University of Illinois at UrbanaChampaign, Urbana, IL, 61801, USA e Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA; f Instituto de Biomedicina (MPPSPS/UCV)/ Instituto Venezolano de los Seguros Sociales (IVSS). Caracas, Venezuela. g Facultad de Farmacia, Universidad Central de Venezuela. b
Abstract
22
Dronedarone and amiodarone are cationic, lipophilic benzofurans used to treat
23
cardiac arrythmias. They also have activity against the parasitic protozoan
24
Trypanosoma cruzi, the causative agent of Chagas disease. They function by
25
disrupting parasite intracellular Ca2+ homeostasis, as well as by inhibiting
26
membrane sterol (ergosterol) biosynthesis. Amiodarone also has activity
27
against Leishmania mexicana, suggesting that dronedarone might likewise be
28
active against this organism. This could be of therapeutic interest since
29
dronedarone is thought to have fewer side-effects in humans than does
30
amiodarone. We show here that dronedarone effectively inhibits the growth of
31
L. mexicana promastigotes in culture and more importantly, has excellent
32
activity against amastigotes inside infected macrophages (the clinically relevant
33
form) without affecting the host-cell, with the IC50 values against amastigotes
34
being three orders of magnitude lower than those obtained previously with T.
35
cruzi amastigotes (0.65 nM vs 0.75 μM). As with amiodarone, dronedarone
36
affects intracellular Ca2+ homeostasis in the parasite, inducing an elevation of
37
intracellular Ca2+. This is achieved by rapidly collapsing the mitochondrial
38
membrane potential and inducing an alkalinization of acidocalcisomes at a rate
39
that is faster than that observed with amiodarone. We also show that
40
dronedarone inhibits parasite oxidosqualene cyclase, a key enzyme in
41
ergosterol biosynthesis, known to be vital for survival. Overall, the results
42
suggest the possibility of a re-purposing dronedarone as a treatment for
43
cutaneous and perhaps other leishmaniases.
44 45
Introduction
46
Leishmania mexicana is a trypanosomatid parasite responsible for cutaneous
47
and muco-cutaneous leishmaniasis. Drugs used to treat these diseases, such
48
as glucantime, amphotericin B and miltefosine, frequently exhibit adverse side-
49
effects (1-2) so there is a growing interest in new drug targets, and leads. We
50
have demonstrated that amiodarone (1, Figure 1), a commonly used anti-
51
arrhythmic, has promising activity against both Leishmania mexicana (3) as well
52
as Trypanosoma cruzi parasites (4-5). Moreover, the combination of
53
amiodarone and miltefosine resulted in the complete parasitological cure of
54
mice infected with L. mexicana (6). It has been proposed that disruption of
55
parasite intracellular Ca2+ homeostasis is the target of action of several drugs,
56
including miltefosine and amiodarone (7). Thus, miltefosine opens a Ca2+
57
channel in the plasma membrane of these parasites (6), probably related to the
58
recently described sphingosine-induced L-type voltage-dependent Ca2+ channel
59
in L.mexicana (8), causing a large increase in the intracellular Ca2+
60
concentration ([Ca2+]i) of the parasites, while amiodarone also increases the
61
[Ca2+]i in L. mexicana promastigotes, by collapsing the mitochondrial
62
electrochemical potential and alkalinizing acidocalcisomes. However, in spite of
63
its frequent use in humans, the use of amiodarone may lead to a number of side
64
effects, due at least in part to the presence of two iodine atoms in its structure,
65
which may contribute, for example, to its thyroid toxicity. For this reason, an
66
analog of amiodarone was developed that does not contain iodine: dronedarone
67
(2, Figure 1). Recently, we found that dronedarone shows even greater potency
68
than does amiodarone against T. cruzi epimastigotes, as well as against
69
amastigotes present in infected VERO cells, which constitute the clinically
70
relevant form of the parasite (9). This opens up the possibility of using
71
dronedarone, already FDA approved for treating arrythmias, against Chagas'
72
disease (4) and perhaps, leishmaniasis. Here, we report the effects of
73
dronedarone on L. mexicana finding that it has potent activity against both
74
promastigotes as well as amastigotes inside macrophages, whose growth is not
75
affected by the drug. As with amiodarone, dronedarone functions by disrupting
76
Ca2+ homeostasis as well as by inhibiting parasite oxidosqualene cyclase
77
(OSC), a key enzyme in ergosterol synthesis. This multi-site targeting is likely to
78
contribute to the potent activity found.
79 80
Chemicals.
81
Amiodarone (1, Figure 1), digitonin, EGTA, carbonylcyanide p-(trifluoromethoxy)
82
phenylhydrazone (FCCP), and nigericin were purchased from Sigma (St. Louis,
83
MO). Fura 2-acetoxymethyl ester (FURA 2-AM), acridine orange, Rhodamine
84
123 and Rhod 2-AM were from Molecular Probes (Eugene, OR).
85 86
Dronedarone extraction
87
At room temperature, two commercial tablets of dronedarone (MULTAQ®
88
Sanofi-Aventi, 400 mg each) were added to 20 ml of methanol. 10 ml of H2O
89
were added and the mixture stirred for 15 minutes. The mixture was extracted
90
with 10 ml of chloroform, four times, the combined chloroform extracts dried
91
over anhydrous magnesium sulphate, filtered and evaporated under reduced
92
pressure to yield colorless crystals. The product was further purified using
93
column chromatography on silica gel (eluent chloroform:hexane 9:1) and
94
fractions containing pure dronedarone were evaporated to obtain pure
95
dronedarone as colorless crystals (mp 65 °C).
96 97
Culture of L. mexicana epimastigotes and growth inhibition by
98
dronedarone.
99
L. mexicana (Bel 21 strain) promastigotes were cultured in LIT (Liver Infusion-
100
Tryptose) medium supplemented with 10% fetal-bovine serum using continuous
101
agitation (100 rpm) at 29 °C, as reported previously (8). A growth curve was
102
performed to evaluate susceptibility of parasites to either dronedarone or
103
amiodarone at different concentrations; live parasites were counted daily using
104
a Neubauer chamber. The initial parasite concentration was 106 parasites/ml
105
and either the drug or the vehicle (DMSO) was added after 24 h. At least 3
106
independent experiments were performed for each drug and dose, and the IC50
107
values were determined using Prism GraphPad 5.0.
108 109
Amastigote growth inhibition.
110
L. mexicana amastigotes were incubated with murine macrophages (J774)
111
maintained in RPMI-1640 (GIBCO), supplemented with 10% fetal-bovine serum
112
and incubated at 37 °C in humidified 95% air/ 5% CO2 (6). 500 macrophages
113
were seeded per cover-slip in a 24-well plate and infected with promasitigotes
114
using 10 parasites per cell, for 12 h. After infection, cells were washed three
115
times to remove the external, non-adherent parasites, and fresh culture medium
116
was added, with or without dronedarone at different concentrations. The
117
infected cells were incubated for 96 h as previously described (6). After 96 h,
118
macrophages were fixed with methanol and stained with Giemsa to determine
119
the percentage of infected cells.
120 121
Determination of the intracellular Ca2+ concentration.
122
L. mexicana promastigotes were loaded with the fluorescent ratiometric
123
indicator Fura 2 as reported previously (8) in order to evaluate the effect of
124
dronedarone on [Ca2+]i. Briefly, 1 x108 parasites were collected by
125
centrifugation at 600g for 2 min and washed twice with a loading buffer (137
126
mM NaCl, 4mM KCl, 1.5 mM KH2PO4, 8.5 mM Na2HPO4, 11 mM glucose, 1 mM
127
CaCl2 0.8 mM, MgSO4 and 20 mM HEPES-NaOH, pH 7.4). The pellet was then
128
re-suspended and loaded with Fura 2-AM (1 μM), probenecid (2.4 mM) and
129
Pluronic acid (0.05 %) in loading buffer. Parasites were incubated at 29 °C in
130
the dark with continuous agitation for 1 h. Fura 2-loaded parasites were washed
131
twice with the same buffer, either in the presence or absence of Ca2+.
132
Fluorescence measurements were carried out on stirred cells at 29 oC using a
133
Perkin Elmer LS 55 spectrofluorimeter using Ex 340 nm/380 nm and Em 510
134
nm.
135
The [Ca2+]i was calculated as described by Grynkiewicz et al. (1985) (10),
136
using the equation:
137
[Ca2+]i = Kd x (R-Rmin/Rmax-R) x Fmin (380)/Fmax(380),
138
where Kd = dissociation constant (244 nM), R = ratio of measured fluorescence
139
intensities (at 340 nm and at 380 nm), Rmin = lowest R value at 0 Ca2+
140
concentration, Rmax = highest R value at saturating concentrations of Ca2+, Fmax
141
= lowest value of measured fluorescence at 380 nm, at 0 Ca2+ concentration,
142
and Fmin = highest value of measured fluorescence at 380 nm, at saturating
143
concentrations of Ca2+.
144
Maximum and minimum values were obtained after the addition of 30 μM
145
digitonin, which allows the flow of Ca2+ to the interior of the cell. Then, 8 mM
146
EGTA was added to chelate the remaining Ca2+.
147 148
Mitochondrial membrane potential.
149
The effect of dronedarone on the mitochondrial membrane potential of L.
150
mexicana promastigotes was evaluated using the fluorescent dye Rhodamine
151
123 as reported previously (9). Briefly, 108 parasites were collected by
152
centrifugation at 600 x g for 2 min and washed in PBS buffer plus 1% glucose.
153
The pellet was re-suspended in the same buffer and loaded with 20 μM of
154
Rhodamine 123 for 45 min. at 29 °C with mild agitation, in the dark.
155
Subsequently, parasites were washed twice and re-suspended in the same
156
buffer and transferred into a magnetically stirred cuvette. Measurements (Ȝext
157
488 nm, Ȝem 530 nm) were made in a HITACHI 7000 spectrofluorimeter at 29
158
°C. FCCP (1 μM) was used as positive control.
159 160
Acidocalcisomes alkalinization.
161
Evaluation of the effect of dronedarone on the alkalinization of acidocalcisomes
162
was performed using acridine orange (9). Promastigotes (108 cells/ml) were
163
collected and washed, then incubated with 2 ȝM acridine orange in PBS buffer,
164
for 5 min at 29 ºC with constant stirring. Measurements were performed at Ȝext
165
488 nm and Ȝem 530 nm at 29 °C in a HITACHI 7000 spectrofluorimeter under
166
magnetic stirring. Nigericin (1 μM) was used as positive control.
167 168
Subcellular fractionation.
169
L. mexicana promastigotes in exponential phase (2 L of culture) were
170
homogenized and fractionated following established procedures (11). Briefly,
171
cells were collected at 3000 x g for 10 min and washed twice in buffer A (25 mM
172
Tris–HCl, pH 7.4, 1 mM EDTA, 250 mM sucrose) and broken by abrasion in a
173
1:1 (wet wt:wt) mixture of cells with silicon carbide (200 mesh) in a chilled
174
mortar to produce an homogenate that was diluted to 100 ml of buffer B (25 mM
175
Tris–HCl, pH 7.2, 250 mM sucrose, 25 mM NaCl, 2 mM EDTANa2, 5 mM
176
dithiothreitol) in the presence of a cocktail of protease inhibitors (10 μM
177
leupeptin, 50 μg ml-1 trypsin inhibitor, 1 mM benzamidine, 50 μM PMSF, 100 μM
178
TLCK, 0.2 μM antipain, 1 μM pepstatin, 1 μM E-64, 1 μM chemostatin, 1 μM
179
bestatin). The homogenate was centrifuged at 1000 x g for 10 min to remove
180
unbroken cells and nuclei and then at 5000 x g for 10 min, which produced a
181
large granule fraction (containing mitochondria and large cellular debris); a
182
small granule pellet was obtained at 33,000 x g for 15 min. The post-small
183
granule supernatant was subjected to 105,000 x g for 90 min to produce a
184
microsomal pellet and a soluble (cytosolic) fraction. The microsomal fraction
185
pellet was re-suspended in 6 ml of 50 mM MOPS/NaOH (pH 7.4), 1 mM EDTA,
186
0.1% Triton X-100 and 0.1% Tween 80, homogenized with a Potter-Elvehjem
187
homogenizer, then centrifuged at 105,000 x g for 90 min. The resulting
188
supernatant was collected and used for the enzyme inhibition assays.
189 190
Determination of protein concentration
191
Proteins were precipitated with 10% (w/v) trichloroacetic acid at 47 ºC. The
192
protein precipitate was washed with cold acetone, re-suspended in distilled
193
water, and finally assayed using a modification (12) of Folin phenol in the
194
presence of 0.1% sodium dodecyl sulfate; the procedure was designed to
195
eliminate the interference from substances present in the fractionation and
196
assay media, including EDTA and sucrose. Bovine serum albumin was used as
197
a standard.
198
199
Enzyme inhibition assay
200
Oxidosqualene cyclase (OSC) was assayed using the radioactive method
201
described by Milla et al. 2002 (13). The microsomal extract (0.7 mg of
202
protein/ml) was incubated with 3S-2,3-[14C]oxidosqualene (35 μM) in the
203
presence of Triton X-100 (0.1%) and Tween 80 (0.1%) in 50 mM MOPS/NaOH
204
(pH 7.4) and 1 mM EDTA for 30 min at 28 ºC under vigorous shaking on a
205
water bath. The reaction was stopped by adding of 20% KOH/ 50%EtOH, and
206
lipid was saponified at 80 ºC for 30 min. The non-saponifiable lipids were
207
extracted twice with 1 ml of petroleum ether and separated on TLC plates
208
(Merck)
209
Radioactivity in 2,3-oxidosqualene and lanosterol were quantified by using an
210
imaging plate on a System 2000 Imaging Scanner (Packard). The amount of
211
lanosterol product formed was used for the calculation of enzyme activity.
212
Chromatographic
213
squalene, 2,3-oxidosqualene and ergosterol. The standards were visualized on
214
the TLC plate with iodine vapor. Enzyme inhibition was carried out in basically
215
the same way, but in the presence of varying concentrations of dronedarone.
216
Lineweaver-Burke plots were utilized to derive apparent Ki values (average of
217
triplicates ± standard deviation).
using
toluene-diethyl
standards
ether
were
(9:1)
as
the
developing
2,3,22,23-dioxidosqualene,
solvent.
lanosterol,
218 219
Computational aspect
220
Docking of dronedarone to a Phyre2 (14) model of LmOSC was carried out by
221
using the Glide program (15) from Schrödinger. The common feature
222
hypothesis was produced using 3-7, known OSC inhibitors (5) using the MOE
223
program (16). All the structural figures were made by using Pymol (17).
224 225
Data analysis
226
Analyses of kinetic data and IC50 results were carried out using non-linear
227
regression methods implemented in the SIGMA-PLOT software packages.
228 229
Results and Discussion
230
We first studied the effect of dronedarone at different concentrations on the
231
growth of L. mexicana promastigotes. Figure 2A shows that dronedarone at
232
concentrations above 0.25 μM has a profound effect on the growth of
233
promastigotes in culture. The calculated IC50 value was of 115 nM (as shown in
234
the insert). This concentration is much smaller than that we previously obtained
235
in L. mexicana with amiodarone, IC50 ~900 nM (6). We also analysed the
236
percentage of growth inhibition induced by dronedarone in promastigotes in
237
Figure 2B, where a clear dose-dependent response was obtained. We then
238
evaluated the effect of dronedarone on the growth of intracellular amastigotes in
239
infected macrophages. As shown in Figure 3, dronedarone has a marked effect
240
on the parasites, with a calculated IC50 value of 0.65 nM; again, significantly
241
less than the IC50 of 8 nM observed using amiodarone under the same
242
conditions (6). Interestingly, this concentration is also dramatically lower than
243
that obtained using dronedarone on T. cruzi amastigotes in infected cells where
244
in previous work (9) we reported an IC50 of 0.75 μM (i.e. a ~1000-fold higher
245
IC50 value). Since amastigotes inside macrophages represent the clinically
246
relevant form of L. mexicana, this result points to the much higher effectiveness
247
of dronedarone in L. mexicana. Additionally, it can be seen (Figure 3) that
248
dronedarone at the concentrations employed does not have any discernible
249
effect on macrophages: over the entire dose range investigated. At therapeutic
250
doses (in humans, to treat arrhythmias) the plasma level of dronedarone is ~0.2
251
μM but in liver, levels are ~10-20x higher (18).
252
The 0.65 nM IC50 value in amastigotes is ~300x less than the 0.2 μM plasma
253
level using normal therapeutic dosing and suggests a potentially good
254
therapeutic index.
255
Since we found in previous work that amiodarone induced an increase in the
256
intracellular, cytoplasmic levels of calcium, [Ca2+]i, we next investigated whether
257
dronedarone had a similar effect on [Ca2+]i using FURA 2-loaded promastigotes.
258
As shown in Figure 4A, the addition of dronedarone (2.5 μM) induces a marked
259
increase in [Ca2+]i which reaches a plateau after a few minutes. To determine
260
whether this increase was due to entry of extra-cellular Ca2+ or as a
261
consequence of its release from intracellular organelles, the same experiment
262
was performed in the absence of external Ca2+ (i.e. a Ca2+-free buffer as well as
263
in the presence of EGTA). As can be seen in Figure 4B, in the absence of
264
external calcium, dronedarone causes the same increase in [Ca2+]i as that
265
obtained in the presence of external calcium, meaning that it was released from
266
intracellular stores. It should be noticed that under both conditions, a biphasic
267
response can be observed that we attribute to the action of dronedarone on at
268
least two intracellular compartments, namely acidocalcisomes and the
269
mitochondrion, given that these are the intracellular organelles known to be
270
involved in Ca2+ accumulation (7;19-20). We calculated the initial slopes of this
271
biphasic response finding that dronedarone acts about twice as fast as does
272
amiodarone (Figure 4D). However, when the experiments were performed using
273
the IC50, instead of 2.5 μM dronedarone (Figure 4C), while there was still a
274
marked increase in [Ca2+]i , the biphasic response was not discernible and the
275
initial slope of the curve with dronedarone was about one third of that obtained
276
in the presence of 2.5 μM of the drug (Figure 4D).
277
In order to test the effect of dronedarone on acidocalcisomes, several
278
experiments were performed using promastigotes loaded with acridine orange,
279
a compound that accumulates in acidic compartments, enabling the
280
visualization of any alkalinization. Figure 4A shows that dronedarone (2.5 μM)
281
induces a rapid alkalinization of acidocalcisomes. In the same experiment,
282
amiodarone, also known to lower the acidity in these organelles (6), was added
283
at 10 μM in order to compare its effect with dronedarone. As can be seen in
284
Figure 5A, dronedarone’s effect was significantly faster than that seen with
285
amiodarone. The subsequent addition of nigericin induced the further
286
alkalinization in these organelles (Figure 5A). Nigericin promotes the
287
electroneutral exchange of H+ and K+, and loading with acridine orange followed
288
by exposure to nigericin leads to the rapid release of the fluorescent dye from
289
the acidocalcisomes with a concomitant increase in fluorescence (9).
290
Performing the same experiment but inverting the order of addition (i.e., adding
291
nigericin first and then dronedarone or amiodarone) shows once again a large
292
rise in acidocalcisome alkalinization (Figure 5B). Subsequent addition of
293
dronedarone or amiodarone induced a further increase in the release of acridine
294
orange. Once again, the effect of dronedarone was more rapid than that seen
295
with amiodarone (Figure 5B), suggesting perhaps better cell membrane
296
permeability. Since nigericin completely disrupts acidocalcisome function, this
297
further effect of the drugs suggests that another separate compartment may be
298
involved in their action. Analysis of the initial slopes in Figure 5A,B indicates
299
(Figure 5C) that the rate of alkalinization of dronedarone is similar to that
300
obtained with nigericin, while that of amiodarone is significantly slower. This
301
could help explain the lower IC50 obtained with dronedarone when compared to
302
amiodarone (Figure 5C). We also observed that dronedarone when added at
303
the IC50, (instead of at 2.5 μM), was again able to induce alkalinization of the
304
acidocalcisomes, albeit with a lower velocity (Figure 5D).
305
The large, unique mitochondrion in L. mexicana is also thought to be involved in
306
[Ca2+]i regulation (21-22), as well as in the leishmanicidal effects of amiodarone
307
(6). We thus next investigated the effects of dronedarone on the mitochondrial
308
membrane potential using Rhodamine 123, which is known to accumulate in
309
energized mitochondria (4,9), resulting in self-quenching of the dye’s
310
fluorescence. An increase in Rhodamine 123 fluorescence corresponds to de-
311
energization. As shown in Figure 6A, dronedarone (2.5 μM) induces a rapid
312
increase in fluorescence, due to a collapse of the mitochondrial membrane
313
potential. Again, the effect is significantly faster with dronedarone than with
314
amiodarone (at 10 μM, Figure 6A). Furthermore, addition of the uncoupler
315
FCCP after adding either amiodarone or dronedarone induces a small
316
additional release of Rhodamine 123, as deduced from the increased
317
fluorescence. When FCCP is added first (Figure. 6B), it induces a rapid but
318
small increase in fluorescence, since FCCP dissipates the mitochondrial
319
membrane potential by permeabilizing the inner mitochondrial membrane to
320
protons. In the same figure it is also evident that the subsequent addition of
321
dronedarone (2.5 μM) causes a rapid release of Rhodamine 123, faster again
322
than that obtained with amiodarone (at 10 μM, Figure. 6B). The calculated initial
323
slope of these curves indicate that the effect of dronedarone is faster than that
324
of amiodarone (about 5-fold), and twice as that obtained with FCCP (Figure
325
6C). We also performed experiments on the mitochondrial electrochemical
326
potential using the IC50 of dronedarone. As can be seen in Figure 6D,
327
dronedarone is able to induce a robust release of Rhodamine 123, even when
328
added after FCCP. This strongly suggests that the action of dronedarone on
329
mitochondria could be part of the improved performance of dronedarone as
330
compared to amiodarone.
331
From these results it is clear that dronedarone induces a rapid collapse of the
332
parasite’s mitochondrial membrane potential, although its specific target is
333
unknown. The effect of dronedarone or amiodarone after FCCP is not easy to
334
explain since it might be expected that the uncoupler would release all the
335
fluorescent dye. In a previous study with epimastigotes of T. cruzi (9) we also
336
observed this behaviour with dronadarone (and amiodarone) after FCCP
337
addition. It is conceivable that FCCP is not properly acting under these
338
experimental conditions. Another possible explanation is that part of the action
339
of dronedarone (and amiodarone) is by affecting also another unknown
340
compartment.
341
We also performed experiments using the IC50 of dronedarone on the
342
electrochemical mitochondrial potential. It can be seen in Figure 6D that this
343
drug is also able to induce a robust release of Rhodamine 123, even when
344
added after FCCP. This strongly suggests that the action of dronedarone on
345
mitochondria could be part of the better performance of dronedarone when
346
compared with amiodarone on these parasites.
347
With amiodarone inhibition of T. cruzi growth (6), we reported previously that
348
not only did amiodarone affect Ca2+i and the mitochondrial membrane potential,
349
it also blocked formation of the essential membrane sterol ergosterol by
350
inhibiting the enzyme oxidosqualene cyclase (OSC). We thus investigated
351
whether dronedarone inhibited the activity of L. mexicana OSC (LmOSC) in a
352
similar fashion.
353
Figure 7A shows that the enzyme exhibits classic Michaelis–Menten kinetics
354
with respect to substrates with Km,app [(3S)-2,3-oxidosqualene] = 36 μM, Vmax,app
355
= 0.82 μM . min-1, and Vmax/Km = 0.023 min-1. Dronedarone behaves as a potent
356
non-competitive inhibitor with respect to [(3S)-2,3-oxidosqualene], with Ki = 0.7
357
μM. The dose-response curves for the activity of dronedarone against LmOSC
358
(Figure 7B) are consistent with noncompetitive inhibition with Ki § IC50; these
359
Ki values are 1 to 2 orders of magnitude lower than the Km of the substrates.
360
Similar experiments were performed with amiodarone, in order to compare it
361
with dronedarone (not shown). The Ki value obtained was of 9.0 μM, much
362
weaker inhibition than with amiodarone.
363
To investigate how dronedarone (2) might bind to LmOSC, we used
364
computational docking with the Glide program (15). The top scoring pose is
365
shown in Figure 8A,B, superimposed on the structure of the OSC inhibitor Ro-
366
48-8071 (3, Figure 1) bound to human OSC (PDB ID code 1W6J). Clearly, it
367
seems very likely that dronedarone binds to the LmOSC substrate binding site
368
with its cationic (trialkylammonium) side-chain located close to the essential
369
D718, corresponding to D455 in Homo sapiens OSC (HsOSC) that catalyzes
370
the protonation-initiated electrocyclization of epoxysqualene. The binding of
371
amiodarone (1) is also similar to that of Ro-48-8071 (3, Figure 7C) and a
372
comparison between the amiodarone (1) and dronedarone (2) docked LmOSC
373
structures is shown in Figure 7D. A common feature pharmacophore obtained
374
using the potent known HsOSC inhibitors (3-7, Figure 1) is shown in Figure 8E
375
superimposed on the structure of dronedarone (2). The key features of this
376
pharmacophore that are also present in dronedarone are the presence of the
377
cationic feature (that presumably mimics the carbo-cation obtained on
378
protonation of epoxysqualene) that is likely to interact with the conserved D718,
379
together with 3 hydrophobic features. In conclusion, the results indicate that
380
dronedarone binds to the same catalytic/hydrophobic pocket as does
381
amiodarone (in T. cruzi OSC), as does the HsOSC inhibitor Ro-48-8071-whose
382
structure is known (PDB ID code 1W6J) and which has a similar
383
pharmacophore. The results also show that the cationic moiety in dronedarone
384
docks very close to the very highly conserved D718 which in human OSC is a
385
key catalytic residue (D455) involved in initial squalene epoxide protonation.
386
Given that amastigotes are inhibited with a very low IC50 (0.65 nM) it seems
387
unlikely that there will be significant OSC inhibition at the sub-micromolar levels
388
that might be anticipated to have therapeutic utility. Nevertheless, the
389
development of more potent analogs that target LmOSC is a potentially
390
important goal since multiple-site targeting would be expected to increase
391
efficacy.
392 393 394
In conclusion, the results we have shown above are of interest for several
395
reasons. First, we show that the anti-arrhythmia drug dronedarone has activity
396
against L. mexicana promastigotes. Second, that it has potent activity against
397
intracellular amastigotes, in macrophages, with an IC50 of 0.65 nM while host
398
cell proliferation is not inhibited. These effects are similar to those observed
399
previously with amiodarone but are more rapid as well as far more pronounced.
400
Third, we find that dronedarone increases intracellular Ca2+ and that this
401
increase in [Ca2+]i arises from Ca2+ release from acidocalcisomes and the
402
mitochondrion. Finally, we find that dronedarone inhibits L. mexicana
403
oxidosqualene cyclase, potentially opening up a future route to blocking
404
formation of the essential membrane sterol, ergosterol. Overall, these results
405
are of considerable interest since not only is dronedarone more potent than is
406
amiodarone against L. mexicana, but also it is known to have less adverse side-
407
effects (in humans, when used as an anti-arrhythmic) since it lacks the iodine-
408
substituents that make amiodarone resemble thyroxine/tri-iodothyronine.
409
From a clinical perspective these findings are of importance because
410
mixed infections involving both Trypanosoma cruzi and different Leishmania
411
species is becoming a common clinical scenario in many countries in Central
412
and South America (23). Thus, having a single drug that can target both
413
parasites is of therapeutic interest. Moreover, Leishmania mexicana is a major
414
causative agent of the difficult-to-treat diffuse cutaneous leishmaniasis (24),
415
where again new drugs are needed.
416
It remains to be seen whether dronedarone is more efficacious than is
417
amiodarone in humans since at least part of the efficacy of amiodarone against
418
cutaneous leishmaniasis is likely due to its unusual, dermal elimination from the
419
body. That is, concentration in the skin may be much higher than with
420
dronedarone, and further work on determining these levels with dronedarone is
421
needed, as are studies aimed at other Leishmania parasites. However, the
422
considerably increased efficacy of dronedarone may offset the decrease in skin
423
elimination.
424
The repositioning of old drugs for their use in other diseases has been a
425
matter of recent interest, since the trials in humans have been already
426
successfully achieved, and most of them are not expensive since their
427
respective patents have expired (25). We postulate that since both dronedarone
428
and
429
clinical trials against both leishmaniasis as well as Chagas’ disease, both drugs
430
could be repurposed in the near future for the treatment of these human
431
infections. It seems likely that the more potent as well as less toxic dronedarone
432
may be more efficacious in humans.
amiodarone are inexpensive and amiodarone has been used in some
433
Because the role of dronedarone in patients with severe heart failure is
434
still controversial, we believe that its use should be avoided in severe cardiac
435
disease. However, its potential use in otherwise healthy patients with cutaneous
436
leishmaniasis and/or mild-to-moderate heart failure or arrhythmias remains a
437
feasible therapeutic option, even more considering its potentially high efficacy.
438 439
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521 522
Acknowledgments: This work was supported by grants from Fondo Nacional
523
de Ciencia, Tecnología e Investigación, Venezuela (FONACIT) (2011000884),
524
and the Consejo de Desarrollo Científico y Humanístico (CDCH-UCV) grant
525
PG-03-8728-2013. Universidad Central de Venezuela to G.B. and in part by
526
NIH grant GM065307 to E.O.
527 528
FIGURES LEGEND
529
Figure 1. Structures of molecules of interest.
530 531
Figure 2. Inhibition of L. mexicana promastigotes growth by dronedarone.
532
A. Susceptibility of promastigotes to dronedarone. Each point represents the
533
mean ± SD of at least three independent experiments. Insert: Dose-response
534
curve from A; IC50 = 115 nM. B. Percentage of growth inhibition induced by
535
dronedarone in promastigotes. All the values were taken from the data of Figure
536
2A using the values obtained at 10 days after addition of the corresponding
537
drug concentration. Each point represents the mean ± SD.
538 539
Figure 3. Effect of dronedarone against intracellular amastigotes.
540
Macrophages (J774 cells) infected with L. mexicana amastigotes were exposed
541
to different concentrations of dronedarone. The percentage of infected
542
macrophages (filled squares) and the effect on non-infected macrophages (filled
543
circles) were determined at 72 hours after the addition of the drug. Around 100
544
cells were counted in every experiment. Each point represents the mean ± SD
545
of at least three independent experiments.
546 547
Figure 4: Effect of dronedarone on the [Ca2+]i of L. mexicana
548
promastigotes. Promastigotes were loaded as described under “Materials and
549
Methods”. A. Effect of 2.5 μM dronedarone (arrow) on the parasite cytoplasmic
550
Ca2+ concentration in the presence of 2 mM external Ca2+. B. Effect of 2.5 μM
551
dronedarone (arrow) on Fura 2-loaded promastigotes in the absence of external
552
Ca2+ (EGTA). C. Effect of 0.65 nM dronedarone (arrow) on the parasite
553
cytoplasmic Ca2+ concentration in the presence of 2 mM external Ca2+. The
554
figures shown are representative of at least three independent experiments. D.
555
Initial slopes (300 sec) of the curves obtained in the presence of 2 mM external
556
Ca2+. The first and second slopes were taken from at least three independent
557
experiments similar to that shown in Figure 4A. The slope named IC50 was
558
taken from at least three independent experiments similar to that shown in
559
Figure 4C. Each point represents the mean ± SD. Asterisks indicate significant
560
differences (measured using a Student’s t test, P
