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SELECTIVE INHIBITION OF HEPATITIS C VIRUS INFECTION BY 1
HYDROXYZINE AND BENZTROPIN. 2
Lidia Mingorance1, Martina Friesland
1, Mairene Coto-Llerena
2, Sofía Pérez-del-3
Pulgar2, Loreto Boix
3, Juan Manuel López-Oliva
3, Jordi Bruix
3, Xavier Forns
2 and 4
Pablo Gastaminza1#. 5
1Centro Nacional De Biotecnología-Consejo Superior de Investigaciones Científicas 6
(CNB-CSIC). 7
2Liver Unit, Hospital Clinic, IDIBAPS, CIBERehd, Barcelona, Spain 8
3Barcelona Clinic Liver Cancer (BCLC) Group, Liver Unit, Hospital Clínic, IDIBAPS, 9
CIBERehd, Barcelona, Spain 10
11
Running Title: Hydroxyzine and benztropin inhibit HCV entry 12
#Address correspondence to: 13 Pablo Gastaminza Ph.D. 14 Departamento de Biología Celular y Molecular 15 Lab. 116 16 Centro Nacional de Biotecnología-C.S.I.C. 17 Calle Darwin, 3 18 28049-Madrid 19 Phone: +34 91 585 4561 20 Fax: +34 91 585 4506 21 Email: [email protected] 22 23
24
AAC Accepts, published online ahead of print on 7 April 2014Antimicrob. Agents Chemother. doi:10.1128/AAC.02619-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Abstract 25
Hepatitis C virus (HCV) infection is a major biomedical problem worldwide as it 26
causes severe liver disease in millions of humans around the world. Despite the recent 27
approval of specific drugs targeting HCV replication to be used in combination with 28
interferon- and ribavirin, there is still an urgent need for pangenotypic, interferon-free 29
therapies to fight this genetically diverse group of viruses. In this study, we have used an 30
unbiased screening cell culture assay to interrogate a chemical library of compounds 31
approved for clinical use in humans. This system enables identifying non-toxic antiviral 32
compounds targeting every aspect of the viral lifecycle, be the target viral or cellular. The 33
aim of this study is to identify drugs approved for other therapeutic applications in 34
humans that could be effective components of combination therapies against HCV. As a 35
result of this analysis we identified twelve compounds with antiviral activity in cell 36
culture, some of which had previously been identified as HCV inhibitors with antiviral 37
activity in cell culture and shown to be effective in patients. We selected two novel HCV 38
antivirals, hydroxyzine and benztropin, to characterize them by determining their 39
specificity and genotype spectrum as well as by defining the step of the replication cycle 40
targeted by these compounds. We found that both compounds effectively inhibited viral 41
entry at a post-binding step of genotypes 1, 2, 3 and 4 without affecting entry of other 42
viruses. 43
44
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45
Introduction 46
It is estimated that 150 million humans are chronically infected by HCV, many of 47
which will suffer from severe liver disease (1). Chronic HCV infection is associated with 48
liver inflammation, fibrosis, cirrhosis and hepatocellular carcinoma (2). Consequently, 49
HCV infection is one of the leading causes of liver transplantation worldwide. The 50
hepatitis C viruses are genetically diverse and are clustered in 7 major genotypes with a 51
large number of subtypes (3). There are important differences among genotypes 52
regarding geographical distribution, pathogenesis and response to treatment (4). There is 53
no vaccine against HCV and the current approved treatments, albeit increasingly 54
effective, are expensive and associated with severe adverse effects (5). 55
HCV replication cycle is initiated by the attachment and entry of the viral 56
particles into the target cells, a multistep process mediated by the viral envelope 57
glycoproteins and host factors that are incorporated into viral particles (6). Virion 58
internalization is triggered by receptor-dependent endocytosis in clathrin-coated pits and 59
subsequent endosomal acidification triggers fusion of the viral and endosomal 60
membranes, a process mediated by HCV glycoprotein complex (E1E2) that results in the 61
release of the viral genome to the cytosol (7). Translation of the incoming viral RNAs 62
leads to expression of the viral proteins, viral RNA replication and assembly of progeny 63
infectious viral particles (reviewed in (8) (9)). 64
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Until 2011, HCV treatment consisted in the combination of interferon alpha 65
(IFN) and ribavirin, which was only partially effective and associated with severe 66
adverse effects (5). The recent approval of viral protease inhibitors to be used as additives 67
to the IFN-based therapy improved significantly the cure rate, at the expense of additional 68
adverse effects and high cost, factors that limit their implementation (10). Nevertheless, a 69
large number of effective and specific HCV inhibitors (Direct Acting Antivirals; DAA) 70
have been developed and display promising efficacy in clinical trials. However, the 71
genetic diversity of HCV viruses and the stage of liver disease (i.e. cirrhosis) are 72
revealing themselves as obstacles for effective, pan-genotypic treatments (5, 11). Thus, 73
the armamentarium against HCV needs to be expanded to combat these diverse viruses, 74
particularly since combination therapy of several drugs will be required to circumvent the 75
selection of escape variants during monotherapy (5). In order to contribute to this task, 76
we used an unbiased screening methodology to interrogate a chemical library for HCV 77
inhibitors (12). This library contains 281 clinically approved drugs prescribed for non-78
HCV applications. Using this strategy, we identified a set of compounds that inhibited 79
HCV infection at non-toxic concentrations. We selected the compounds with the highest 80
therapeutic index (hydroxyzine and benztropin) and characterized the step(s) of the viral 81
replication cycle inhibited by these compounds. All the compounds inhibit HCV entry in 82
various human hepatoma cell lines at a post-adsorption step. Interestingly, we observed 83
that genotype 2 envelope glycoproteins are more susceptible to hydroxyzine than 84
genotypes 1, 3 and 4, an observation that was not evident for other compounds. Our study 85
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identifies hydroxyzine and benztropin as effective, selective inhibitors of HCV entry in 86
cell culture. It also reveals important intergenotypic differences in the susceptibility of 87
HCV to the antiviral action of hydroxyzine, indicating that this compound might also be 88
useful for the study of basic aspects of HCV entry. 89
90
Materials and Methods 91
Reagents 92
NIH Collection 2 compound library was purchased from Evotec (San Francisco, 93
CA). Hydroxyzine pamoate, fluoxetine and perphenazine were purchased from Sigma-94
Aldrich (St. Louis, MO) and benztropin mesylate was purchased from Sellekchem 95
(Houston, TX). 2´-c-methyladenosine (2mAde) was purchased from BOC Sciences 96
(Shirley, NY). All compounds, including those in the library were dissolved in DMSO at 97
a final 10 mM concentration. Except when stated otherwise the compounds were used at 98
the following concentrations: hydroxyzine and perphenazine (5 µM), fluoxetine (3 µM) 99
and benztropin mesylate (7.5 µM). 100
101
Cells 102
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Human hepatoma Huh-7 cells and derived subclone Huh-7.5.1 clone2 (hereafter 103
clone 2 cells) as well as human embryonic kidney cells (HEK293T) have been previously 104
described (13-15). These cells were cultured in Dulbecco´s Modified Eagles´s Medium 105
supplemented with 100 mM HEPES, non-essential aminoacids (Gibco) and 106
Penicillin/Streptomycin (Gibco) and 10% Fetal Bovine Serum (FBS). Hepatocarcinoma 107
cell lines BCLC5, BCLC6 and BCLC10 have previously been described (16). 108
109
Viruses 110
JFH-1 virus was produced from cloned cDNA using previously described 111
methodology (17). D183 virus (D183v) was previously described (18). Infectious, 112
defective HCV particles produced by trans-complementation (HCVtcp) were generated 113
using the methodology and reagents described in (19) and kindly provided by Dr. Ralf 114
Bartenschlager. 115
116
Library Screening 117
Compounds were screened at a final 10µM concentration using a cell-based 118
ELISA- colorimetric readout of the infection approach in a 96-well format as described in 119
(20)(12). Briefly, this assay allows determining viral spread by measuring the total viral 120
antigen (E2) present in a given well using specific antibodies. Relative infection values 121
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are determined using a standard curve generated by serial dilution of the virus inoculum 122
as previously described (12). Once infection efficiency is determined, wells are 123
extensively washed to determine remaining cell biomass in order to evaluate compound 124
cytoxicity. Cell biomass is measured by crystal violet staining and colorimetry at 570 nm 125
as described previously (21). Relative biomass values are expressed as percentage of the 126
values found in the control wells. Since cells are plated at low density (approximately 127
30%), the cells proliferate until reaching confluency in the control wells by the end of the 128
experiment 72 hours post-infection. Thus, biomass estimation allows identification of 129
false-positives in the screening. 130
Primary hits were identified as those reducing by more than 20-fold HCV 131
infection but maintaining 85% of the cell biomass at 10µM. Primary hits were 132
subsequently counterscreened at the same concentration for confirmation. Compounds 133
significantly reducing HCV infection but displaying detectable toxicity (biomass <85%) 134
were counterscreened at 2µM. Compounds displaying more than 80% reduction in HCV 135
infection but no significant toxicity (biomass>85%) were further considered in the study. 136
137
Determination of potency (EC50) and toxicity (CC50). 138
In order to determine potency and cytotoxicity indexes, 104 clone 2 cells are 139
seeded in 96-well plates (approximately 30% confluency). Potency of the compounds 140
(EC50, EC90) was calculated by determining relative infection efficiency in the presence 141
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of serial compound dilutions (ranging from 50 µM to 2.5 nM), 72 hours post infection, 142
using the colorimetric method described above and as previously described (20) (12). 143
Parallel uninfected cultures, treated with the same compound dilutions were used to 144
determine CC50 values by MTT-formazan cellular viability assays 72 hours post-145
treatment using standard procedures (22). EC50, EC90 and CC50 values were interpolated 146
graphically from the dose-response curves. 147
148
Infection experiments 149
Multiple cycle infections 150
Huh-7.5.1. clone 2 cells (5 X 105 cells; 6-well plate) were inoculated (moi 0.01) 151
with JFH-1 virus (17) in the presence of perphenazine (5µM), hydroxyzine (5µM), 152
fluoxetine (3µM) or benztropin (7.5µM) and incubated for 6 days at 37oC. Cells were 153
split (1:3) at day 3 post-inoculation, time at which the cells were replenished with fresh 154
medium containing the compounds. Intracellular HCV RNA levels in the infected cells 155
were determined at day 6 by real time RT-qPCR on total cellular RNA extracted using 156
the guanidinium thiocyanate (GTC)-acid phenol extraction method (23), as previously 157
described (17). 158
159
Single cycle infections 160
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Huh-7 cells (5 X 104 cells/well; 24-well plates) were seeded in the presence of 5 161
X105 f.f.u./well (500µl) of the D183 virus (18)and perphenazine (5µM), hydroxyzine 162
(5µM) or benztropin (7.5µM) Five hours post-infection, cells were washed twice with 163
warm PBS and further incubated for 24 and 48 hours in complete medium at 37oC. 164
Samples of the cells and supernatants were collected at 24 and 48 hours postinfection for 165
HCV RNA quantification by RT-qPCR and infectivity analyses by titration. 166
167
Infection with HCVtcp 168
Cell supernatants containing HCVtcp were mixed 1:1 with compound dilutions 169
required to obtain the desired final concentrations of perphenazine (5µM), hydroxyzine 170
(5µM) or benztropin (7.5µM). Mixtures were used to inoculate Huh-7 cells, which were 171
cultured for 48 hours before measuring intracellular luciferase levels using a 172
commercially available kit (Luciferase Assay System, Promega- Madison, WI). 173
174
Persistent HCV infections 175
Establishment of persistent JFH-1 infections was previously described (24). Cells 176
were treated for 48 hours (replenishing medium and inhibitors at 24 hours) with 177
perphenazine (5µM), hydroxyzine (5µM) or benztropin (7.5µM) using DMSO and 178
2mAde (10µM) as negative and positive inhibition controls. 179
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180
Time-of-addition experiments. 181
In order to discriminate inhibition of particle binding and post-binding events, we 182
incubated pre-chilled target cells (Huh-7) for 1 hour at 4oC in the presence of the 183
perphenazine (5µM), hydroxyzine (5µM) or benztropin (7.5µM). After this adsorption 184
period, cells were washed twice with cold PBS and replenished with warm DMEM 10% 185
FBS and incubated for 72 hours at 37oC. Parallel cultures inoculated with virus at 4
oC in 186
the absence of inhibitors (1 h), were washed twice with cold PBS and replenished with 187
DMEM 10%FBS containing the compounds. Cultures were incubated in DMEM 10% 188
FBS for additional 72 hours. Relative infection efficiency was determined by 189
immunostaining and infection foci counting as previously described (25) using a 190
monoclonal antibody against E2 (AR3A) kindly provided by Dr. Law (TSRI-La Jolla, 191
CA). 192
193
HCV pseudotype particle infectivity inhibition assay. 194
HCV E1/E2-pseudotyped retroviral particles bearing the luciferase reporter gene 195
were generated as described (26) using the reagents kindly provided by Dr. F.L. Cosset 196
(INSERM-Lyon, France). As controls, pseudotypes bearing vesicular stomatitis virus 197
(VSV), feline retrovirus (RD114) or influenza envelope glycoproteins were produced in 198
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parallel. For the inhibition assays, particles were mixed 1:1 with medium containing 199
(DMSO) or with compound dilutions to achieve the desired final concentration of 200
perphenazine (5µM), hydroxyzine (5µM) or benztropin (7.5µM). The mixture was used 201
to inoculate Huh-7 or BCLC-5, 6, 10 cells at 37oC for another 72-hour period after which 202
infection efficiency was evaluated by measuring reporter gene expression using a 203
commercial kit (Luciferase Assay System, Promega- Madison, WI). Relative infection 204
values were obtained using DMSO-treated cells as control (100%). Background levels 205
were established by measuring luciferase expression in cells transduced with HCVpp 206
lacking envelope glycoproteins, as previously described (26). 207
208
In vitro transcription and HCV RNA electroporation 209
In vitro transcribed subgenomic HCV replicon RNA was electroporated as 210
described previously (27, 28). Five hours post-electroporation compounds were added at 211
the indicated concentrations and left throughout the experiment. Firefly luciferase 212
activities were measured in the sample using a commercial kit (Dual Luciferase Assay 213
System; Promega- Madison, WI) at different times post-transfection. 214
215
216
Western-Blot Analysis 217
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Quantification of NS3 protein and human beta actin by Western-Blot was 218
performed as described previously (28). 219
220
221
Results 222
Identification of novel compounds inhibiting HCV infection. 223
In order to identify clinical compounds with antiviral potential against HCV 224
infection, we screened a chemical library composed of 281 clinically approved 225
compounds for non-HCV therapeutic purposes. The screening methodology was identical 226
to that described previously (12). Briefly, highly susceptible human hepatoma cells (Huh-227
7.5.1-clone 2) were inoculated at low multiplicity of infection (m.o.i. 0.01) with HCV 228
(D183; (18)). Infected cultures were incubated in the presence of the compounds (10 µM) 229
for 72 hours at 37oC, time after which relative infection efficiency and cytotoxicity were 230
determined as previously described in Materials and Methods. We identified 12 231
compounds that reduced HCV infection by more than one order of magnitude without 232
significantly reducing cell biomass (Table I). Figure 1 displays the chemical structure of 233
the primary hits. Interestingly, five of the primary hits are tricyclic antidepressants 234
(Figure 1; Group 1), very similar to those we previously identified as HCV inhibitors 235
using a similar approach (12). Another family of compounds is composed by three 236
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synthetic estrogen receptor modulators (SERM): tamoxifen, clomifene and raloxifene 237
(Group 2; Figure 1) which have been shown to inhibit several aspects of HCV infection 238
(29), similarly to what we had previously described for another SERM, toremifene citrate 239
(12). Finally, we found hydroxyzine pamoate, a histamine H1 receptor antagonist that is 240
effective in the treatment of chronic dermatological disorders and anxiety; fluoxetine, a 241
highly specific serotonin uptake inhibitor used as antidepressant and benztropin mesylate, 242
a muscarinic receptor antagonist used for symptomatic treatment of Parkinson´s disease. 243
Since others and we have previously characterized compounds similar to those described 244
in Group 1 as HCV entry inhibitors (12) and since the antiviral activity of SERMs (Group 245
2) against HCV has been studied previously (12, 29), we focused our attention on the 246
compounds in Group 3. In addition, we included in this study perphenazine (from Group 247
1) as a control, since we expect it to block viral entry as others and we have shown 248
previously for compounds with nearly identical structure (12, 30). 249
We first determined their potency by evaluating the antiviral activity of serial 250
compound dilutions using the colorimetric readout described above. The same compound 251
concentrations were tested for cytotoxicity using an MTT-based colorimetric assay (31). 252
The results of this analysis are listed in Table II and show that all the selected compounds 253
display EC50 values below 1µM, while CC50 values are significantly higher, with indexes 254
(CC50/EC50) higher than 10. We note that hydroxyzine displays the highest potency and 255
the lowest toxicity with a therapeutic index of 178. 256
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We set out to confirm that these compounds are capable of inhibiting HCV spread 257
in cell culture using an alternative readout of infection. Huh-7.5.1. clone 2 cells were 258
inoculated with JFH-1 virus (genotype 2a) at low multiplicity (m.o.i. 0.01) in the 259
presence of perphenazine (5µM), hydroxyzine (5µM), fluoxetine (3µM) or benztropin 260
(7.5µM). Cell lysates were prepared at day 6 post inoculation and processed for Western-261
Blot against NS3. Figure 2 shows how JFH-1 spread was inhibited by perphenazine, 262
hydroxyzine and benztropin by more than 10-fold as compared with the virus spreading 263
in the presence of the vehicle (DMSO), reinforcing the notion that these compounds are 264
effective HCV inhibitors. Despite its apparent potency, fluoxetine only inhibits HCV 265
spread by less than one order of magnitude (Figure 2), indicating that it is poorly 266
effective at controlling viral spread as compared with the other compounds. Therefore 267
fluoxetine was excluded from further studies. 268
269
Selected compounds inhibit early aspects of HCV infection. 270
In order to determine which aspect(s) of the virus replication cycle are affected 271
by the different compounds, we performed single cycle infection experiments (m.o.i. of 272
10) in the presence of the different compounds as described in Materials and Methods. 273
Analysis of extracellular infectivity titers 48 hours postinfection confirmed inhibition of 274
the infection, as extracellular infectivity titers were reduced by one order of magnitude in 275
the cultures treated with benztropin and 2 orders of magnitude for those treated with 276
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perphenazine or hydroxyzine (Figure 3A). Intracellular HCV RNA levels display a 277
parallel reduction of more than two orders of magnitude in the cells treated with 278
perphenazine and hydroxyzine and more than 30-fold for those treated with benztropin 279
(Figure 3A), indicating that the selected inhibitors interfere with an early step of the 280
infection leading to HCV RNA accumulation. 281
Similar results were obtained with defective reporter viruses produced by trans-282
encapsidation (HCVtcp) that produce a single round of infection (19). Figure 3B shows a 283
reduction of more than 10-fold for the cells treated with perphenazine or hydroxyzine and 284
more than 5-fold for those treated with benztropin in infection efficiency determined 48 285
hours postinfection. These results reinforce the notion that all the selected compounds 286
inhibit an early step of the infection leading to viral RNA accumulation. 287
In order to determine if the selected compounds are targeting initial aspects of 288
HCV RNA replication, Huh-7 cells were electroporated with a subgenomic replicon 289
bearing a luciferase reporter gene to study primary translation and HCV RNA replication 290
independently of viral entry. Treatment of these cells with perphenazine (5µM), 291
hydroxyzine (5µM) or benztropin (7.5µM) did not have any impact on HCV RNA 292
replication as shown by similar accumulation of luciferase 24 hours post electroporation 293
(Figure 3C) in comparison with cells treated with DMSO and in contrast with the marked 294
reduction observed in cells treated with the polymerase inhibitor 2´-C-m-adenosine 295
(2mAde; 10µM) (32). These results suggest that early HCV RNA replication is not 296
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affected by the selected compounds and indicate that these compounds might target a step 297
upstream of primary translation, likely at the level of viral entry. 298
299
Analysis of the impact of the selected inhibitors on persistent infections. 300
Huh-7 cells persistently infected with JFH-1 virus were treated for 48 hours with 301
the antiviral concentrations of the compounds or with DMSO as control. Intracellular 302
HCV RNA and supernatant infectivity titers were measured to determine the impact of 303
the inhibitors on HCV RNA replication and on virus production. Figure 4 shows that, as 304
expected, 2mAde caused a profound reduction in intracellular HCV RNA and a 305
comparable reduction in extracellular infectivity titers, indicating that the experimental 306
setup is suitable to identify HCV RNA replication inhibitors. In contrast with 2mAde, 307
none of the selected inhibitors reduced significantly HCV RNA contents in persistently 308
infected cells, indicating that they do not inhibit HCV RNA replication in a persistent 309
infection. Since the analysis of the extracellular infectivity titers could be interfered by 310
the presence of the compounds in the supernatants, we purified the infectious virions 311
present in the supernatants of the infected cells using microfiltration devices. As shown in 312
Figure 4, neither hydroxyzine nor perphenazine has any impact on virus production and 313
only a marginal (2-fold) reduction was observed in cells treated with benztropin. Overall, 314
these data suggest that neither persistent HCV RNA replication nor infectious virus 315
production is severely impaired by the selected compounds. Together with the data 316
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obtained in single cycle infection and RNA electroporation experiments (Figure 3), these 317
results reinforce the notion that the selected inhibitors inhibit an early step of the 318
infection preceding HCV RNA replication. 319
320
Selected compounds inhibit entry of several HCV genotypes. 321
In order to determine if the selected compounds target initial steps of HCV 322
infection, we studied whether they inhibit infection by HCV-pseudotyped retroviral 323
particles (HCVpp). HCVpp infection is a suitable model to study entry inhibitors because 324
it recapitulates early events of the infection mediated by the HCV envelope glycoprotein 325
complexes, such as receptor recognition, particle internalization and low pH-triggered, 326
E1E2-mediated membrane fusion (26). This system enables measuring the infectivity of 327
HCVpp bearing envelopes of different HCV genotypes. Thus, we tested the ability of the 328
selected compounds to inhibit the infection of HCVpp from genotypes 1a, 1b, 2a, 2b, 3a, 329
4 and 5. Interestingly, we observed that perphenazine displays antiviral activity against 330
genotypes 1 through 4 (Figure 5). Genotype 2a JFH-1 strain, the one used in the original 331
screening, was particularly susceptible to inhibition (Figure 5). Strikingly, hydroxyzine 332
(5µM) displayed only marked antiviral activity against HCVpp from genotype 2, while 333
the rest of the genotypes appeared to be only marginally (genotypes 1, 3 and 4) or not 334
susceptible at all (genotype 5). Nevertheless, pronounced inhibition of the HCVpp 335
infection of genotypes 1, 3 and 4, but not genotype 5, was observed with 25µM 336
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hydroxyzine. Finally, benztropin inhibited infection by HCVpp from genotypes 1 through 337
4, although JFH-1 appears to be particularly susceptible to the antiviral activity of this 338
compound as compared with the other HCV envelopes. On the other hand, genotype 5 339
was not susceptible to inhibition by any of the compounds. The infectivity of retroviruses 340
pseudotyped either with vesicular stomatitis virus G protein (VSV), feline retrovirus 341
glycoprotein (RD114) or influenza envelope HA and NA glycoproteins (FLU) was not 342
significantly affected by the presence of the antiviral compounds (Figure 5), indicating 343
that they specifically inhibit HCV entry of genotypes 1-4 at the assayed concentrations. 344
In order to verify if inhibition by the selected compounds occurred in non-Huh7 345
cells, we took advantage of several human hepatocellular carcinoma cell lines, which 346
support efficient infection by HCVpp (Coto-Llerena M. et al; unpublished results). 347
Infection of BCLC5, BCLC6 and BCLC10 cell lines with HCVpp (JFH-1) was 348
performed in the presence of the selected compounds or in the presence of vehicle 349
(DMSO). Significant inhibition of HCVpp infection was observed for all three 350
compounds in all cell lines, including the reference cell line Huh-7 (Figure 6). Although a 351
small reduction in the effectiveness was observed for hydroxyzine in BCLC5 and BCLC6 352
cells, these results indicate that inhibition of HCV entry is not restricted to Huh-7 cells. 353
The foregoing data suggest that HCVpp bearing genotype 2 envelopes are 354
particularly susceptible to the antiviral activity of hydroxyzine. These results were 355
confirmed in multiple cycle infection experiments with chimeric JFH-1 viruses bearing 356
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the structural regions from genotype 1a (H77) and genotype 1b (Con1), where 357
hydroxyzine (5 µM) efficiently inhibited the spread of genotype 2a (JFH-1) and only 358
slightly inhibited infection by chimeric viruses expressing the structural regions from 359
genotype 1 (Figure 7). This differential susceptibility was not observed in the case of 360
perphenazine and benztropin. These results confirm the differential susceptibility of 361
genotype 2 to hydroxyzine and reinforce the notion that this compound targets a step of 362
the replication cycle driven by the viral envelope glycoproteins. 363
364
JFH-1 infection is blocked at a post-attachment step. 365
The results described above suggest that all the selected compounds inhibit 366
infection at the level of viral entry, a multistep process that requires attachment to the 367
target cell and internalization of the incoming viral particles. In order to determine 368
whether the compounds target adsorption or internalization of the virion into the target 369
cell, we inoculated naive Huh-7 cells with JFH-1 virus, in the presence of perphenazine 370
(5µM), hydroxyzine (5µM) or benztropin (7.5µM), using DMSO as control. Inoculation 371
was performed at low temperature (4oC), to allow virion adsorption but not 372
internalization (7, 33). After one hour, the compounds and unbound virus were washed 373
with PBS and the cells were replenished with fresh media without inhibitors (Figure 8A; 374
adsorption). Parallel cultures were inoculated similarly but in the absence of inhibitors, 375
washed and incubated in the presence of the selected inhibitors for the rest of the 376
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experiment (Figure 7A; post-adsorption). Relative infection efficiency in the different 377
conditions was determined by immunofluorescence microscopy and infection foci 378
counting (25). The results are shown in Figure 8B, where the relative infectivity levels 379
are comparable between the control and the selected compounds, with the exception of 380
perphenazine, when compounds are present during viral adsorption. These results suggest 381
that hydroxyzine and benztropin do not interfere with viral particle stability, or with 382
physical attachment of the virion to the target cells. In contrast, the relative infectivity 383
levels are significantly different when the compounds are added post-adsorption, 384
indicating that they block infection at a step downstream of particle attachment. 385
386
Discussion 387
In this study we describe the screening of a library of compounds used in the 388
clinical practice for non-HCV applications. Using an unbiased screening technology, we 389
have identified 12 non-toxic compounds with antiviral activity against HCV in cell 390
culture. The primary hits were divided into three groups (Figure 1) based on their 391
structure and known primary pharmacological utility. The first group is constituted by 392
“tricyclic antidepressants” both phenothiazines (chlorpromazine and perphenazine) and 393
dibenzazepines (clomipramine, desipramine and imipramine). Interestingly, in a previous 394
screening, we found that several compounds of this class inhibit HCV entry (12). 395
Mechanistic studies have shown that phenothiazines interfere with E1E2-induced 396
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membrane fusion by changing the properties of the endosomal membrane (30). We 397
confirmed that this class of inhibitors interferes with viral entry of different HCV 398
genotypes (Figure 5) using as a representative member of the group perphenazine, which 399
has also been recently identified as an HCV inhibitor by others (34). We have also 400
described previously that dibenzazepines inhibit HCV entry (12). The structural 401
similarities between dibenzazepines and phenothiazines; i.e. planar molecules with 402
aromatic rings and a tertiary amine suggest a similar mechanism of inhibition of HCV 403
entry. 404
The second group of compounds includes selective estrogen receptor modulators 405
(SERMs). This class of compounds has previously been characterized as HCV inhibitors 406
initially in the context of the replicon system, identifying tamoxifen as an inhibitor of 407
HCV RNA replication by interfering with proviral functions of the estrogen receptor 408
alpha (ERa) (35). Others and we subsequently showed that SERMs could also inhibit 409
viral entry and virion production (12, 29), indicating that SERMs may interfere with 410
multiple steps of the viral lifecycle. In this sense, raloxifene, a SERM which displays 411
anti-HCV activity in cell culture (36), ameliorates the cure rates when used in 412
combination with IFN- and ribavirin in postmenopausal women (37), suggesting that 413
this class of inhibitors may have some therapeutic value in combination with other 414
antivirals. In this study, we focused our attention in hydroxyzine and benztropine, which 415
are benzhydryl-containing compounds with antihistaminic properties. While it is 416
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tempting to speculate that these characteristics are responsible for their antiviral activity, 417
further studies will be necessary to determine the structural and pharmacological 418
properties required for antiviral activity. 419
Study of perphenazine, the putative entry inhibitor, hydroxyzine and benztropin 420
revealed that they effectively inhibit HCV infection in multiple cycle (Figure 2 and 7) 421
and single cycle (Figure 3A, 3B) infection experiments at concentrations in the low-422
micromolar range. Interestingly, none of these compounds interfere with HCV RNA 423
replication after RNA transfection (Figure 3C) or in persistently infected cell cultures 424
(Figure 4), and only benztropin displays a marginal inhibitory effect on virus production. 425
These results indicate that the selected compounds inhibit HCV infection by blocking an 426
early step preceding HCV RNA replication. This hypothesis was confirmed using 427
HCVpp as a surrogate model for HCV entry, as the three compounds inhibit HCVpp 428
infection for genotypes 1, 2, 3 and 4 (Figure 5). Time of addition and low temperature 429
inoculation experiments show that all three compounds were most active when added 430
post-adsorption (Figure 8), indicating that they target a step downstream of virion 431
attachment. 432
Intriguingly, a high concentration (>40-fold the EC90) of hydroxyzine was 433
required for inhibition of entry of genotypes 1, 3 and 4, indicating that genotype 2 strains 434
are particularly susceptible to inhibition by this compound (Figure 5 and 7). These results 435
may suggest that there are important intergenotypic differences in the susceptibility to 436
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similar entry inhibitors. The differential inhibition by hydroxyzine is not unique, as other 437
reported HCV entry inhibitors display important intergenotypic differences. In this sense, 438
two recent reports describe 1,3,5-triazine derivatives as HCV entry inhibitors that 439
selectively block entry of genotype 1a and 1b but not that of other tested genotypes (38, 440
39). These results may be interpreted as that the inhibitors target directly the envelope 441
glycoproteins, which display very high genetic variability among HCV genotypes (40) 442
and that genetic differences account for the differential susceptibility. Future studies, 443
including selection of resistant mutants, determination of the genetic determinants 444
leading to resistance and comparison with genotypes displaying reduced or no 445
susceptibility, like the genotype 5 isolate in our study (Figure 5), may shed light onto the 446
molecular basis for this differential susceptibility. Alternatively, the different 447
susceptibility among different isolates (Figure 5) may suggest that, while the route of 448
entry of different HCV isolates requires similar host cell factors (6), there might be 449
differences in the usage of such factors. In fact, it has recently been reported that different 450
HCV isolates may use different members of the claudin family for viral entry as an 451
alternative to claudin-1 (41), which was originally shown to play an essential role in viral 452
entry (42). In the study by Haid et al., when virus infection is neutralized with anti-453
claudin1 antibodies, some isolates appear to escape inhibition by using claudin-6 as an 454
alternative receptor (41). These results illustrate the complexity of inhibiting entry of 455
different genotypes and reinforce the notion that a large assortment of entry inhibitors 456
might be required for preventing infection by different isolates. 457
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HCV entry inhibitors are not currently used in clinics for the treatment of chronic 458
HCV infection (5). This is in part due to the fact that it is not clear that viral entry is 459
required to maintain chronic HCV infection, as HCV could spread from cell-to-cell using 460
mechanisms that are different from entry of the incoming virions (43, 44). Nevertheless, 461
it has been recently shown that HCV entry inhibitors contribute to viral suppression in 462
persistently infected cell cultures when co-administered with replication inhibitors (45), 463
underscoring the potential utility of entry inhibitors in combination treatments. While the 464
utility of entry inhibitors in chronic HCV is still under debate, the use of such inhibitors 465
in the transplant setting appears to be a promising approach to prevent liver graft 466
reinfection, which occurs in nearly 100% of the cases. The combination of sofosbuvir and 467
ribavirin has recently shown to prevent hepatitis C recurrence in around two thirds of 468
HCV-infected patients treated while awaiting liver transplantation (46). Nevertheless, 469
relapse occurred in patients who had undetectable HCV-RNA for less than 30 days 470
before transplantation. It has been proposed that infection of the graft in this situation 471
occurs because residual circulating virions infect the new graft during transplantation 472
(47). Therefore, addition of a drug inhibiting HCV entry might prevent or decrease the 473
incidence of relapse in this liver transplant setting. 474
While translation of our results to the clinic should be taken cautiously, it is 475
noteworthy that there are no reports of drug-induced liver toxicity neither by hydroxyzine 476
nor benztropine. Moreover, hydroxyzine is indicated for the treatment of pruritus in 477
patients with liver disease and the peak plasma concentration achieved in these patients 478
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falls into the range of active concentrations in cell culture against HCV (Table II) (48, 479
49). On the other hand, benztropine displays a narrow therapeutic window and peak 480
plasma concentrations that are lower than those required for antiviral activity in cell 481
culture (50). Despite these potential limitations, we believe these compounds deserve 482
further investigation as potential anti-HCV antivirals, being hydroxyzine a better 483
candidate a priori. 484
Our results show that two drugs approved for clinical applications in humans, 485
hydroxyzine pamoate or benztropin mesylate, are effective against HCV infection in cell 486
culture. We believe these and other related compounds deserve further investigation for 487
their potential applications for treatment of chronic HCV infection as well as in the 488
transplant setting. In addition of their therapeutic potential, they constitute excellent tools 489
to study basic aspects of HCV entry, including the expected intergenotypic differences in 490
terms of susceptibility to this class of inhibitors. These studies will certainly contribute to 491
designing better HCV entry inhibitors, which might be important components of future 492
therapeutic strategies in combination with inhibitors targeting other aspects of the 493
infection. 494
495
Acknowledgements 496
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We are indebted to Takaji Wakita (National Institute of Infectious Diseases, Tokyo, 497
Japan) for providing JFH-1 cDNA, Francois Loic Cosset for providing the reagents 498
necessary for the studies with HCVpp, Ralf Bartenschlager (University of Heidelberg) for 499
providing reagents to produce HCVtcp and Mansun Law (The Scripps Research Institute) 500
for providing antibodies against E2. We are thankful to Juan Ortín, Ana Montero, Sara 501
Landeras and Urtzi Garaigorta for critically reading the manuscript. L.M. is funded by a 502
JAE-Pre fellowship from Consejo Superior de Investigaciones Científicas and M.C. by 503
the Roche Organ Transplantation Research Foundation. This work was supported by the 504
grants Plan Nacional De Investigación Científica, Desarrollo e Innovación Tecnológica 505
from the Spanish Ministry of Science and Innovation (SAF2010-19270) and a Marie 506
Curie Career Integration Grant (PCIG-9-GA-2011-293664) from the European Union 7th 507
Framework Programme for Research (P.G.). X.F. and S. P. P. received a grant from the 508
Roche Organ Transplantation Research Foundation (ROTRF, CI: 442035057). J.B. is 509
supported by a grant of the Instituto de Salud Carlos III (PI11/01830). CIBERehd is 510
funded by Instituto de Salud Carlos III. 511
512
Figure Legends: 513
Figure 1: Chemical structure of the primary screening hits. 514
Figure 2: Selected compounds inhibit JFH-1 propagation. 515
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Huh-7.5.1 clone 2 cells were inoculated at a multiplicity of 0.01 in the presence of 516
perphenazine (5µM), hydroxyzine (5µM), benztropin (7.5µM) or DMSO as a control. A-517
Samples of the cells were harvested at day 6 post-inoculation and analyzed by Western-518
blot against NS3 and beta-actin as loading control. B-Average values and standard 519
deviation of the normalized NS3 signal are indicated (n=3).. 520
521
Figure 3: Selected compounds inhibit early aspects of HCV infection. A- Huh7 cells 522
were inoculated at a moi of 10 in the presence of perphenazine (5µM), hydroxyzine 523
(5µM), benztropin (7.5µM) or DMSO as control. Samples of cells and supernatants were 524
collected to determine infectivity titers as well as intracellular HCV RNA by titration and 525
RT-qPCR respectively at the indicated time points. B-Inhibition of HCVtcp infection. 526
Huh-7 cells were inoculated with HCVtcp in the presence of perphenazine (5µM), 527
hydroxyzine (5µM), benztropin (7.5µM) and samples of the cells were assayed for 528
luciferase activity 48 hours post-inoculation. Data are shown as average and standard 529
deviation of two independent experiments performed in triplicate (n=6). C-Treatment of 530
Huh-7 cells electroporated with a subgenomic HCV RNA replicon bearing a luciferase 531
gene with perphenazine (5µM), hydroxyzine (5µM), benztropin (7.5µM) or DMSO and 532
2mAde (10µM) as negative and positive controls respectively. Data are shown as average 533
and standard deviation of two independent experiments performed in duplicate (n=4). 534
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Statistical significance of the differences with the control dataset was determined using 535
Student´s t-test (* p<0.05; ** p<0.01). 536
537
Figure 4: Impact of the selected compounds on persistently infected cell cultures. 538
Persistently infected cells were treated for 48 hours with perphenazine (5µM), 539
hydroxyzine (5µM), benztropin (7.5µM) or DMSO and 2mAde (10µM) as negative and 540
positive controls respectively. Samples of cells and supernatants were collected to 541
determine infectivity titers as well as intracellular HCV RNA by titration and RT-qPCR 542
respectively. Viruses present in the supernatant were partially purified using 543
microfiltration devices to eliminate residual compound present in the supernatant. 544
Infectivity titers were determined in the purified material. Data are shown as average and 545
standard deviation of two independent experiments performed in triplicate (n=6). 546
Statistical significance of the differences with the control dataset was determined using 547
Student´s t-test (* p<0.05; ** p<0.01). 548
Figure 5: Inhibition of HCV entry by perphenazine, hydroxyzine and benztropin. 549
Huh-7 cells were infected with pseudotyped retroviruses bearing envelope glycoproteins 550
from different HCV genotypes in the presence of perphenazine (5µM), hydroxyzine 551
(5µM), benztropin (7.5µM) or DMSO as a control. In addition, control pseudotypes 552
bearing glycoproteins from VSV, influenza (FLU) and feline retrovirus (RD114) were 553
used as controls to determine the specificity of the inhibition. Data are shown as average 554
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and standard deviation of two independent experiments performed in triplicate (n=6). 555
Statistical significance of the differences with the control dataset was determined using 556
Student´s t-test (* p<0.05; ** p<0.01). 557
558
Figure 6: Inhibition of viral entry by the selected compounds is not restricted to 559
Huh-7 cells. 560
BCL5, BCL6 and BCL10 were infected with HCVpp in the presence of perphenazine 561
(5µM), hydroxyzine (5µM) or benztropin (7.5µM) or DMSO as a control. Huh-7 cells 562
were infected in parallel as control. Luciferase activity was measured in cell lysates forty-563
eight hours postinfection. Data are shown as average and standard deviation of two 564
independent experiments performed in triplicate (n=6). Statistical significance of the 565
differences with the control dataset was determined using Student´s t-test (* p<0.05; ** 566
p<0.01). 567
568
Figure 7: Hydroxyzine displays selective antiviral activity against genotype 2 569
viruses. Huh-7.5.1 clone 2 cells were inoculated at a moi of 0.01 with JFH-1 and 570
recombinant H77C3 and Con1C3 -JFH1 chimeras in the presence of perphenazine 571
(5µM), hydroxyzine (5µM) or benztropin (7.5µM) or DMSO as a control. Samples of the 572
cells were collected at day 6 post-inoculation and intracellular HCV RNA levels were 573
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determined by RT-qPCR. Data are shown as average and standard deviation of two 574
independent experiments performed in triplicate (n=6). Statistical significance of the 575
differences with the control dataset was determined using Student´s t-test (* p<0.05; ** 576
p<0.01). 577
578
Figure 8: Hydroxyzine and benztropin inhibit viral entry at a post-attachment step. 579
A-Huh7 cells were inoculated with JFH-1 virus dilutions in the presence of perphenazine 580
(5µM), hydroxyzine (5µM) or benztropin (7.5µM) or vehicle (DMSO) as a control at 4oC 581
for 1 hour in the presence or absence of the compounds. Cells were washed and 582
replenished with fresh media with or without the compounds, depending on whether the 583
cells had been exposed to the antivirals during the adsorption phase. B-Relative 584
infectivity titers were determined by immunofluorescence microscopy. Data are shown as 585
average and standard deviation of two independent experiments performed in triplicate 586
(n=6). Statistical significance of the differences with the control dataset was determined 587
using Student´s t-test (* p<0.05; ** p<0.01). 588
Table I: Primary hits in the compound library screening. 589
Relative infection efficiency values were determined by colorimetry, using a standard 590
curve with serial virus inoculum dilutions. Relative biomass was subsequently 591
determined in the same wells, also using a colorimetric readout, setting the biomass 592
measured in the control wells to 100%. Data are shown as average and SD of two 593
independent experiments (n=2). 594
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Table II: Potency and toxicity of the selected compounds. 595
Potency (EC50 and EC90) indexes are shown as average and standard deviation of a 596
minimum of four independent determinations (n=4). Citotoxicity (CC50) was calculated 597
in parallel uninfected cell cultures and is shown as average and SD of three independent 598
determinations (n=3). Selectivity indexes (S.I.) were calculated by determining the ratio 599
of the average CC50 and EC50 values. 600
601
602
603
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REFERENCES 604
1. Maasoumy B, Wedemeyer H. 2012. Natural history of acute and chronic 605 hepatitis C. Best Pract Res Clin Gastroenterol 26:401-412. 606
2. Guidotti LG, Chisari FV. 2006. Immunobiology and pathogenesis of viral 607 hepatitis. Annu Rev Pathol 1:23-61. 608
3. Smith DB, Bukh J, Kuiken C, Muerhoff AS, Rice CM, Stapleton JT, 609 Simmonds P. 2013. Expanded classification of hepatitis C Virus into 7 genotypes 610 and 67 Subtypes: updated criteria and assignment web resource. Hepatology. 611
4. Ripoli M, Pazienza V. 2011. Impact of HCV genetic differences on pathobiology 612 of disease. Expert Rev Anti Infect Ther 9:747-759. 613
5. Pawlotsky JM. 2013. Hepatitis C virus: standard-of-care treatment. Adv 614 Pharmacol 67:169-215. 615
6. Ploss A, Evans MJ. 2012. Hepatitis C virus host cell entry. Curr Opin Virol 2:14-616 19. 617
7. Tscherne DM, Jones CT, Evans MJ, Lindenbach BD, McKeating JA, Rice 618 CM. 2006. Time- and temperature-dependent activation of hepatitis C virus for 619 low-pH-triggered entry. J Virol 80:1734-1741. 620
8. Scheel TK, Rice CM. 2013. Understanding the hepatitis C virus life cycle paves 621 the way for highly effective therapies. Nat Med 19:837-849. 622
9. Bartenschlager R, Lohmann V, Penin F. 2013. The molecular and structural 623 basis of advanced antiviral therapy for hepatitis C virus infection. Nat Rev 624 Microbiol 11:482-496. 625
10. Afdhal NH, Zeuzem S, Schooley RT, Thomas DL, Ward JW, Litwin AH, 626 Razavi H, Castera L, Poynard T, Muir A, Mehta SH, Dee L, Graham C, 627 Church DR, Talal AH, Sulkowski MS, Jacobson IM. 2013. The new paradigm 628 of hepatitis C therapy: integration of oral therapies into best practices. J Viral 629 Hepat 20:745-760. 630
11. Lange CM, Jacobson IM, Rice CM, Zeuzem S. 2013. Emerging therapies for 631 the treatment of hepatitis C. EMBO Mol Med. 632
12. Gastaminza P, Whitten-Bauer C, Chisari FV. 2010. Unbiased probing of the 633 entire hepatitis C virus life cycle identifies clinical compounds that target multiple 634 aspects of the infection. Proc Natl Acad Sci U S A 107:291-296. 635
13. Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J. 1982. Growth of 636 human hepatoma cells lines with differentiated functions in chemically defined 637 medium. Cancer Res 42:3858-3863. 638
14. Pedersen IM, Cheng G, Wieland S, Volinia S, Croce CM, Chisari FV, David 639 M. 2007. Interferon modulation of cellular microRNAs as an antiviral 640 mechanism. Nature 449:919-922. 641
on June 19, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
Page 33
- 33 -
15. Graham FL, Smiley J, Russell WC, Nairn R. 1977. Characteristics of a human 642 cell line transformed by DNA from human adenovirus type 5. J Gen Virol 36:59-643 74. 644
16. Armengol C, Tarafa G, Boix L, Sole M, Queralt R, Costa D, Bachs O, Bruix 645 J, Capella G. 2004. Orthotopic implantation of human hepatocellular carcinoma 646 in mice: analysis of tumor progression and establishment of the BCLC-9 cell line. 647 Clin Cancer Res 10:2150-2157. 648
17. Zhong J, Gastaminza P, Cheng G, Kapadia S, Kato T, Burton DR, Wieland 649 SF, Uprichard SL, Wakita T, Chisari FV. 2005. Robust hepatitis C virus 650 infection in vitro. Proc Natl Acad Sci U S A 102:9294-9299. 651
18. Zhong J, Gastaminza P, Chung J, Stamataki Z, Isogawa M, Cheng G, 652 McKeating JA, Chisari FV. 2006. Persistent hepatitis C virus infection in vitro: 653 coevolution of virus and host. J Virol 80:11082-11093. 654
19. Steinmann E, Brohm C, Kallis S, Bartenschlager R, Pietschmann T. 2008. 655 Efficient trans-encapsidation of hepatitis C virus RNAs into infectious virus-like 656 particles. J Virol 82:7034-7046. 657
20. Gastaminza P, Pitram SM, Dreux M, Krasnova LB, Whitten-Bauer C, Dong 658 J, Chung J, Fokin VV, Sharpless KB, Chisari FV. 2011. Antiviral Stilbene 1,2-659 Diamines Prevent Initiation Of Hepatitis C Viral RNA Replication At The Outset 660 of Infection. J Virol. 661
21. Bernhardt G, Reile H, Birnbock H, Spruss T, Schonenberger H. 1992. 662 Standardized kinetic microassay to quantify differential chemosensitivity on the 663 basis of proliferative activity. J Cancer Res Clin Oncol 118:35-43. 664
22. Hoh A, Maier K, Dreher RM. 1987. Multilayered keratinocyte culture used for 665 in vitro toxicology. Mol Toxicol 1:537-546. 666
23. Chomczynski P, Sacchi N. 1987. Single-step method of RNA isolation by acid 667 guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-668 159. 669
24. Gastaminza P, Cheng G, Wieland S, Zhong J, Liao W, Chisari FV. 2008. 670 Cellular determinants of hepatitis C virus assembly, maturation, degradation, and 671 secretion. J Virol 82:2120-2129. 672
25. Cheng G, Montero A, Gastaminza P, Whitten-Bauer C, Wieland SF, Isogawa 673 M, Fredericksen B, Selvarajah S, Gallay PA, Ghadiri MR, Chisari FV. 2008. 674 A virocidal amphipathic {alpha}-helical peptide that inhibits hepatitis C virus 675 infection in vitro. Proc Natl Acad Sci U S A 105:3088-3093. 676
26. Bartosch B, Dubuisson J, Cosset FL. 2003. Infectious hepatitis C virus pseudo-677 particles containing functional E1-E2 envelope protein complexes. J Exp Med 678 197:633-642. 679
27. Krieger N, Lohmann V, Bartenschlager R. 2001. Enhancement of hepatitis C 680 virus RNA replication by cell culture-adaptive mutations. J Virol 75:4614-4624. 681
on June 19, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
Page 34
- 34 -
28. Friesland M, Mingorance L, Chung J, Chisari FV, Gastaminza P. 2013. 682 Sigma-1 receptor regulates early steps of viral RNA replication at the onset of 683 hepatitis C virus infection. J Virol 87:6377-6390. 684
29. Murakami Y, Fukasawa M, Kaneko Y, Suzuki T, Wakita T, Fukazawa H. 685 2013. Selective estrogen receptor modulators inhibit hepatitis C virus infection at 686 multiple steps of the virus life cycle. Microbes Infect 15:45-55. 687
30. Chamoun-Emanuelli AM, Pecheur EI, Simeon RL, Huang D, Cremer PS, 688 Chen Z. 2013. Phenothiazines inhibit hepatitis C virus entry, likely by increasing 689 the fluidity of cholesterol-rich membranes. Antimicrob Agents Chemother 690 57:2571-2581. 691
31. Levitz SM, Diamond RD. 1985. A rapid colorimetric assay of fungal viability 692 with the tetrazolium salt MTT. J Infect Dis 152:938-945. 693
32. Carroll SS, Tomassini JE, Bosserman M, Getty K, Stahlhut MW, Eldrup AB, 694 Bhat B, Hall D, Simcoe AL, LaFemina R, Rutkowski CA, Wolanski B, Yang 695 Z, Migliaccio G, De Francesco R, Kuo LC, MacCoss M, Olsen DB. 2003. 696 Inhibition of hepatitis C virus RNA replication by 2'-modified nucleoside analogs. 697 J Biol Chem 278:11979-11984. 698
33. Koutsoudakis G, Kaul A, Steinmann E, Kallis S, Lohmann V, Pietschmann 699 T, Bartenschlager R. 2006. Characterization of the early steps of hepatitis C 700 virus infection by using luciferase reporter viruses. J Virol 80:5308-5320. 701
34. Hu Z, Lan KH, He S, Swaroop M, Hu X, Southall N, Zheng W, Liang TJ. 702 2013. A Novel Cell-Based Hepatitis C Virus Infection Assay for Quantitative 703 High Throughput Screening of Anti-Hepatitis C Virus Compounds. Antimicrob 704 Agents Chemother. 705
35. Watashi K, Inoue D, Hijikata M, Goto K, Aly HH, Shimotohno K. 2007. 706 Anti-hepatitis C virus activity of tamoxifen reveals the functional association of 707 estrogen receptor with viral RNA polymerase NS5B. J Biol Chem 282:32765-708 32772. 709
36. Takeda M, Ikeda M, Mori K, Yano M, Ariumi Y, Dansako H, Wakita T, 710 Kato N. 2012. Raloxifene inhibits hepatitis C virus infection and replication. 711 FEBS Open Bio 2:279-283. 712
37. Furusyo N, Ogawa E, Sudoh M, Murata M, Ihara T, Hayashi T, Ikezaki H, 713 Hiramine S, Mukae H, Toyoda K, Taniai H, Okada K, Kainuma M, 714 Kajiwara E, Hayashi J. 2012. Raloxifene hydrochloride is an adjuvant antiviral 715 treatment of postmenopausal women with chronic hepatitis C: a randomized trial. 716 J Hepatol 57:1186-1192. 717
38. Coburn GA, Fisch DN, Moorji SM, de Muys JM, Murga JD, Paul D, 718 Provoncha KP, Rotshteyn Y, Han AQ, Qian D, Maddon PJ, Olson WC. 2012. 719 Novel small-molecule inhibitors of hepatitis C virus entry block viral spread and 720 promote viral clearance in cell culture. PLoS One 7:e35351. 721
on June 19, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
Page 35
- 35 -
39. Baldick CJ, Wichroski MJ, Pendri A, Walsh AW, Fang J, Mazzucco CE, 722 Pokornowski KA, Rose RE, Eggers BJ, Hsu M, Zhai W, Zhai G, Gerritz SW, 723 Poss MA, Meanwell NA, Cockett MI, Tenney DJ. 2010. A novel small 724 molecule inhibitor of hepatitis C virus entry. PLoS Pathog 6:e1001086. 725
40. Simmonds P. 2004. Genetic diversity and evolution of hepatitis C virus--15 years 726 on. J Gen Virol 85:3173-3188. 727
41. Haid S, Grethe C, Dill MT, Heim M, Kaderali L, Pietschmann T. 2013. 728 Isolate-dependent use of Claudins for cell entry by hepatitis C virus. Hepatology. 729
42. Evans MJ, von Hahn T, Tscherne DM, Syder AJ, Panis M, Wolk B, 730 Hatziioannou T, McKeating JA, Bieniasz PD, Rice CM. 2007. Claudin-1 is a 731 hepatitis C virus co-receptor required for a late step in entry. Nature 446:801-805. 732
43. Brimacombe CL, Grove J, Meredith LW, Hu K, Syder AJ, Flores MV, 733 Timpe JM, Krieger SE, Baumert TF, Tellinghuisen TL, Wong-Staal F, Balfe 734 P, McKeating JA. 2011. Neutralizing antibody-resistant hepatitis C virus cell-to-735 cell transmission. J Virol 85:596-605. 736
44. Ramakrishnaiah V, Thumann C, Fofana I, Habersetzer F, Pan Q, de Ruiter 737 PE, Willemsen R, Demmers JA, Stalin Raj V, Jenster G, Kwekkeboom J, 738 Tilanus HW, Haagmans BL, Baumert TF, van der Laan LJ. 2013. Exosome-739 mediated transmission of hepatitis C virus between human hepatoma Huh7.5 740 cells. Proc Natl Acad Sci U S A 110:13109-13113. 741
45. Bush CO, Greenstein AE, Delaney WEt, Beran RK. 2013. Hepatitis C viral 742 entry inhibitors prolong viral suppression by replication inhibitors in persistently-743 infected Huh7 cultures. PLoS One 8:e65273. 744
46. Curry MP, Forns X, Chung RT, Terrault N, Brown RS, J.M. F, Gordon FD, 745 O´Leary JG, Kuo A, Schiano TD, Everson GT, Schiff ER, Befeler A, 746 McHutchison JG, Symonds WT, Denning JM, McNair L, Arterburn S, 747 Moonka D, Gane EJ, Afdhal NH. 2013. Pretransplant Sofosbuvir and Ribavirin 748 to Prevent Recurrence of HCV Infection after Liver Transplantation. Hepatology 749 58:314A. 750
47. Ramirez S, Perez-Del-Pulgar S, Carrion JA, Costa J, Gonzalez P, Massaguer 751 A, Fondevila C, Garcia-Valdecasas JC, Navasa M, Forns X. 2009. Hepatitis C 752 virus compartmentalization and infection recurrence after liver transplantation. 753 Am J Transplant 9:1591-1601. 754
48. Simons FE, Simons KJ, Frith EM. 1984. The pharmacokinetics and 755 antihistaminic of the H1 receptor antagonist hydroxyzine. J Allergy Clin Immunol 756 73:69-75. 757
49. Simons FE, Watson WT, Chen XY, Minuk GY, Simons KJ. 1989. The 758 pharmacokinetics and pharmacodynamics of hydroxyzine in patients with primary 759 biliary cirrhosis. J Clin Pharmacol 29:809-815. 760
50. Carranza M, Snyder MR, Shaw JD, Zesiewicz TA. 2013. Parkinson´s Disease. 761 Seed Medical Publishers. 762
on June 19, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
Page 36
- 36 -
763
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.org/D
ownloaded from
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.org/D
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.org/D
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.org/D
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.org/D
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Group NCC_Structure_ID Screening Concentration Name HCV Infection
(% of control) Biomass (%)
Group1: Tricyclic antidepressants
CPD000058254 10 µM Chlorpromazine 0.14 ± 0.20 90.19 ± 3.54 CPD000058295 10 µM Clomipramine 0.22 ± 0.10 93.48 ± 0.76 CPD000036827 10 µM Desipramine 0.22 ± 0.17 88.56 ± 5.99 CPD000058180 10 µM Perphenazine 0.60 ± 0.52 88.22 ± 10.84 CPD000058388 10 µM Imipramine 2.25 ± 1.87 106.96 ± 4.58
Group 2: SERMs CPD000058508 10 µM Raloxifene 0.09 ± 0.12 91.82 ± 20.05 CPD001491671 10 µM Tamoxifen 1.09 ± 1.53 96.05 ± 4.91 CPD001317855 2 µM Clomifene 5.04 ± 0.87 149.75 ± 2.62
Group 3: Others CPD001370751 10 µM Hydroxyzine 4.59 ± 6.48 87.51 ± 6.8 CPD000394012 10 µM Benztropin 0.51 ± 0.50 93.87 ± 3.11 CPD000058452 2 µM Fluoxetine 15.41 ±2.47 159.34 ± 2.00
Table I: Primary hits in the compound library screening.
Relative infection efficiency values were determined by colorimetry, using a standard curve with serial virus inoculum dilutions. Relative biomass was subsequently determined in the same wells, also using a colorimetric readout, setting the biomass measured in the control wells to 100%. Data are shown as average and SD of two independent experiments (n=2).
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Compound Name EC50 (µM) EC90 (µM) CC50 (µM) S.I. Hydroxyzine 0.26 ± 0.13 0.55 ± 0.25 46.25 ± 6.25 178 Benztropine 0.67 ± 0.29 2.75 ± 1.08 26.25 ± 1.76 39 Fluoxetine 0.45 ± 0.27 1.50 ± 1.05 10.25 ± 1.76 23
Perphenazine 1.00 ± 0.45 4.50 ± 1.44 10.50 ± 0.70 10
Table II: Potency and toxicity of the selected compounds.
Potency (EC50 and EC90) indexes are shown as average and standard deviation of a minimum of four independent determinations (n=4). Citotoxicity (CC50) was calculated in parallel uninfected cell cultures and is shown as average and SD of three independent determinations (n=3). Selectivity indexes (S.I.) were calculated by determining the ratio of the average CC50 and EC50 values.
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