aac accepts, published online ahead of print on 7 april...

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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.

on June 19, 2018 by guesthttp://aac.asm

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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



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



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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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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