Accepted Manuscript Novel antiviral activity and mechanism of bromocriptine as a Zika virus NS2B-NS3 protease inhibitor Jasper Fuk-Woo Chan, Kenn Ka-Heng Chik, Shuofeng Yuan, Cyril Chik-Yan Yip, Zheng Zhu, Kah-Meng Tee, Jessica Oi-Ling Tsang, Chris Chung-Sing Chan, Vincent Kwok-Man Poon, Gang Lu, Anna Jinxia Zhang, Kin-Kui Lai, Kwok-Hung Chan, Richard Yi-Tsun Kao, Kwok-Yung Yuen PII:
S0166-3542(16)30725-2
DOI:
10.1016/j.antiviral.2017.02.002
Reference:
AVR 4004
To appear in:
Antiviral Research
Received Date: 28 November 2016 Accepted Date: 5 February 2017
Please cite this article as: Chan, J.F.-W., Chik, K.K.-H., Yuan, S., Yip, C.C.-Y., Zhu, Z., Tee, K.-M., Tsang, J.O.-L., Chan, C.C.-S., Poon, V.K.-M., Lu, G., Zhang, A.J., Lai, K.-K., Chan, K.-H., Kao, R.Y.-T., Yuen, K.-Y., Novel antiviral activity and mechanism of bromocriptine as a Zika virus NS2B-NS3 protease inhibitor, Antiviral Research (2017), doi: 10.1016/j.antiviral.2017.02.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Research Article
Title: Novel antiviral activity and mechanism of bromocriptine as a Zika virus NS2B-NS3
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protease inhibitor
Authors: Jasper Fuk-Woo Chana,b,c,d,#,*, Kenn Ka-Heng Chikb,#, Shuofeng Yuanb, Cyril Chik-Yan
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Yipb, Zheng Zhub, Kah-Meng Teeb, Jessica Oi-Ling Tsangb, Chris Chung-Sing Chanb, Vincent Kwok-Man Poonb, Gang Lue, Anna Jinxia Zhangb, Kin-Kui Laib, Kwok-Hung Chana,b,c,d, Richard
#
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Yi-Tsun Kaoa,b,c,d, Kwok-Yung Yuena,b,c,d,f,*
J.F.W. Chan and K.K.H. Chik contributed equally to the manuscript.
Affiliations:
State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong
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a
Kong Special Administrative Region, China b
Department of Microbiology, Queen Mary Hospital, The University of Hong Kong, Hong Kong
c
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Special Administrative Region, China
Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong
d
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Special Administrative Region, China Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong Special
Administrative Region, China e
Department of Pathogen Biology, Hainan Medical University, Haikou, Hainan, China
f
The Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The
University of Hong Kong
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*Corresponding authors at: State Key Laboratory of Emerging Infectious Diseases, Carol Yu Centre for Infection, Department of Microbiology, The University of Hong Kong, Queen Mary
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Hospital, 102 Pokfulam Road, Pokfulam, Hong Kong Special Administrative Region, China. Tel.: +852-22554892; fax: +852-28551241. Email address:
[email protected] (J.F.W.C.) or
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[email protected] (K.Y.Y.)
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Word count: abstract = 209, text = 3618
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Keywords: Zika; antiviral; treatment; protease; bromocriptine; interferon.
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ABSTRACT Zika virus (ZIKV) infection is associated with congenital malformations in infected fetuses and severe neurological and other systemic complications in adults. There are currently limited anti-
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ZIKV treatment options that are readily available and safe for use in pregnancy. In this drug repurposing study, bromocriptine was found to have inhibitory effects on ZIKV replication in cytopathic effect inhibition, virus yield reduction, and plaque reduction assays. Time-of-drug-
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addition assay showed that bromocriptine exerted anti-ZIKV activity between 0-12 hours postZIKV inoculation, corroborating with post-entry events in the virus replication cycle prior to
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budding. Our docking model showed that bromocriptine interacted with several active site residues of the proteolytic cavity involving H51 and S135 in the ZIKV-NS2B-NS3 protease protein, and might occupy the active site and inhibit the protease activity of the ZIKV-NS2BNS3 protein. A fluorescence resonance energy transfer-based enzymatic assay confirmed that
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bromocriptine inhibited ZIKV protease activity. Moreover, bromocriptine exhibited synergistic effect with interferon-α2b against ZIKV replication in cytopathic effect inhibition assay. The availability of per vagina administration of bromocriptine as suppositories or vaginoadhesive
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discs and the synergistic anti-ZIKV activity between bromocriptine and type I interferon may make bromocriptine a potentially useful and readily available treatment option for ZIKV
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infection. The anti-ZIKV effects of bromocriptine should be evaluated in a suitable animal model.
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1. Introduction Zika virus (ZIKV) is a human-pathogenic flavivirus that has been previously neglected because of its limited geographical distribution and apparently mild clinical disease (Chan et
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al.,2016a;Zhu et al., 2016). However, ZIKV has recently emerged as a public health threat because of its rapid spread in multiple regions, sexual and vertical human-to-human transmissions, and its newly recognized association with congenital malformations in infected
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fetuses (Duffy et al.,2009;de Paula Freitas et al.,2016;Leal et al.,2016;Mlakar et al.,2016;Musso and Gubler,2016). Moreover, an increasing number of serious complications have recently been
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reported in ZIKV-infected adults. These include severe neurological complications, such as Guillain-Barré syndrome, menigoencephalitis, and myelitis, thrombocytopenia and disseminated intravascular coagulation with hemorrhagic complications, hepatic dysfunction, acute respiratory distress syndrome, shock, multi-organ dysfunction syndrome, and death (Arzuza-Ortega et
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al.,2016;Azevedo et al.,2016;Cao-Lormeau et al,2016;Carteaux et al.,2016;Chraibi et al,2016;Mecharles et al,2016;Sarmiento-Ospina et al.,2016;Soares et al.,2016). Moreover, ZIKV is implicated in causing orchitis and possible long term male subfertility (Chan et al.,2016c).
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Effective antivirals are therefore urgently needed for treating ZIKV-infected pregnant patients and those with severe complications.
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As in the case of other emerging viral infections, the de-novo development of novel anti-
ZIKV agents, such as monoclonal antibodies and antiviral peptides, inevitably lags behind the rapidly expanding epidemic (Dai et al.,2016;Sapparapu et al.,2016). Repurposed drug programmes have therefore been used to identify existing clinically approved drugs that may be immediately used to treat ZIKV infection (Barrows et al.,2016;Xu et al.,2016). Based on a literature review, we selected 11 drugs for the initial screen in this study. Similar to a previous
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report, these drugs were chosen as they met several of the following selection criteria: (i) a known safety profile in humans and pregnancy; (ii) recognized broad-spectrum antiviral activities; (iii) recognized antiviral activities against other flaviviruses or emerging RNA viruses;
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and (iv) coverage of a broad range of indications and drug classes (Gehring et al.;2014). Among them, we identified bromocriptine and recombinant types I and II interferons to be inhibitors of
effects of combination bromocriptine/interferon treatment.
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2. Materials and methods
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ZIKV in-vitro. We further evaluated the mechanism of bromocriptine’s anti-ZIKV effects and the
2.1. Virus and cell lines
A clinical isolate of ZIKV (Puerto Rico strain PRVABC59) obtained from a patient in the recent South American epidemic was kindly provided by Brandy Russell and Barbara Johnson,
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Centers for Disease Control and Prevention, USA. The virus was amplified by three additional passages in Vero cells (ATCC) in minimum essential medium (MEM) supplemented with 1% fetal bovine serum (FBS, GibcoTM, Life Technologies Corporation, Massachusetts, USA) and
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100units/ml penicillin plus 100µg/ml streptomycin to make working stocks of the virus at 4×106 tissue culture infectious dose (TCID50)/ml. For virus titration, aliquots of ZIKV were applied on
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confluent Vero cells in 96-well plates for TCID50 assay as we previously described with slight modifications (Zhou et al.,2014). Briefly, serial 10-fold dilutions of ZIKV were inoculated in a Vero cell monolayer in quadruplicate and cultured in penicillin/streptomycin-supplemented MEM and 1% FBS. The plates were observed for cytopathic effect (CPE) for 6 days. Viral titer was calculated with the Reed and Münch endpoint method. One TCID50 was interpreted as the amount of virus that causes CPE in 50% of inoculated wells.
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2.2. Drug compounds and cytotoxicity assay Based on a literature review, a total of 11 commercially available drugs that fulfilled the criteria
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described above were selected for evaluation. These included amiodarone (Hikma Pharmaceuticals, London, UK), artesunate (Guilin Pharmaceutical Co., Ltd., Shanghai, China), bromocriptine mesylate (Sigma-Aldrich, Inc., Missouri, USA), flufenamic acid (Sigma-Aldrich),
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lopinavir-ritonavir (AbbVie Inc., Illinois, USA), minocycline (Pfizer, New York City, USA), Intron A (interferon-α2b, Schering-Plough, New Jersey, USA), Avonex (interferon-β1a, Biogen,
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Massachusetts, USA), Rebif (interferon-β1a, Merck Serono, Darmstadt, Germany), Betaferon (interferon-β1b, Bayer Schering Pharma AG, Berlin, Germany), and Imukin (interferon-γ1b, Boehringer Ingelheim, Germany) (Inglot,1969;Dutta and Basu,2011;Chan et al.,2013;Cheng et al.,2013;Gehring et al.,2014;Ho et al.,2014;Chan et al.,2015;Kato et al.,2016;Zumla et al.,2016).
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The 50% effective cytotoxic concentration (CC50) of the selected drugs were determined by thiazolyl blue tetrazolium bromide (MTT) assay according to manufacturer's instructions and as
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we previously described (Chan et al.,2013).
2.3. CPE inhibition assay
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The ZIKV CPE inhibition assay was performed as previously described with slight
modifications (Chan et al.,2013). Briefly, the drug compounds were diluted with serum-free MEM and added to confluent Vero cells in 96-well culture plates (4×104cells/well) in triplicate for 2h at 37oC. After incubation, the drug-containing media were removed, and ZIKV (multiplicity of infection, MOI=0.0001) was added together with fresh drug-compound media to each well. Following 1h adsorption at 37oC, the virus-compound mixture was removed and the
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cells were washed twice with MEM to remove unbound virus. Subsequently, media with antiviral compounds were added to the cells for further incubation for 6 days at 37oC in 5% CO2. CPE was examined by inverted light microscopy, and 50µl of supernatant was collected for virus
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quantification, as we previously described with modifications (Chan et al.,2016b). Thereafter, 40µl of serum free MEM and 10µl of 5mg/ml MTT solution (prepared in 1×PBS, filtered) were added to the wells. The monolayers were incubated as above for 4h (away from light). Finally,
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100µl of 10% sodium dodecyl sulfate with 0.01M HCl was added and further incubated at 37°C in 5% CO2 overnight. The activity was read at OD570 with reference wavelength at OD640. The
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half maximal inhibitory concentration (IC50) and CC50 were calculated using Sigma plot (SPSS) in an Excel add-in ED50V10. Drug compounds (non-interferons) with anti-ZIKV activity were selected for combination studies with interferon-α2b using the CPE inhibition assay. Loewe
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additivity index was calculated as previously described (Tallarida RJ, 2011;Delaney et al.,2004).
2.4. Virus yield reduction assay
Virus yield reduction assay was performed by quantitative reverse transcription-PCR
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(qRT-PCR) using total nucleic acid extracted from culture supernatants of the Vero cells infected by ZIKV with different drugs or dimethyl sulfoxide (DMSO) control at 6 day post-inoculation
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(dpi) as previously described (Chan et al.,2016b). Internal control β-actin gene was measured as previously described (Chan et al.,2016c).
2.5. Plaque reduction assay For the drug compounds with antiviral activity in the CPE inhibition and/or virus yield reduction assays, further evaluation by plaque reduction assay (PRA) was performed as we
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previously described with modifications (Chan et al.,2013). Briefly, Vero cells were seeded at 2×105cells/well in 24-well tissue culture plates in MEM (Invitrogen, California, USA) with 10% FBS on the day before carrying out the assay. After 24h of incubation, 20-40 plaque-forming
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units of ZIKV were added to the cell monolayer with or without the addition of drug compounds and the plates further incubated for 1h at 37°C in 5% CO2 before removal of unbound viral particles by aspiration of the media and washing once with MEM. Monolayers were then
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overlaid with media containing 1% low melting agarose (Cambrex Corporation, New Jersey, USA) in MEM and appropriate concentrations of drug compounds and incubated as above for
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96h. Next, the wells were fixed with 10% formaldehyde (BDH, Merck, Darmstadt, Germany) overnight. After removal of the agarose plugs, the monolayers were stained with 0.7% crystal violet (BDH, Merck) and the plaques counted. The percentage of plaque inhibition relative to the control (without compound addition) plates were determined for each drug compound
confirmation.
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concentration. The PRA experiments were performed in triplicate and repeated twice for
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2.6. Time-of-drug-addition assay
Bromocriptine time-of-drug-addition assay was performed as previously described with some
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modifications (Kato et al.,2016). Briefly, Vero cells were seeded in 24-well plates (2×105cells/well). The cells were infected with ZIKV (MOI=2) and then incubated for 1h. The viral inoculum was then removed and the cells were washed twice with PBS. At 0,3,6,9,12, and 14 hours post-inoculation (hpi), 20µM of bromocriptine was added to the infected cells, followed by incubation at 37°C in 5% CO2 until 18hpi. For the time point of “-1 to 0hpi”, 20µM of bromocriptine was added at 1h before ZIKV inoculation and removed at 0hpi, which was
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followed by ZIKV inoculation and incubation of the cells until 18hpi. For the time point “01hpi”, 20µM of bromocriptine was added together with ZIKV inoculation at 0hpi, followed by drug removal at 1hpi and incubation of the cells incubated until 18hpi. At 18hpi, the cell culture
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supernatant of each time point experiment was collected for viral yield measurement using qRTPCR. DMSO (0µM bromocriptine) was included as negative control. The experiments were
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performed in triplicate and repeated twice for confirmation.
2.7. Protein expression and purification
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The recombinant ZIKV-NS2B-NS3 protease protein was produced as previously described with some modifications (Lei et al.,2016). Briefly, coding regions of the NS2B (residues 49-95) and NS3 (residues 1-170) genes were fused and cloned into the pET-28b(+) expression vector (Novagen, Merck). The plasmid was transformed into Escherichia coli strain BL21 (DE3) and
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overexpressed in 2×YT medium. When the OD600 of the culture reached 0.8, overexpression of ZIKV-NS2B-NS3 protease gene was induced for overnight by addition of 0.5mM IPTG at 20°C. ZIKV-NS2B-NS3 protease protein was purified by His-tag affinity chromatography from soluble
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lysate and dialyzed overnight in 10mM Tris-HCl, 20% glycerol, 1mM CHAPS, pH 8.5. The protease protein was further concentrated through Vivaspin 20 centrifugal concentrator (GE
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Healthcare, Little Chalfont, UK) and stored at -80°C. The purified protein was detected by Western blot using anti-His-tag antibodies. Concentration of each protein was determined by Bradford protein assay kit (Bio-Rad Laboratories, California, USA) using bovine serum albumin as standard.
2.8. Molecular modeling
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For docking experiments, the dimeric ZIKV-NS2B-NS3 crystal structure (Protein Data Bank [PDB] entry: 5lc0) was used (Lei et al.,2016). To prepare the bromocriptine and ZIKV-NS2BNS3 protein structures for docking, the three-dimensional structure of bromocriptine was
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obtained from PubChem Compound database (NCBI) and ligands on the ZIKV-NS2B-NS3 protein were removed and polar hydrogen atoms and Gasteiger charges were added using Autodock tools. Docking experiments with the compounds were performed on the whole dimeric
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protein with a grid size of 65×52×46 points and 1.0Å resolution using Autodock vina software (Trott and Olson,2010). After docking, the conformation with the lowest energy level was
UCSF Chimera (Pettersen et al.,2004).
2.9. Protease inhibition assay
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selected. The 3D models and protein-ligand interactions were generated and calculated with
et
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To detect the NS2B-NS3 protease activity, a fluorescence-based enzymatic assay (Lei
al.,2016) was conducted in 96-well black micro-plates (Greiner Bio-One, Kremsmünster, Germany) utilizing benzoyl-norleucinelysine-lysine-arginine 7-amino-4-methylcoumarine
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(Bz-Nle-Lys-Lys-Arg-AMC) as a substrate. The fluorescence signal from the released AMC was measured at 460nm with excitation at 360nm, using a VICTOR™ X3 multilabel plate reader
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(Perkin Elmer, Massachusetts, USA). To detect if the drug inhibited protease activity, 5nM ZIKV-NS2B-NS3 protease was incubated for 10min with different concentrations of bromocriptine or aprotinin (as positive control) at 37°C (Phoo et al.,2016). This mixture of protease and bromocriptine or aprotonin was prepared in 50µl reaction buffer and dispersed into each well, followed by the addition of substrate in 50µl buffer to initiate the cleavage. After 10min, the fluorescence intensity was measured. The reaction and dilution buffer were consisted
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of 10mM Tris-HCl, 20% glycerol, 1mM CHAPS, 5% DMSO, pH 8.5. DMSO (0µM
2.10. Statistical analysis
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bromocriptine) was included as negative control.
Statistical comparison between different groups was performed by the Student’s t-test using
3. Results
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3.1. Identification of drugs with anti-ZIKV activity
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GraphPad Prism 6. P3.07
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(bromocriptine) to >32327.59 (Avonex) (Table 1). In the CPE inhibition assay, ZIKV-induced CPE was completely absent at 6dpi in Vero cells treated with bromocriptine or recombinant interferon in their tested concentration ranges (Table S1). In the virus yield reduction assay, dose-
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dependent reduction in virus titer was observed in cell culture supernatants inoculated with any one of the 6 drugs. The reduction in mean virus titer was >4 log10copies/ml for bromocriptine
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and type I interferons (Intron A, Avonex, Rebif, and Betaferon), and >2 log10copies/ml for type II interferon (Imukin) (Fig. 1). The largest virus titer reduction was a nearly 5 log10copies/ml reduction at ≥195.31IU/ml of Betaferon. In the PRA, bromocriptine and all of the interferons achieved near-100% or 100% plaque reduction (Fig. 2&S1).
3.2. Bromocriptine and interferon-α2b exhibited synergistic activity against ZIKV in-vitro
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As the peak serum concentration (Cmax):IC50 ratio achieved with routine clinical dosage of oral bromocriptine was relatively low, we further assessed if there were additive or synergistic effects between bromocriptine and recombinant interferons against ZIKV replication in-vitro (Fløgstad
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et al.,1994). We selected Intron A for the combination studies because the drug has been commonly used to treat hepatitis C virus (another Flaviviridae member) infection and we have recently shown its in-vivo effects against ZIKV in a mouse model (Chan et al.,2016c;Reichard et
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al.,1993). Combination studies showed that the mean Loewe additivity index was 0.78±0.19 (Table 2). The IC50 of bromocriptine was lowered by 15.26-58.51% with 0.31-1.25IU/ml of
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Intron A (Loewe additivity index, 0.88-0.53), and that the IC50 of Intron A was lowered by 20.3491.39% with 1.25-5.00µM of bromocriptine (Loewe additivity index, 1.01-0.48). These findings suggested that the two drugs inhibited the in-vitro replication of ZIKV synergistically.
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3.3 Bromocriptine inhibited post-entry events of ZIKV replication cycle As shown in Fig. S2, the cell lysate virus titer sharply increased at ~10-16hpi, suggesting that viral RNA synthesis was completed during this stage in a single ZIKV replication cycle.
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Concurrently, assembly and budding of the virions into culture supernatant started to occur at around 12-14hpi as indicated by the commencement of increasing culture supernatant virus titer
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at this stage. The duration of a single ZIKV life cycle of around 12-14h is compatible with that of other flaviviruses, in which the onset of intracellular viral RNA production was determined to occur at around 10-12hpi, and progeny virions were assembled and released after 12hpi (Chambers et al.,1990;Qing et al.,2009;Wang et al.,2011;Zmurko et al.,2016). We then performed a time-of-drug-addition assay by exposing ZIKV-infected cells to bromocriptine at different times during the virus replication cycle and measuring virus titers at 18hpi to determine the
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phase of ZIKV replication cycle interrupted by bromocriptine. When bromocriptine was added during the virus replication stage (0,3,6,9, or 12hpi), virus titers were significantly reduced (P
