Antiviral Research 126 (2016) 43e54

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Antiviral Research

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Resistance analysis and characterization of NITD008 as an analog inhibitor against virus

* Jie Qing a, , Rui Luo a, Yaxin Wang b, Junxiu Nong a, Ming Wu a, Yan Shao a, Ruoyi Tang a, ** *** Xi Yu a, Zheng Yin b, , Yuna Sun c, a Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China b Center of Basic Molecular Science, Department of Chemistry, Tsinghua University, Beijing 100084, China c National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Science, Beijing 100101, China article info abstract

Article history: Hepatitis disease caused by (HCV) is a severe threat to global public health, affecting Received 19 June 2015 approximately 3% of the world's population. (PSI-7977), a uridine nucleotide analog inhibitor Received in revised form targeting the HCV NS5B polymerase, was approved by FDA at the end of 2013 and represents a key step 19 December 2015 towards a new era in the management of HCV infection. Previous study identified NITD008, an adenosine Accepted 22 December 2015 nucleoside analog, as the specific inhibitor against dengue virus and showed good antiviral effect on Available online 24 December 2015 other flaviviruses or enteroviruses. In this report, we systematically analyzed the anti-HCV profile of NITD008, which was discovered to effectively suppress the replication of different strains of HCV in Keywords: HCV human hepatoma cells with a low nanomolar activity. On genotype 2a virus, or 2a, 1a, and 1b replicon > NITD008 cells, EC50 values were 8.7 nM, 93.3 nM, 60.0 nM and 67.2 nM, and selective index values were 2298.9, Nucleoside analog >214.4, >333.3, >298.5 respectively. We demonstrated that resistance to NITD008 was conferred by Polymerase mutation in NS5B (S282T) in the HCV infectious virus genotype 2a (JFH-1). Then, we compared the resistant profiles of NITD008 and PSI-7977, and found that the folds change of EC50 of NITD008 to full replicon cells containing mutation S282T was much bigger than PSI-7977(folds 76.50 vs. 4.52). Analysis of NITD008 cross-resistance against previously reported NS5B drug-selected mutations showed that the resistance pattern of NITD008 was not completely similar to PSI-7977, and meanwhile, S282T resistant mutation to NITD008 emerge more easily in cell culture than PSI-7977. Interestingly, NITD008 displayed significant synergistic effects with the NS5B polymerase inhibitor PSI-7977, however, only additive ef- fects with alpha (IFNa-2b), , and an NS3 protease inhibitor. These results verify that NITD008 is an effective analog inhibitor against hepatitis C virus and a good research tool as a supple- ment to other types of nucleoside analogs. © 2015 Elsevier B.V. All rights reserved.

1. Introduction year due to HCV-related complications (Lavanchy, 2009; Pol et al., 2012). Chronic infection with hepatitis C virus (HCV) has been reported Chronic HCV infection in many patients is curable; however, the to affect 170 million people worldwide, and hepatitis C not only existing standard of care (SOC) that is based on parenteral admin- seriously affects the function of the patient's liver but also triggers istration of pegylated interferon-a (PEG-IFN-a), together with the liver fibrosis and cirrhosis that may eventually lead to hepatocel- oral broad-spectrum antiviral ribavirin (RBV), has a number of lular carcinoma (Lavanchy, 2009). The World Health Organization deficiencies (Devogelaere et al., 2012). The treatment requires a estimates that there are 350,000 people in the world who die each period of up to 48 weeks, depending on the HCV genotype, and the efficacy shows distinct variations across HCV genotypes. Genotype 1, which predominates in Europe, Japan, and the United States, is * Corresponding author. particularly difficult to treat, and the rates of sustained virologic ** Corresponding author. response (SVR) post treatment seldom exceed 50% for genotype 1 *** Corresponding author. with IFN-a/RBV (Strader et al., 2004). Two direct-acting antiviral E-mail addresses: [email protected] (J. Qing), [email protected]. cn (Z. Yin), [email protected] (Y. Sun). (DAA) inhibitors of the HCV NS3 protease, and http://dx.doi.org/10.1016/j.antiviral.2015.12.010 0166-3542/© 2015 Elsevier B.V. All rights reserved. 44 J. Qing et al. / Antiviral Research 126 (2016) 43e54

Fig. 1. The chemical structures of the HCV NS5B polymerase inhibitors, NITD008, PSI-7977 and the NS3/4A protease inhibitor . telaprevir, were approved in 2011 for use in combination with IFN- et al., 2012; Jacobson et al., 2011). a/RBV for genotype 1 treatment. The using of either of these in- The U.S. Food and Drug Administration approved two further hibitors with IFN-a/RBV elevated the SVR rates to approximately DAA inhibitors in late 2013. Sofosbuvir (PSI-7977), a uridine analog 70% in treatment-naive patients (Bacon et al., 2011; Devogelaere inhibitor of the HCV NS5B RNA-dependent RNA polymerase (RdRp),

Fig. 2. Antiviral and cytotoxic activities of NITD008 and telaprevir against HCV genotypes. The inhibitory effect of NITD008 and telaprevir to the proliferation of the JFH-1 virus (A), JFH-1 full replicon (B), H77 replicon (C), and Con1 replicon (D) by dose-dependence was evaluated at 48 h posttreatment by detecting the luciferase expression of the reporter gene, and (E) The cytotoxicity was evaluated by the incubation of Huh7.5.1 cells with the indicated concentration of NITD008. At 48 h posttreatment, the viability of the treated cells was measured by a WST-1 assay and presented as a percentage of the untreated cells. The results are reported as the average ± SD from three independent experiments that were performed in triplicate. J. Qing et al. / Antiviral Research 126 (2016) 43e54 45

Fig. 3. Selection and characterization of NITD008-resistant HCV virus. (A) The scheme used for the selection of a NITD008-resistant JFH-1 recombinant virus, which is con- structed by inserting an EGFP reporter gene into the C-terminus of NS5A of the JFH-1 genome. The Huh7.5.1 cells were infected by a JFH-1 recombinant virus in the presence of gradually increasing concentrations of NITD008 and passaged every 2 or 3 days, and the arrow symbol indicated that the supernatant treated by NITD008 would infect the native Huh7.5.1 cells. The concentrations of NITD008 that correspond to different passages during the selection are indicated. (B) A characterization of the resistance phenotypes of the JFH-1 resistant virus. The indicated concentrations of NITD008 were added to the Huh7.5.1 cells infected by HCV at an MOI of 0.5. (C) The HCV proliferation in the presence or absence of NITD008 was quantified at 48 h p.i. by the EGFP expression. (D) The frequency of amino acid changes in the HCV genome that emerged during the selection with NITD008 in the Huh7.5.1 cells infected by the JFH-1 recombinant virus. is used in combination with IFN-a/RBV for the treatment of geno- However, both IFN-a/RBV and DAA have severe and treatment type 1 and 4, with RBV alone for genotype 2 and 3(Dieterich et al., limiting side effects, for example IFN-a/RBV results in a range of 2014; Jonckers et al., 2014; Lam et al., 2012; Lawitz and Gane, 2013). side effects, including anemia, flu-like symptoms and depression in , another NS3 protease inhibitor, is used with IFN-a/RBV a significant minority of patients; meanwhile, telaprevir and for genotype 1 treatment (Dieterich et al., 2014; Zeuzem et al., boceprevir are associated with a number of toxicities, such as rash, 2014). Both of these newer agents, in combination with IFN-a/ pruritus and the exacerbation of treatment-related anemia (Manns RBV, have further improved the genotype 1 treatment responses, et al., 2007). Although sofosbuvir as a uridine nucleotide analog has with treatment-naive SVR rates of approximately 80% for sime- become commercially available for the treatment of chronic HCV previr and approximately 90% for sofosbuvir. infection, we are highly interested to know whether other types of 46 J. Qing et al. / Antiviral Research 126 (2016) 43e54 nucleotide analogs could exert a good inhibitory effect on HCV and inhibitors. to study the drug resistance and other differences between PSI- 7977 and other potential nucleotide analogs. 2. Materials and methods NITD008 is an adenosine nucleoside analog that contains a carbon substitution for N-7 of the purine and an acetylene at the 2' 2.1. Compounds and reagents position of ribose (Yin et al., 2009). NITD008 has been reported to inhibit viruses within the family Flaviviridae, such as dengue virus NITD008 and PSI-7977 were synthesized according to a pub- (DENV), (WNV), virus (YFV), or lished procedure (Jonckers et al., 2014; Yin et al., 2009). Telaprevir Enterovirus 71 (EV71) (Deng et al., 2014; Yin et al., 2009). Notably, was generously provided by GinkgoPharma, China. Recombinant the selection of resistant virus was attempted by continuous human Interferon alpha-2b (IFNa-2b, An Dafen) was purchased culturing of DENV or WNV on BHK-21 or Vero cells, and no resistant from Anhui Anke Biotechnology Group, China. Ribavirin was pur- virus was discovered (Yin et al., 2009). In this study, we charac- chased from Sigma. The inhibitors were initially dissolved in DMSO, terized the anti-HCV activity of NITD008 across broad HCV geno- and stock solutions were stored at 20 C. Immediately before types and systematically studied the resistance to NITD008 and the addition, these compounds were diluted to the desired concen- synergistic effects of NITD008, PSI-7977 and other anti-HCV trations using Dulbecco's modified Eagle's medium (DMEM, Gibco)

Fig. 4. Phenotypic and NITD008 resistance analysis of the NS5BS282T or L384M mutation in the JFH-1 full replicon containing a luciferase reporter gene. Replicons con- taining S282T or L384M were generated by site-directed mutagenesis and tested for susceptibility to NITD008 using a transient assay. (A) A sequencing chromatogram of the mutated JFH-1 NS5B region. (B) The replication fitness of the S282T or L384M replicons from JFH-1 to the corresponding wild type. The effect of S282T or L384M on the NITD008 (C), telaprevir (D) and PSI-7977 (E) activity. (F) The EC50-fold change was determined by normalizing the EC50s of the mutated replicons to the corresponding wild type. The results are reported as the average ± SD from three independent experiments that were performed in triplicate. J. Qing et al. / Antiviral Research 126 (2016) 43e54 47

Fig. 5. Phenotypic and NITD008 resistance analysis of the NS5B M289L mutation in the JFH-1 full replicon containing a luciferase reporter gene. (A) A sequencing chro- matogram of the mutated JFH-1 M289L site. (B) The replication fitness of the M289L or S282T/M289L replicons from JFH-1 to the corresponding wild type. The effect of M289L on the NITD008 (C), telaprevir (D) activity was measured. (E) The EC50-fold change was determined by normalizing the EC50s of the mutated replicons to the corresponding wild type. The results are reported as the average ± SD from at least two independent experiments that were performed in triplicate.

with 10% fetal bovine serum (FBS, Gibco). Huh7.5.1 cells containing virus transcripts were seeded in a 10 cm The MEGA script T7 High Yield Transcription kit was purchased dish. After the cells were cultured for 10 days and passaged every 3 from Ambion. The kit of the Renilla-Glo™ Luciferase Assay System days, the supernatant was collected and filtered to obtain the stock was purchased from Promega. The cell viability and proliferation solution of the virus hRluc-JFH-1. The JFH-1 virus with an EGFP assay (WST-1) was purchased from Roche. reporter gene was a gift from Xinwen Chen (Wuhan Institute of Virology, Chinese Academy of Science), which is an infectious HCV 2.2. HCV virus and replicon cell lines monocistronic reporter virus constructed by inserting an EGFP re- porter gene into the C-terminus between amino acid 399 and 400 The HCV virus assay (JFH-1-Luc) was constructed by using a of NS5A of the JFH-1 genome (Han et al., 2009). fi All Huh7.5.1-based replicon cell lines were grown in DMEM method developed as previously described with some modi ca- tions (Cui et al., 2013; Han et al., 2009). Briefly, the pRluc-JFH-1 supplemented with 10% FBS at 37 C under 5% CO2. Stable replicon plasmid was constructed as follows. Based on the pJFH-1 plasmid, cell lines were selected and maintained in medium containing as a gift from Apath, L.L.C, a humanized Renilla luciferase reporter 0.5 mg/ml G418 (Geneticin; Invitrogen). The creation of Huh7.5.1 gene was introduced into the C-terminus between amino acid 399 stable genotype 1a (H77), 1b (Con-1) subgenomic replicon cells has and 400 of NS5A in the JFH-1 genome. The plasmid phRluc-JFH-1 been reported previously (Hao et al., 2007; Lohmann et al., 1999). was made through a digestion with the XbaI restriction enzyme The JFH-1 full replicons were created by electroporating and used as a template for RNA transcription. The transcripts were Huh7.5.1 cells with RNA transcribed from pRluc-JFH-1 that con- prepared in vitro by using the Ambion MEGAscript Kits, and then tained a humanized Renilla luciferase (hRLuc) gene. After an elec- 10 mg RNA was mixed with 400 ml Huh7.5.1, which was gifted by Jin troporation of 3 days, replicon-containing cell clones were used for Zhong (Institute Pasteur of Shanghai, Chinese Academy of Science), the antiviral activity test. at a concentration of 1 107 cells/ml. After electroporation, the 48 J. Qing et al. / Antiviral Research 126 (2016) 43e54

cells. The plates were then incubated at 37 Cin5%CO2. The EGFP expression levels were observed under the microscope using an epifluorescence microscope to visualize the expression of EGFP after 3e4 days. The determination of the virus titer, the 50% tissue culture infectious dose (TCID50), was performed by using EPDA.

2.4. HCV virus or replicon inhibition assay

Huh7.5.1 cells or Replicon cell lines were seeded in 96-well plates at a density of 2 104 cells per well at 37 C overnight. All of the compounds were diluted with DMSO to 10 mM of stock solution. The initial concentration of the compounds was 20 mM and then diluted at a gradient. For the HCVcc system, the serially diluted compounds were mixed with a certain titer of the hRluc- JFH1 virus, and the final concentration of the hRluc-JFH1 virus titer was diluted to the numbers of relative luminescence units (RLU), ranging from 20,000 to 50,000 RLU and then added to the Huh7.5.1 cells. For the replicon cell lines, the serially diluted com- pounds were mixed with cells. Then, the cells were cultured for 2 days at 37 C and were harvested. The luminescence was detected according to the manufacturer's protocol of the Renilla-Glo™ Luciferase Assay System. The EC50 is the concentration of the compound at which the HCV luminescence level in the Huh7.5.1 cells is reduced by 50%. The values of EC50 were plotted by the GraphPad Prism 5 software.

2.5. Cell proliferation assay

Huh7.5.1 cells were seeded in 96-well plates at a density of 2 104 cells per well overnight. The cells were incubated with the serially diluted compounds for 48 h. The viability of the Huh7.5.1 cells was determined in 96-well tissue culture plates using the cell proliferation reagent WST-1 (Roche), and the absorbance (OD450/ reference OD630) was measured to detect the cytotoxicity of the compounds, according to the manufacturer's protocol of the Cell Proliferation Reagent WST-1. The CC50 is the concentration of the compound that inhibited cell growth by 50%. The values of CC Fig. 6. Comparison of emergence rate of the resistant mutation S282T (A) or HCV 50 clearance ability in NITD008 or PSI-7977 treatment (B). (A)Comparison of emer- were plotted by the GraphPad Prism 5 software. gence rate of amino acid changes S282T in the HCV genome that emerged during the selection with NITD008 or PSI-7977. The Huh7.5.1 cells were infected by a JFH-1 re- 2.6. Selection of NITD008 or PSI-7977-resistant viruses combinant virus and then were treated for four passages with NITD008 (0.5 mM) or m m m PSI-7977(2.5 M) and next eight passages with NITD008(2 M) or PSI-7977(10 M). 5 Total RNA was extracted from NITD008 or PSI-7977-selected cells from fourth, eighth, Huh7.5.1 cells were seeded at 5 10 cells/well in 6-well plates. and twelfth passages. The JFH-1 NS5B region was reverse transcribed into cDNA and In the next day, the medium was removed and replaced with amplified for clonal analysis. (B) HCV clearance and rebound experiment in DMEM containing 10% FBS and 0.5 moi JFH-1 with an EGFP reporter Huh7.5.1 cells. Cells were infected with an HCV cell culture (JFH-1) system with a gene in the presence of gradually increasing concentrations of the e fi luciferase reporter gene for three passages (passage 1 3) and then treated for ve compound (Fig. 3A). To keep the virus growing in the treatment consecutive passages (passage 4e8) with either NITD008(0.5 mMor2mM) or PSI- 7977(2.5 mMor10mM). During rebound (passage 9e13), NITD008 or PSI-7977 were with the compound, a method of supernatant-collected reinfection omitted from the culture medium and cells were cultured. The luciferase activity was was used in the experiment. The Huh7.5.1 cells were passaged measured at different passages. every 2 or 3 days in the presence of the compound. We then omitted the compound and after the Huh7.5.1 cells were infected completely by detecting the EGFP expression, the cell supernatants 2.3. Virus titration were collected following centrifugation at 4000 g for 5 min and were stored at 80 C as JFH-R virus. The Huh7.5.1 cells were To obtain the hRluc-JFH-1 virus titers, the virus stocks were reinfected with the JFH-R virus and treated with compound under diluted at a gradient of 1:10 and incubated with Huh7.5.1 cells for the same conditions described above. The experiment was repeated 48 h at 37 C. Then, the cells were harvested, and the luminescence for 3 cycles, and the cell supernatants were collected as JFH-R virus. was detected according to the manufacturer's protocol of the Renilla-Glo™ Luciferase Assay System. 2.7. Identification of NITD008-resistant mutations The titer of the JFH-1 virus containing the EGFP reporter gene was determined by endpoint dilution assays (EPDA) using focus- For the Huh7.5.1 RNA resistant mutation analysis, a cellular RNA forming units (ffu) as the read-out (Zhong et al., 2006). Briefly, extraction was performed using the TRIzol reagent (Invitrogen), the measurement was performed by seeding 1 104 Huh7.5.1 cells according to the manufacturer's instructions. For the reverse tran- per well in 96-well microtiter plates. After an overnight culture, the scription PCR, first strand cDNA was synthesized using random virus was serially diluted in 10-fold dilutions with DMEM con- primers and the Super Script III First-strand Synthesis System for taining 10% FBS (10 1e 10 8 fold dilutions) and added to Huh7.5.1 the RT-PCR kit (Invitrogen), according to the manufacturer's J. Qing et al. / Antiviral Research 126 (2016) 43e54 49 instructions. The HCV protein coding region of JFH-1 NS5B was 3. Results amplified by PCR: NS5B-7614-sense: 50 GCTCGGGGTCTTGGTCTAC 30, NS5B-9475- anti-sense: 50 GTTAGCTATGGAGTGTACCTAG 30. The 3.1. In vitro antiviral activity and cytotoxicity of NITD008 on HCV short RT-PCR product of the resistant JFH-R virus or the control JFH- genotypes 1 virus was ligated into the TA cloning vector PMD18-T (Takara). For each time point, multiple individual bacterial colonies were iso- The chemical structures of three direct-acting antiviral (DAA) lated, and the purified plasmid DNA was sequenced. The sequences inhibitors, NITD008, PSI-7977 and telaprevir, were shown in Fig. 1. were aligned using the Sequencher 5.0 and the BioEdit software. To determine the inhibitory activities of NITD008, we prepared an HCV cell culture system (hRluc-JFH1), a genotype 2a HCV virus containing a luciferase reporter gene. NITD008 inhibited the JFH-1 2.8. Generation of a mutational JFH-1 recombinant full replicon virus in a dose-responsive manner, with an EC50 value of 8.7 nM; meanwhile, telaprevir, as the positive control, showed an EC50 The mutational JFH-1 recombinant full replicon clone was value of 5.1 nM, indicating NITD008 exhibited an antiviral activity constructed based on the phRluc-JFH-1 plasmid. Site-directed against HCV with as low of a nanomolar activity as telaprevir (Fig. 2 mutagenesis was performed using a QuikChange Lighting Site- A). Directed Mutagenesis kit (Stratagene). The mutagenic primers To further confirm the anti-HCV activity of NITD008, we pre- were designed as follows: NS5BS282T-Sense: 50 CAG 0 pared HCV replicon systems, including JFH-1 full replicon (geno- ACGTTGCCGCGCCACCGGGGTGCTAACCACTAG 3 , NS5BS282T- type 2a), H77 replicon (genotype 1a), and Con1 replicon (genotype Antisense: 50 CTAGTGGTTAGCACCCCGGTGGCGCGGCAACGTCTG 30, 0 1b), containing a luciferase reporter gene. In addition to the JFH-1 NS5B-M289L-sense: 5 GGTGCTAACCACTAGCCTGGGTAACACCAT virus, NITD008 exhibited activity against all three of the geno- CAC 30, NS5B-M289L-antisense: 50 GTGATGGTGTTACCCAGGC- 0 0 types of the HCV replicons, with EC50 values of 93.3 nM, 60.0 nM TAGTGGTTAGCAC C3 ,NS5B-L384M-sense: 5 GGCCGCCGCA and 67.2 nM, respectively, for the JFH-1 full replicon, H77 replicon GATACTACATGACCAGAGACCC AACCAC30,NS5B-L384Mantisense:50 0 and Con1 replicon on the Huh7.5.1 cell lines (Fig. 2B, C, D). No GTGGTTGGGTCTCTGGTCATGTAGTA TCTGCGGCGGCC 3 . For the obvious cytotoxicity was observed from compound NITD008 or JFH-1 M289L recombinant full replicon, the method of overlap PCR telaprevir at concentrations of 20,000 nM, as demonstrated by the fi was also used for the clone. The constructs were con rmed by WST-1-based assay (Fig. 2E), indicating that the inhibition of HCV sequencing.

Fig. 7. Analysis of the interaction of 2a HCV NS5B with the primer-template RNA and nucleotide analog inhibitor. (A) A model of HCV NS5B with the primer-template RNA and GS-607596 (diphosphate metabolite of sofosbuvir) based on the structure of Appleby et al. (PDB ID codes 4TWG) (Appleby et al., 2015) (color coding: the fingers are red, palm is grey, thumb is blue), and the positions of the mutations S282T, M289L, L384M are indicated by their a carbons (yellow). GS-607596 was shown by an arrow. (B) Detailed structure of the 282 Serine and GS-607596. The black triangle indicated a methyl group at the 20 position of ribose. (C) Detailed structure of NITD008. The black triangle indicated an acetylene group at the 20 position of ribose. (D) A structure-based sequence alignment of the RNA-dependent RNA polymerase in different viruses. The residues playing key roles in the active site and conserved are colored red. The positions of mutations V63A, S282T, M289L, L384M, and M392L are indicated below. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 50 J. Qing et al. / Antiviral Research 126 (2016) 43e54 J. Qing et al. / Antiviral Research 126 (2016) 43e54 51 proliferation was specific. As summarized (Fig. 2F), NITD008 (Fig. 3D). In contrast, among the clones from the WT virus, no inhibited the replication of these replicons with low nanomolar mutation was observed. This result indicated that S282T was the EC50 values, which led to a selectivity index of >2298.9, >214.4, most predominant single amino acid change selected by NITD008. >333.3, >298.5, respectively, for the JFH-1 virus, JFH-1 full replicon, To identify whether the non-synonymous mutations in NS5B H77 replicon and Con1 replicon. We also assessed the activities of contributed to the resistant capability of JFH-1, the two mutations telaprevir to compare them with the genotype coverage of in NS5B were engineered into the plasmid of pJFH-Luc to produce NITD008, which were 5.1 nM, 186.0 nM, 129.2 nM, and 284.9 nM, JFH-1 S282T and JFH-1 L384M full replicons (Fig. 4A). After the respectively, which led to a selectivity index of >3921.6, >107.5, transfection of Huh7.5.1 cells with the RNA transcripts, transient >154.8, and >70.2. To our surprise, there was distinct difference in assays were performed to examine the effect of S282T on the antiviral activity between the JFH-1 virus and JFH-1 full replicon. It replication fitness and NITD008 antiviral activity. Consistent with may be because that the cells infected by JFH-1 virus at 48 h p.i. had previous findings (Lam et al., 2012), our results showed that the lower amount of virus than the replicon cells. Similar phenomenon JFH-1 2a S282T full replicon replicated at approximately 2% of its could be found before in nucleoside or nonnucleoside Inhibitors of wild type level, and JFH-1 L384M has the same fitness compared hepatitis C virus NS5B polymerase (Feng et al., 2014; Shih et al., with its wild type (Fig. 4B). No significant effect on the activity of 2011). Taken together, these results show that NITD008 is a NITD008 was observed with L384M, while S282T showed a sig- potent inhibitor of three HCV genotypes. nificant increase (approximately 76.5-fold) in the EC50 values (Fig. 4C). 3.2. Selection of resistant viruses using the HCV JFH-1 recombinant Next, we combined the amino acid substitutions within the virus finger and palm domains of the NS5B polymerase, and the double mutation (S282T/L384M) full replicon contained the same fold shift To confirm that NS5B polymerase is the target of inhibition by in EC50 as the S282T mutatant, and the addition of the thumb NITD008, resistance selection studies were performed using the domain L384M change to the S282T full replicon did not appear to JFH-1 recombinant virus, an infectious HCV monocistronic reporter further enhance the EC50-fold shifts for NITD008. As a control, no virus that is constructed by inserting an EGFP reporter gene into the significant effect on the activity of telaprevir was observed with C-terminus of NS5A of the JFH-1 genome, through multiple cell L384M, while S282T showed a slight decrease (approximately 0.45- culture passages in the presence of compound NITD008. fold) in the EC50s(Fig. 4D and F). This result may be because the To keep the JFH-1 recombinant virus growing in the treatment S282T full replicon was unfit for proliferation and therefore it was with NITD008, Huh7.5.1 cells was infected by virus in the presence more sensitive to the compound telaprevir. In conclusion, the of gradually increasing concentrations of the compound and a S282T mutation in NS5B contributed to the resistant capability of method of supernatant-collected reinfection was used in the the JFH-1 full replicon to NITD008. experiment (Fig. 3A). After a selection of 5 weeks, we observed that there were no obvious differences of the fluorescent foci between 3.4. Cross-resistance studies to NITD008 and PSI-7977 the treatment with or without compound NITD008. Three inde- pendent selections were performed to test the reproducibility of The phenotype of the drug resistant NS5B mutants is associated the resistance results. Three strains of the JFH-1 resistant virus with the binding sites of different inhibitory classes (Devogelaere were collected, JFH-R1, JFH-R2, and JFH-R3. All of the resistant vi- et al., 2012; Jiang et al., 2014). S282T is a resistant mutation in ruses exhibited significant resistance to NITD008 (Fig. 3B). Specif- the active site that is selected by a series of nucleotide analogs (Lam ically, at a 2 mM concentration of NITD008, the JFH-R1, JFH-R2, and et al., 2012). In this study, the replicon with S282T remained JFH-R2 viral titers were almost unchanged, whereas the WT viral resistant to PSI-7977, by a 4.52-fold EC50 change, consistent with titers were suppressed significantly (Fig. 3C). These results previous findings (Fig. 4E and F). No significant effect on the activity demonstrate that resistant HCV virus can be obtained in cell of PSI-7977 was observed with L384M, and the double mutation culture. (S282T/L384M) full replicon has a similar fold shift (7.15-fold) in the EC50s to PSI-7977 as the S282T mutant. Therefore, the S282T mu- 3.3. Sequence analyses of the NITD008 selected JFH-1 recombinant tation but not L384M in NS5B contributed to the resistance capa- virus bility of the JFH-1 full replicon to PSI-7977, somewhat similar to NITD008. Our results also showed that S282T contributed more To confirm our speculation that the strong resistance of the JFH- resistance to NITD008 than PSI-7977 (76.5-fold change vs. 4.52-fold 1 recombinant virus to NITD008 was due to the generation of change). adaptive mutations, the viral RNA of JFH-Rs as well as the control To further study the cross-resistance of NITD008 and PSI-7977, JFH-1 recombinant virus was extracted, and the genomes were the M289L mutation, which is in the palm domain of NS5B, re- sequenced. Compared with the wild type (WT) virus, two point ported as a resistant mutation selected by PSI-7977 (Lam et al., mutations were identified in the JFH-1 genome, including one 2012), was engineered into the plasmid of pJFH-Luc to produce located in the fingers domain (S282T) and another one in the the JFH-1 M289L or S282T/M289L full replicons (Fig. 5A). The re- thumb domain (L384M) of NS5B. Our sequencing analysis showed sults from our fitness analysis showed that the JFH-1 M289L full that among the whole nine cDNA clones of NITD008 selection with replicon replicated at approximately 0.2% of its wild type and JFH-1 five, two, and two clones, respectively, all of the clones contained S282T/M289L was the most unfit replicon (0.03%) (Fig. 5B) and the S282T change, whereas only 2 cDNA clones contained L384M almost did not replicate. Therefore, we only studied the JFH-1

Fig. 8. A comparison of the drugedrug interactions of NITD008 or PSI-7977 with other inhibitors of HCV proliferation. The anti-HCV effects of the combination of NITD008eIFNa-2b (A), PSI-7977eIFNa-2b (B), NITD008eribavirin (C), PSI-7977eribavirin (D), NITD008etelaprevir (E), PSI-7977etelaprevir (F) and PSI-7977-NITD008 (G). Huh7.5.1 cells infected by the JFH-1 virus with a luciferase reporter gene were treated with various concentrations of NITD008, IFNa-2b, ribavirin or telaprevir alone, or in combinations with each other for 48 h. A 3D-analysis of the combinations on the JFH1 infection was evaluated using the method of Prichard and Shipman (Prichard and Shipman, 1990). The combination studies for each pair of compounds were performed in triplicate. The theoretical additive surface is subtracted from the actual experimental surface, resulting in a horizontal surface that equals the zero plane when the combination is additive. A surface rising more than 25% above the zero plane indicates a synergistic effect of the combination, and a surface dropping lower than 25% below the zero plane indicates antagonism (H). 52 J. Qing et al. / Antiviral Research 126 (2016) 43e54

M289L full replicon. In a previous study, replicons with the S289L recognize the incoming nucleotide 2'-hydroxy (Appleby et al., changes remained resistant to PSI-7977 by a 2.9-fold EC50 change 2015). Previous biochemical studies revealed that analogs are (Lam et al., 2012); however, NITD008 showed no significant in- readily incorporated into the growing chain, similarly to the natural crease in the EC50 against the M289L full replicon (Fig. 5C and E). As ribonucleotide substrates (Dutartre et al., 2006). In contrast, the we previously explained, the JFH-1 full replicon with the S289L trapped elongation assembly containing 20-F/20-CH3-UDP (GS- change remained more sensitive to telaprevir than the wild type 607596) as the incoming nucleotide reveals that the hydrogen JFH-1 full replicon by a 0.5-fold change because of its unfitness bonding network is disrupted (Appleby et al., 2015). As previously (Fig. 5D and E). In conclusion, the M289L mutation in NS5B did not shown (Appleby et al., 2015; Jin et al., 2013), residue 282 Serine is contribute to the resistance capability of JFH-1 to NITD008. located close to the NS5B polymerase active cavity. M289 is located on the alpha helix in the palm domain, and L384 is within the 3.5. Comparison of emergence rate of the resistant mutation S282T thumb. Nucleoside or nucleotide analogs exert activity during or HCV clearance ability in NITD008 or PSI-7977 treatment initiation or elongation. Drug susceptibility studies and selection experiments have shown that a single mutation, S282T, confers a To further examine emergence rate of the resistant mutation reduced susceptibility to some 20-C-Me nucleotides (Fenaux et al., S282T selected by NITD008 in parallel with the PSI-7977, we per- 2013; Migliaccio et al., 2003). Residue 282 Serine contacts closely formed clonal sequencing of the NS5B region from various passages with the incoming nucleotide of the growing RNA strand (Fig. 7B). It of the selection to determine the progression and frequency of the gives an insight into the selection of a threonine as a potential mutation. The method of Gradually increasing concentrations of resistant mutation to 20-C-Me nucleotides. Based on the structure the compound and supernatant-collected reinfection was used in NS5B with 20-OH/20-CH3-UDP, it has been predicted that there is a this experiment as before. In the first four passages, selection was steric clash between the T282 side chain and the 20-CH3(Appleby 0 0 performed at compound concentrations equivalent to 6 EC50s, et al., 2015). Compared with a PSI-7977 derivate from 2 -F/2 - respectively 0.5 mM NITD008 or 2.5 mM PSI-7977, and followed by CH3-UDP, NITD008 contains a carbon substitution for the N-7 of the 0 selection at compound concentrations equivalent to 24 EC50s in the purine and an acetylene at the 2 position of ribose (Fig. 7C). The next eight passages, respectively 2 mM NITD008 or 10 mM PSI-7977. steric clash between 2'- acetylene and the T282 side chain may be At passage 4, no substitutions in the NS5B polymerase domain were bigger than that of the 20-CH3; therefore, compared with PSI-7977, observed either treated by NITD008 or PSI-7977 (Fig. 6A). At pas- the mutation S282T has a greater effect on the antiviral activity of sage 8, three of the whole seven cDNA clones of NITD008 selection NITD008. A sequence alignment shows that S282T is completely contained the S282T change, and no substitutions were observed in conserved among various members of positive RNA viruses PSI-7977 selection (Fig. 6A). At passage 12, the whole seven cDNA (Fig. 7D). Therefore, residue Serine 282 in HCV virus is important clones of NITD008 selection contained the S282T change, and only for regulating RNA synthesis by the polymerase, and it could be three substitutions of the whole eight cDNA clones of PSI-7977 used by the virus to overcome the selective pressure of NITD008. selection was observed (Fig. 6A). These results demonstrate that resistant mutant S282T selected by NITD008 emerges more easily 3.7. Combination effects of NITD008 with IFN, ribavirin or an NS3 in cell culture than PSI-7977. protease inhibitor To study the potent anti-HCV activity of NITD008 as compared with PSI-7977, we detected the ability of the compound to clear Because the inhibition of each step of the virus life cycle can Huh7.5.1 cells from JFH-1 virus. Huh7.5.1 cells were infected by JFH- affect the proliferation of a virus, we tested whether NITD008 could 1 virus with a luciferase reporter, and after three passages, the be combined with different anti-HCV drugs currently used in relative luminescence units (RLU) has a high increase (Fig. 6B). Then hepatitis C treatment. To test this hypothesis, we selected IFNa-2b, these cells were cultured in the presence of either NITD008 (0.5 mM ribavirin, telaprevir and PSI-7977. The assay of combinations was or 2.5 mM) or PSI-7977(2 mMor10mM). After another five passages, evaluated with an HCV cell culture (JFH-1) system with a luciferase a rapid decrease in HCV protein was already noted (Fig. 6B). When reporter gene. The results revealed a concentration-dependent the relative luminescence units dropped to the detection limit, we inhibition of HCV replication with NITD008, IFNa-2b, ribavirin, omitted the compound and only those cells that still carry the HCV telaprevir, or PSI-7977 alone, or with any two in combination. The virus will be able to proliferate under these conditions. All cultures data were analyzed using the method of Prichard and Shipman to that had been treated with NITD008 or PSI-7977 were able to determine whether the effect of the combinations was synergistic, proliferate in the absence of compound (Fig. 6B). But at a high additive, or antagonistic (Prichard and Shipman, 1990). The results concentration of NITD008 (2 mM) or PSI-7977(10 mM), the rebound revealed that the combination of NITD008eIFNa-2b (Fig. 8A), PSI- of JFH-1 will be more difficult than at a low concentration. These 7977eIFNa-2b (Fig. 8B), NITD008eribavirin (Fig. 8C), PSI- results suggested that anti-HCV activity of NITD008 is similar to 7977eribavirin (Fig. 8D), NITD008etelaprevir (Fig. 8E), and PSI- PSI-7977. 7977etelaprevir (Fig. 8F) had antiviral effects that were not significantly more potent than the theoretical additive effects, as 3.6. A model of the NITD008 interaction site in the HCV RNA- the maximal percent synergy was below the range of 25%(H), dependent RNA polymerase which supported that this combination was additive. No evidence of a significant antiviral antagonism was observed with the tested Despite the absence of a viral polymerase inhibition in doses, as the maximal percent antagonism did not exceed 25%. biochemical assays, the overlap in the resistant mutations led us to These results were consistent with previous reported HCV NS5B probe for potential NITD008 interaction sites in the NS5B cavity. We inhibitors (Fenaux et al., 2013). In contrast, the combination of PSI- constructed a model of JFH-1 NS5B with primer-template RNA and 7977-NITD008 showed a considerably more synergistic effect a nucleotide analog inhibitor (GS-607596, diphosphate metabolite compared with the theoretical additive effects (Fig. 8G), as the of sofosbuvir) based on the structure of Appleby et al. (Appleby maximal percent antagonism extended to a range of 36%, while the et al., 2015) and the resistant mutants were mapped to the NS5B antagonism did not extend outside a range of 4%. In our experi- polymerase of JFH-1, S282T, M289L and L384M (Fig. 7A). The HCV ments, no significant cytotoxicity was observed with the combi- NS5B RNA-dependent RNA polymerase displays stringent selec- nation of NITD008 or any of the other two compounds (data not tivity for nucleotides by using a hydrogen bond network to shown). Therefore, the observed synergy is indeed specific and J. Qing et al. / Antiviral Research 126 (2016) 43e54 53 does not reflect synergistic toxicity. 7977-NITD008 at various concentrations resulted in a greater in- hibition than any compound alone at the same concentration, and 4. Discussion PSI-7977-NITD008 showed a significant synergistic effect. In conclusion, NITD008 is an effective analog inhibitor against hepa- At present, three identified types of polymerase inhibitors, titis C virus with a broad genotype coverage and although JFH-1 including substrate analogs (nucleoside and nucleotide analogs), mutant resistant to NITD008 emerged more easily in cell culture product analogs (pyrophosphate analogs) and allosteric inhibitors than PSI-7977, it is a good research tool for developing nucleoside are under development as antiviral drugs (Lou et al., 2014; Zhou analogs. et al., 2013). Nucleoside or nucleotide analogs occupy the domi- nant position of all antiviral reagents. Except PSI-7977 which is the Acknowledgments RNA polymerase inhibitor as a drug against HCV infection approved by the U.S. Food and Drug Administration, all other nucleoside or This work was supported by the National Natural Science nucleotide analogs target the DNA polymerase and reverse tran- Foundation of China (Grant no. 81322023, 91313301, 31370733, scriptase (Oberg, 2006). A nucleoside or nucleotide analog will be 31170678, 31100208, 31000332 and 21202087), the National Basic fi rst converted to the triphosphorylated form by host or viral en- Research Program of China (973 program, Grant No. 2013CB911104 zymes, and then compete with the natural nucleoside tri- and 2014CB542800), the Fundamental Research Funds for the phosphates as a chain terminator of the growing viral genome (Lou Central Universities (Grant No. 65124002), the Specialized Research fi et al., 2014). Both the ef ciency of conversion to the triphos- Fund for the Doctoral Program of Higher Education Ministry of phorylated form and the blockage of viral genome elongation are Education of China (Grant No. 20120031120049), the Tianjin Sci- selective and determine the inhibitory activity (Oberg, 2006). ence and Technology Program (Grant No. 13JCYBJC24300, NITD008 was reported to selectively inhibit viruses within the 13JCQNJC13100) and the “111” Project of the Ministry of Education Flaviviridae family, including Dengue virus, West Nile virus, yellow of China (Project No. B06005) and Tsinghua University Initiative fever virus, or EV71, both in vitro and in vivo (Deng et al., 2014; Scientific Research Program (2009THZ01, 20141081129) and The Shang et al., 2014; Yin et al., 2009), but did not show antiviral ac- Importation and Development of High-Caliber Talents Project of tivity to Western equine encephalitis virus or vesicular stomatitis Beijing Municipal Institutions (grant no. YETP0166). virus (Yin et al., 2009). In this study, we demonstrated the efficacy of NITD008 as an inhibitor of HCV infection. NITD008 exhibits References antiviral activity against different HCV genotypes. As an adenosine analog, NITD008 has a function as polymerase active site inhibitor Appleby, T.C., Perry, J.K., Murakami, E., Barauskas, O., Feng, J., Cho, A., Fox 3rd, D., in dengue virus (Yin et al., 2009). Its triphosphate form competi- Wetmore, D.R., McGrath, M.E., Ray, A.S., et al., 2015. Viral replication. Structural tively inhibits polymerase activity, probably through binding at the basis for RNA replication by the hepatitis C virus polymerase. Science 347, 771e775. nucleoside triphosphate site and incorporating into the RNA Bacon, B.R., Gordon, S.C., Lawitz, E., Marcellin, P., Vierling, J.M., Zeuzem, S., product chain. The same mechanism may be inferred to inhibit HCV Poordad, F., Goodman, Z.D., Sings, H.L., Boparai, N., et al., 2011. Boceprevir for RNA synthesis by NITD008. The selection of resistant virus was previously treated chronic HCV genotype 1 infection. N. Engl. J. Med. 364, 1207e1217. attempted on DENV or WNV; however, no resistant virus was Cui, H.K., Qing, J., Guo, Y., Wang, Y.J., Cui, L.J., He, T.H., Zhang, L., Liu, L., 2013. Stapled discovered in viral genome (Yin et al., 2009). In a previous study, peptide-based membrane fusion inhibitors of hepatitis C virus. Bioorg. Med. enterovirus 71 (EV71) with the V63A and M392L changes remained Chem. 21, 3547e3554. Deng, C.L., Yeo, H., Ye, H.Q., Liu, S.Q., Shang, B.D., Gong, P., Alonso, S., Shi, P.Y., resistant to the NITD008, which were mapped to the 3D polymer- Zhang, B., 2014. Inhibition of enterovirus 71 by adenosine analog NITD008. ase of EV71 (Fig. 7D) (Deng et al., 2014). Notably, our experiment J. 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