Novel Antiviral Agent DTriP-22 Targets RNA-Dependent RNA Polymerase of Enterovirus 71

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 2009, p. 2740–2747 0066-4804/09/$08.00⫹0 doi:10.1128/AAC.00101-09 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Vol. 53, No. 7

Novel Antiviral Agent DTriP-22 Targets RNA-Dependent RNA Polymerase of Enterovirus 71䌤 Tzu-Chun Chen,1,2,5 Hwan-You Chang,5 Pei-Fen Lin,2 Jyh-Haur Chern,4 John Tsu-An Hsu,4 Chu-Yi Chang,6 and Shin-Ru Shih1,2,3,4* Research Center for Emerging Viral Infections, Chang Gung University, Taoyuan, Taiwan1; Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan, Taiwan2; Clinical Virology Laboratory, Chang Gung Memorial Hospital, Taoyuan, Taiwan3; Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Chunan, Taiwan4; Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan5; and Department of Computer Science and Information Engineering, Chang Gung University, Taoyuan, Taiwan6

Enterovirus 71 (EV71) has emerged as an important virulent neurotropic enterovirus in young children. DTriP-22 (4{4-[(2-bromo-phenyl)-(3-methyl-thiophen-2-yl)-methyl]-piperazin-1-yl}-1-pheny-1H-pyrazolo[3,4d]pyrimidine) was found to be a novel and potent inhibitor of EV71. The molecular target of this compound was identified by analyzing DTriP-22-resistant viruses. A substitution of lysine for Arg163 in EV71 3D polymerase rendered the virus drug resistant. DTriP-22 exhibited the ability to inhibit viral replication by reducing viral RNA accumulation. The compound suppressed the accumulated levels of both positive- and negative-stranded viral RNA during virus infection. An in vitro polymerase assay indicated that DTriP-22 inhibited the poly(U) elongation activity, but not the VPg uridylylation activity, of EV71 polymerase. These findings demonstrate that the nonnucleoside analogue DTriP-22 acts as a novel inhibitor of EV71 polymerase. DTriP-22 also exhibited a broad spectrum of antiviral activity against other picornaviruses, which highlights its potential in the development of antiviral agents. assays (25). A pharmacologically active drug library has been employed to identify anti-EV71 compounds (4). Ribavirin used in combination with interferon to treat patients with hepatitis C virus infection reduces the mortality rate of EV71-infected mice by reducing viral replication (26). We previously discovered piperazine-containing pyrazolo[3,4d]pyrimidine derivatives as a novel class of anti-EV71 compounds (9). DTriP-22 (4{4-[(2-bromo-phenyl)-(3-methyl-thiophen-2-yl)methyl]-piperazin-1-yl}-1-pheny-1H-pyr-azolo[3,4-d]pyrimidine) is one such compound, containing a diacrylmethyl group at the piperazine and a phenyl group at the pyrazolo[3,4-d]pyrimidine (9). Although DTriP-22 has a pyrazolo[3,4-d]pyrimidine structure and is thus similar to pyrazolo[3,4-d]pyrimidine nucleoside analogues, DTriP-22 differs from these analogues in that it lacks an appropriate carbocyclic ring, such as a ribose, for incorporation into the growing viral RNA chains (39). Moreover, the size of DTriP-22 differs markedly from those of the nucleoside analogues (39). In this study, we identified DTriP-22, a nonnucleoside analogue which targets EV71 3D polymerase, by analysis of DTriP22-resistant viruses. Inhibition of 3D polymerase activity in vitro by DTriP-22 was also investigated.

Enterovirus 71 (EV71), a positive-stranded RNA virus, belongs to the genus Enterovirus in the family Picornaviridae. EV71 infection typically causes hand, foot, and mouth disease or herpangina, followed by severe central nervous system complications, including aseptic meningitis, encephalitis, poliomyelitis-like paralysis, neurogenic cardiopulmonary failure, and even death in some young children. Infants, following infection by EV71 with central nervous system complications, reportedly suffer from neurologic sequelae and delayed neurodevelopment (6). In 1998, a large outbreak of EV71 infection occurred in Taiwan, resulting in almost 80 fatalities and 405 severe cases (7, 21). Subsequently, many outbreaks of EV71 infection in Taiwan have been reported, with a total of 51 verified fatal cases in 2000 and 2001 (29). Severe EV71 infections continued to occur for several years thereafter. EV71 infection also has been reported to occur in many countries, such as Malaysia, Singapore, Australia, Japan, the United States, Germany, and mainland China (1–3, 5, 13, 22, 33, 34). The development of anti-EV71 agents is important because EV71 is regarded as the most important neurotropic enterovirus, after poliovirus control (34). A novel series of pyridyl imidazolidinones targeting VP1 protein, based on the skeletons of WIN compounds, has been developed using computerassisted drug design (37). The new EV71 3C protease inhibitors, based on rhinovirus 3C protease inhibitor AG7088, exhibit inhibitory activities in both enzymatic and cell-based

MATERIALS AND METHODS Cells, viruses, and chemicals. RD (rhabdomyosarcoma) cells (American Type Culture Collection [ATCC] accession no. CCL-136) and MDCK (Madin-Darby canine kidney) cells (ATCC accession no. CCL-34) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS). Vero (African green monkey kidney) cells (ATCC accession no. CCL-81) and HeLa cells (ATCC accession no. CCL-2) were maintained in modified Eagle’s medium (Gibco) supplemented with 10% FBS. EV71 TW/ 1743/98 and TW/2231/98; coxsackieviruses A9, A10, A16, A24, B1, B2, B3, B4, B5, and B6; echovirus 9; human rhinovirus 2 (HRV-2); herpes simplex virus type 1 (HSV-1); and HSV-2 were isolated from clinical specimens in the Clinical Virology Laboratory of Chang Gung Memorial Hospital (Linkou, Taiwan).

* Corresponding author. Mailing address: Department of Medical Biotechnology and Laboratory Science, Chang Gung University, 259 Wen-Hua 1st Rd., Kwei-Shan, Taoyuan 333, Taiwan. Phone: 886-3-2118800, ext. 5497. Fax: 886-3-2118174. E-mail: srshih@mail .cgu.edu.tw. 䌤 Published ahead of print on 4 May 2009. 2740

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Received 23 January 2009/Returned for modification 17 March 2009/Accepted 28 April 2009

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T256A mutation in the 2C protein and the R163K and S264L (and R163KS264L) mutations in the 3D protein were introduced using mutagenic primer pairs comprising the sequences 5⬘-GATTCCTATAAGGCAGAGCTGGGCA G-3⬘ and 5⬘-CTGCCCAGCTCTGCCTTATAGGAATC-3⬘, 5⬘-GTTAAAGAT GAACTTAAAGCCATCGACAAGATC-3⬘ and 5⬘-GATCTTGTCGATGGCT TTAAGTTCATCTTTAAC-3⬘, and 5⬘-CCGAAGACGCAGTGTTACTCATA GAAGGGATC-3⬘ and 5⬘-GATCCCTTCTATGAGTAACACTGCGTCTTCG G-3⬘, respectively (mutated nucleotides are underlined). The mutant clones were verified by sequencing and further used to generate virus, as described below. The plasmid was linearized with EcoRI and MluI and then transcribed using a MEGAscript T7 kit (Ambion) in accordance with the manufacturer’s instructions. Vero cells in six-well plates were transfected with the transcribed RNA by using Lipofectamine 2000 reagent (Invitrogen) in accordance with the manufacturer’s instructions and then incubated at 35°C for 72 h. The mutant viruses from the culture supernatant were further plaque purified and confirmed by sequencing. Analysis of viral RNA accumulation. RD cells (6 ⫻ 105 cells/well in a six-well plate) were infected with EV71 at an MOI of 1 PFU/cell. After 1 h of absorption at room temperature, the cells were washed twice and supplemented with medium containing 2 ␮M DTriP-22. The cells were further incubated at 35°C for the indicated periods. The intracellular RNA was then extracted using an RNeasy kit (Qiagen). The viral RNA was further detected using quantitative real-time reverse transcriptase PCR (RT-PCR) and slot blot analysis. Quantitative RT-PCR was performed with a TaqMan RT-PCR kit (Applied Biosystems), using an ABI Prism 7000 apparatus. The oligonucleotide primers and the TaqMan probe for detecting EV71 RNA, designed by W. A. Verstrepen, were as follows: sense, 5⬘-CCCTGAATGCGGCTAATC-3⬘; antisense, 5⬘-ATT GTCACCATAAGCAGCCA-3⬘; and probe, FAM (6-carboxyfluorescein)-AAC CGACTACTTTGGGTGTCCGTGTTTC-TAMRA (6-carboxytetramethylrhodamine) (46). 18S rRNA probe and primers obtained from TaqMan (Applied Biosystems) were used as internal controls. Each sample was assayed in triplicate, and the experiment was performed three times independently. The obtained data were analyzed using ABI Prism 7000 sequence detection system software. The yield of EV71 RNA was normalized to that of 18S rRNA. Slot blot analysis for detecting viral RNA was performed as described previously (27). Briefly, denaturing RNA was loaded onto a nylon membrane in the slot blot manifold. The membrane was then cross-linked. Digoxigenin-labeled RNA probes, specific for the genome or antigenome of EV71, were produced using a DIG Northern starter kit (Roche). The hybridization and detecting procedures were performed according to the manufacturer’s instructions. Dicistronic expression assay. pRHF-EV71-5⬘ UTR, containing the EV71 5⬘ untranslated region (UTR) between the Renilla and firefly luciferase genes, was used to evaluate the internal ribosome entry site (IRES)-dependent translation of EV71, as described elsewhere (28). Briefly, RD cells (2.5 ⫻ 105 cells/well in a 12-well plate) were grown to 90% confluence and transfected with the plasmid in the presence of DTriP-22. After 2 days, cell extracts were assayed for Renilla and firefly luciferase activity with a Lumat LB9507 bioluminometer, using a dualluciferase reporter assay kit (Promega) in accordance with the manufacturer’s instructions. Expression and purification of EV71 3D polymerase. To construct pET26bUb-EV71-3D, the EV71 3D region of pEV71 was subcloned into pET26b-Ub3D-GSSG-6H and used to replace the poliovirus 3D region of pET26b-Ub-3DGSSG-6H, which encodes ubiquitin (Ub)-poliovirus 3D (a gift from C. E. Cameron) (16). pCG1 (from C. E. Cameron) encodes an Ub-specific carboxyterminal protease (Ubp1). Expression of Ub-3D fusion protein in the presence of Ubp1 has glycine at the amino terminus of polymerase, not methionine. Plasmids pET26b-Ub-EV71-3D and pCG1 were cotransformed into BL21(DE3). Expression of EV71 3D was induced by adding 50 ␮M isopropyl-␤-D-thiogalactopyranoside at 25°C for 4 h. The protein expressed from lysed cells was suspended in buffer A (50 mM Tris, pH 8.0, 20% glycerol, 1 mM dithiothreitol, 0.1% NP-40, and 60 ␮M ZnCl2) and loaded onto a HisTrap column (GE Healthcare Biosciences), which was then washed with buffer A containing 30, 50, 70, or 90 mM imidazole; the protein was then eluted with buffer A containing 500 mM imidazole. The eluted product was dialyzed against buffer B (50 mM HEPES, pH 7.5, 500 mM NaCl, 20% glycerol, 1 mM dithiothreitol, 0.1% NP-40, and 60 ␮M ZnCl2) and stored at ⫺70°C. Polymerase elongation assay. The elongation assay was performed with 1 ␮M polymerase in 50 mM HEPES, pH 7.5, 10 mM 2-mercaptoethanol, 5 mM MgCl2, 60 ␮M ZnCl2, 5 ␮M UTP, 0.4 ␮Ci/␮l [␣-32P]UTP, 1.8 ␮M dT15-0.15 ␮M poly(rA)300 (primer/template), and DTriP-22. The reaction mixtures were incubated for 10 min at 30°C and the reactions terminated by adding EDTA to give a concentration of 83 mM. The quenched reactants were spotted onto DE81 filter paper discs (Whatman) and air dried. The discs were washed with 5%

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EV71 Tainan/4643/98 was obtained from Jen-Ren Wang, National Cheng Kung University (Tainan, Taiwan). EV71 BrCr, the prototype of EV71 (ATCC accession no. VR 784), was obtained from the ATCC. EV71; coxsackieviruses A9, A10, A16, A24, B1, B2, B3, B4, B5, and B6; and echovirus 9 were propagated in RD cells. HRV-2 was propagated in HeLa cells. Influenza A virus (A/WSN/33) and influenza B virus (B/HK/72) were propagated in MDCK cells. HSV-1 and HSV-2 were propagated in Vero cells. DTriP-22 was synthesized at the National Health Research Institutes (Taiwan) and dissolved in dimethyl sulfoxide (DMSO). Antiviral assay of DTriP-22 activity against EV71. RD cells (6 ⫻ 105 cells/well) were seeded in a six-well plates and incubated for 24 h. Cells were washed and then infected with EV71 TW/2231/98 at a multiplicity of infection (MOI) of 0.001, 0.1, or 1 PFU/cell. After 1 h of absorption at room temperature, the infected cells were washed twice, covered with medium containing 2% FBS and various concentrations of DTriP-22 (0, 0.005, 0.1, 0.2, 1, 2, and 2.5 ␮M), and further incubated at 35°C. At 16 h postinfection (p.i.), the supernatant and debris were collected together and the total virus yield was quantified by a plaque assay. Plaque assay. RD cells (6 ⫻ 105 cells/well) were seeded in a six-well plate and incubated for 24 h. Cells were washed and infected with or without virus with a 10-fold series dilution. After absorption at room temperature for 1 h, the infected cells were washed twice and covered with medium containing 2% FBS and 0.3% agarose gel. The infected cells were further incubated at 35°C for 4 days. The plates were fixed with 0.5% formaldehyde and then stained with 0.1% crystal violet. The plaques were then counted, and the viral titers were presented as numbers of PFU/milliliter. Neutralization test. The neutralization assay measured the ability of a test compound to inhibit the cytopathic effects induced by viruses, as described previously (37). Briefly, 96-well tissue culture plates were seeded with 3 ⫻ 104 cells/well in DMEM with 10% FBS. After 24 h of incubation at 37°C, RD cells were infected with the virus at an MOI of 0.005 PFU/cell. After adsorption, the infected cells were covered with medium containing 2% FBS and 0.5% DMSO or DTriP-22 at various concentrations (twofold dilutions from 25 ␮M). The infected cells were further incubated at 35°C for 64 h. The plates were fixed with 0.5% formaldehyde and then stained with 0.1% crystal violet. The density of the well at 570 nm was measured. Each experiment was performed in triplicate and repeated at least two times. The 50% effective concentrations (EC50s) were calculated according to the formula EC50 ⫽ [(Y ⫺ B)/(A ⫺ B)] ⫻ (H ⫺ L) ⫹ L], where Y represents half of the mean optical density at 570 nm (OD570) of the cell control without the compound, B represents the mean OD570 of wells with the compound dilution nearest to and below Y, A represents the mean OD570 of wells with the compound dilution nearest to and above Y, and L and H are the compound concentrations at B and A, respectively. Cytotoxicity assay. Cell viability was evaluated using the MTS {tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]}/PMS (phenazine methosulfate) method (Cell Titer96 AQueous cell proliferation kit; Promega) in accordance with the manufacturer’s instructions. RD cells (6.5 ⫻ 103 cells/well) were seeded into each well of a 96-well microtiter plate. Following incubation for 24 h, the medium was replaced with fresh medium with 2% FBS and a twofold serial dilution of the test compound (each dilution in triplicate). The cells grew at 35°C for 64 h and reached 90% confluence. The culture medium was then replaced with 100 ␮l phenol red-free medium containing MTS (Promega) and PMS (Sigma). The plate was incubated at 35°C for 1 to 4 h, and the absorbance at 490 nm was then recorded using a microplate reader. The survival rate of the cells that had been treated with the compound was determined using the following formula: percent cell viability ⫽ (OD490 of treated cells ⫺ OD490 of blank)/(OD490 of control cells ⫺ OD490 of blank). The 50% cytotoxic concentration (CC50) was determined as the concentration of the compound at which cell viability was reduced to 50%. Data were analyzed using GraphPad Prism 4.0 software. Generation of DTriP-22-resistant viruses. RD cells (6 ⫻ 105 cells/well) were seeded in a six-well plate and incubated for 24 h. The cells were infected with EV71 TW/2231/98 at an MOI of 0.001 PFU/cell. After virus absorption for 1 h, the cells were washed twice and incubated for 3 days in 3 ml of DMEM-2% FBS containing 0.2 ␮M DTriP-22. The clear supernatant was collected and termed passage 1. The passage 1 virus was used to infect a new cell monolayer, which was further incubated in the presence of a compound. The procedure was repeated for 14 passages. The susceptibility to DTriP-22 of passage 14-resistant variants was confirmed using a neutralization test. Ten isolates of DTriP-22-resistant viruses from passage 14 virus were plaque purified. The drug susceptibilities of these 10 isolates were further verified using a neutralization test. Generation of EV71 mutants. A pEV71 plasmid containing the full-length genome of EV71 TW/2231/98 was used (37). The mutations in pEV71 were generated using a QuikChange site-directed mutagenesis kit (Stratagene). The

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dibasic sodium phosphate and rinsed in absolute ethanol. The radioactivity of the sample was quantified using scintillation fluid. In vitro uridylylation assay. VPg uridylylation was assayed with a reaction mixture containing 50 mM HEPES, pH 7.5, 2.5 mM manganese(II) acetate, 8% glycerol, 0.5 ␮g poly(rA)300 RNA, 2 ␮g EV71-synthesized VPg peptide, 1 ␮M polymerase, 0.04 ␮M [␣-32P]UTP, 10 ␮M unlabeled UTP, and DTriP-22. The reaction mixture was incubated at 33°C for 60 min. The sample was analyzed by Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 13.5% polyacrylamide. The radioactivity of the sample was exposed to X-ray film. Data analysis. Data were plotted and statistical significance was determined using the GraphPad Prism 4 program. The curves in the figures were plotted using nonlinear regression analysis. The related elongation activity of 3D polymerase is shown in the bar graph. Significance of differences among groups was assessed by one-way analysis of variance followed by Tukey’s post hoc multiplecomparison test.

RESULTS Antiviral activity of DTriP-22 against EV71. The chemical structure of DTriP-22, shown in Fig. 1A, contains pyrazolo[3,4d]pyrimidine. According to a previous investigation of structure-activity relationship, the phenyl group at the pyrazolo[3,4d]pyrimidine and the hydrophobic diarylmethyl group at the piperazine in a series of pyrazolo[3,4-d]pyrimidine-containing compounds significantly influence antienterovirus activity (9). DTriP-22 exhibited better anti-EV71 activity than the other derivatives (9). Therefore, it was used to select drug-resistant viruses. The anti-EV71 activity of DTriP-22 was evaluated with high and low viral titers. RD cells were infected with EV71 at an MOI of 0.001, 0.1, or 1 PFU/cell in the presence of DTriP22. The total viral yields at 16 h p.i. were then detected using

TABLE 1. Drug susceptibilities and sequence changes observed for viruses harvested from different passages Passage no.

EC50 (␮M)a

0 5 10 14

0.74 ⫾ 0.03 0.25 ⫾ 0.02 ⬎25 ⬎25

Amino acid(s) at indicated position VP1 106

VP1 243

2C 256

3D 163

3D 264

Y C C C

P S S S

T A A A

R R K K

S S S/Lb L

a Data are presented as means ⫾ standard deviations of results from at least two independent experiments. b Two kinds of amino acid (serine and leucine) at position 264 in the 3D region were observed in passage 10 virus. A leucine residue at position 264 in the 3D region was predominant.

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FIG. 1. (A) Structural formula of DTriP-22. (B) Anti-EV71 activity of DTriP-22. RD cells (6 ⫻ 105 cells/well) were seeded in a six-well plate and incubated for 24 h. RD cells were infected with EV71 (MOI of 0.001, 0.1, or 1 PFU/cell, separately). After absorption, the infected cells were treated with various concentrations of the compound (0, 0.05, 0.1, 0.2, 1, 2, and 2.5 ␮M) for 16 h. The infected cells were lysed into culture medium, and the total virus yield was quantified by a plaque assay. Data are presented as means ⫾ standard errors of the means (SEM) of results from three independent experiments. The 50% inhibitory concentrations were calculated using nonlinear regression analysis.

a plaque assay. The result, shown in Fig. 1B, demonstrates that DTriP-22 has the ability to inhibit EV71 at both high and low MOI. DTriP-22 at 0.1 ␮M reduced the viral yield by 95% when the cells were infected with an MOI of 0.001 PFU/cell. The EC50s were 0.023 and 0.16 ␮M for 0.001 MOI and 1 MOI (PFU/cell), respectively. The cytotoxicity of the compound was evaluated using an MTS-based assay. The CC50 of the compound was larger than 100 ␮M. The selective index (ratio of CC50 to EC50) of DTriP-22 for EV71 exceeds 625 for an MOI of 1 PFU/cell. Identification of mutations that confer resistance to DTriP22. To understand the antiviral mechanism of DTriP-22, the molecular target that can render the virus resistant to this compound was initially identified. EV71 TW/2231/98 at an MOI of 0.001 PFU/cell was cultivated to select resistant viruses at increasing concentrations (0.2 to 2 ␮M, representing an approximately 8.7- to 87-fold increase in EC50) of DTriP-22. Virus pools in passages 5, 10, and 14 were tested to evaluate their drug susceptibilities by using a neutralization test. Viruses harvested from passages 10 and 14 were found to have EC50s of over 25 ␮M (Table 1), indicating that those viruses had become resistant to the compound. Ten plaque-purified viruses from passage 14 were at least 187 times more resistant to DTriP-22 than were the parental viruses (data not shown). Full-genome sequence analysis of the plaque-purified resistant viruses revealed consistent mutations at five amino acids in these resistant viruses relative to the sequence for the parental strain. They are Y106C and P243S in the VP1 region, T256A in the 2C region, and R163K and S264L in the 3D region. To identify the mutations that are involved in drug resistance, the amino acid mutations at these five positions in various passages of viruses were monitored. The data revealed that the sensitive viruses became resistant in passage 10; two mutations (R163K and S264L) in the 3D region were observed in this passage (Table 1). However, one mutation in the 2C region (T256A) and two mutations in the VP1 region (Y106C and P243S) were observed in passage 5, and those mutations were also observed in passages 10 and 14. These results indicate that R163K and S264L mutations may render EV71 drug resistant but that T256A, Y106C, and P243S mutations do not. The above-mentioned mutations were introduced into the EV71 infectious clone to confirm that the resistance markers are located in the 3D region. The other mutation in the 2C region was also used, as a control. The drug susceptibilities of these recombinant viruses were tested by using a neutralization

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TABLE 2. Plaque formation and drug susceptibilities for recombinant EV71 viruses Virus

Wild type 2C T256A 3D R163K 3D S264L 2C T256A-3D S264L 3D R163K-S264L VP1 V192Mc

EC50 (␮M)a

Plaque formation

DTriP-22

Pyridyl imidazolinoned

⫹ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹

0.308 ⫾ 0.032 0.279 ⫾ 0.014 ⬎25 NTb NT ⬎25 0.240 ⫾ 0.030

0.0021 ⫾ 0.0002 0.0041 ⫾ 0.0010 0.0034 ⫾ 0.0002 NT NT 0.0035 ⫾ 0.0001 ⬎25

test (Table 2). An arginine-to-lysine amino acid substitution at residue 163 (R163K) in the 3D polymerase reduced the drug susceptibility of the mutant virus (EC50 ⬎ 25 ␮M). A change at position 264 in the 3D region from serine to leucine (S264L) was a lethal mutation for EV71. This lethality can be rescued by a compensatory mutation at the R163K residue in the 3D region rather than at the T256A residue in the 2C region. Both the 3D R163K and the 3D R163K-S264L mutants had sensitivities to DTriP-22 at least 81 times lower than that observed for the wild-type virus. However, the 2C T256V mutant and the wild-type virus had similar drug susceptibilities. The VP1 V192M mutant virus, resistant to pyridyl imidazolidinones (an EV71 capsid binder), was also sensitive to DTriP-22 (37). In summary, the change at residue 163 in the 3D polymerase is crucial to drug resistance. However, DTriP-22-resistant viruses exhibited sensitivity to pyridyl imidazolidinone. Time-of-addition experiments with EV71-infected cells. The results of the genetic approach indicated that 3D-involving viral replication is likely to be the target of DTriP-22. To determine which stage(s) of the EV71 replication cycle is affected by DTriP-22, the time course of a single viral replication cycle was first determined. The growth curve for the viral progeny production initially rose exponentially, reaching a stationary phase (approximately 8 ⫻ 107 PFU/ml) at 12 to 14 h p.i. (Fig. 2A). Therefore, DTriP-22 was added to the culture medium 2 h prior to the end of EV71 absorption, at the end of EV71 absorption, or at 2-h intervals until 14 h p.i. At 16 h p.i., the total virus yield was quantified by a plaque assay. DTriP-22 inhibited progeny virus production by 79 to 97% when it was added before 2 h p.i. (Fig. 2B). A significant increase in virus yield (to 76%) was observed when the compound was added at 4 h postadsorption. Almost no inhibition occurred when the compound was added after 6 h postadsorption. This result differs from that obtained by adding pyridyl imidazolidinone, a capsid inhibitor of EV71. Pyridyl imidazolidinone exhibited a loss of antiviral activity when it was added to EV71-infected cells later than 1 h p.i. (37). These observations indicate that the molecular target of DTriP-22 differed from that of the capsid binder. The results of the time-of-addition assay with EV71-infected cells supported the involvement of DTriP-22 in viral replication.

DTriP-22 decreased the level of accumulated EV71 RNA. To verify that DTriP-22 inhibits viral replication, we first monitored viral RNA production in EV71-infected cells following DTriP-22 treatment. Virus-infected cells (at an MOI of 1 PFU/ cell) were treated with 2 ␮M DTriP-22 after virus absorption. Intracellular RNA was isolated at different intervals p.i. The amounts of EV71 RNA were measured using both quantitative real-time RT-PCR and slot blot analysis. The results of realtime RT-PCR showed that the presence of 2 ␮M DTriP-22 reduced viral RNA production at 6 to 14 h p.i. by 63% to 87% from that obtained by DMSO treatment at each time point (Fig. 3A). The reduction in the amount of viral RNA due to DTriP-22 treatment was also observed with slot blot analysis (Fig. 3B). Treating infected cells with DTriP-22 reduced yields of both positive- and negative-stranded viral RNA. Also, we monitored the effect of DTriP-22 on IRES-medicated translation. EV71 IRES activity was detected when a dicistronic plasmid with Renilla and firefly luciferase reporter genes was used. No significant decrease in IRES-driven translation was observed in the presence of DTriP-22 (Fig. 3C). These results

FIG. 2. (A) Single step growth curve of EV71. RD cells (6 ⫻ 105 cells/well) were seeded in a six-well plate and incubated for 24 h. RD cells were infected with EV71 at an MOI of 1 PFU/cell. After 1 h of absorption at room temperature, cells were covered with 2 ml medium containing 2% FBS. At the indicated times, total virus yield was quantified by a plaque assay. (B) Time-of-addition assay. The condition of virus infection was the same as that described above. After 1 h of absorption at room temperature, the infected cells were treated with 2 ␮M DTriP-22 at the indicated times (2 h before the end of viral absorption and at 0, 2, 4, 6, 8, 10, 12, and 14 h postabsorption). At 16 h p.i., the culture supernatant and cell lysate were collected together. Total viral yield was measured by a plaque assay. Data are presented as means ⫾ SEM of results from three independent experiments.

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a Data are presented as means ⫾ standard deviations of results from at least two independent experiments. b NT, not tested. c The VP1 V192M mutant is a pyridyl imidazolinone-resistant virus. d Pyridyl imidazolinone is an EV71 inhibitor targeting the VP1 capsid protein.

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FIG. 3. Effect of DTriP-22 on level of accumulated viral RNA. (A) RD cells (6 ⫻ 105 cells/well) were seeded in a six-well plate and incubated for 24 h. RD cells were infected with EV71 at an MOI of 1 PFU/cell. After absorption, the virus-infected cells were treated with or without 2 ␮M DTriP-22. The intracellular viral RNA was isolated at 2, 4, 6, 8, 10, 12, and 14 h p.i. The amount of viral RNA was determined by real-time RT-PCR. The amount of viral RNA at 12 h p.i. in the absence of the compound was set to 100%. The relative amount of viral RNA isolated at each time point is presented as a percentage on the vertical axis. Data are presented as means ⫾ SEM. (B) The condition of the treatment of the infected cells with DTriP-22 was as described above. Cytoplasmic RNA was extracted at the indicated

times. The amounts of positive- and negative-strand viral RNA were detected by slot blot analysis. For the loading control, the total RNA in each test was measured using a ␤-actin probe. The data are from one representative experiment of three. (C) Effect of DTriP-22 on IRES activity of EV71. RD cells were transfected with pRHF-EV71-5⬘UTR or pRHF-EV71-5⬘UTR-AS (antisense EV71 5⬘ UTR) in the presence of DTriP-22 or DMSO. After 48 h posttransfection, the Renilla luciferase (RLuc) and firefly luciferase (FLuc) activities were analyzed. The FLuc/RLuc activity of pRHF-EV71-5⬘UTR without DTriP-22treatment is set to 100%, and the other relative FLuc/RLuc activities are presented. Data are presented as means ⫾ SEM.

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demonstrated that DTriP-22 inhibits EV71 RNA accumulation during virus infection but that it does not reduce IRES-driven translation. DTriP-22 inhibited the poly(U) polymerase activity of recombinant EV71 3D protein. To investigate the effect of DTriP-22 on in vitro EV71 3D polymerase activity, EV71 3D polymerase activity was evaluated by detecting the amount of radiolabeled UMP incorporated into poly(U) RNA in the presence of poly(A) templates and oligo(dT) primers. The recombinant EV71 3D polymerase in the presence of 5% DMSO exhibited an activity of 110 pmol UMP incorporated/ min/␮g. A dose-dependent decrease in polymerase activity was observed in the presence of DTriP-22 (P ⬍ 0.001; one-way analysis of variance) (Fig. 4A). As a negative control, pyridyl imidazolidinone, a capsid inhibitor of EV71, did not inhibit 3D polymerase activity. The experiments were repeated several times, and the results consistently demonstrated that the EV71 3D polymerase activity was reduced in the presence of DTriP22. The activity of the mutant EV71 3D polymerase with a resistance marker (R163K) treated with DTriP-22 was assessed. The mutant polymerase exhibited 28 to 29% more activity than wild-type polymerase in the presence of DTriP-22 at 250 and 500 ␮M (P ⬍ 0.01 and P ⬍ 0.05, respectively; one-way analysis of variance followed by Tukey’s post hoc test) (Fig. 4A). The 3D polymerase of enterovirus has been observed to have another function, VPg uridylylation, in virus-infected cells (41). Therefore, an in vitro uridylylation assay was performed to detect the ability of EV71 polymerase to generate VPgpU(pU) in the presence of DTriP-22. Recombinant 3D polymerase uridylylated VPg in the presence of poly(A) RNA templates (Fig. 4B, lane 3). As a control, the uridylylation activity of 3D polymerase was inhibited in the presence of 0.1 M NaCl (Fig. 4B, lane 7). DTriP-22 could not inhibit the VPg-pU(pU) synthesis at the indicated concentrations (Fig. 4B, upper panel, lanes 4 to 6). However, a capsid inhibitor of EV71, pyridyl imidazolidinone, also did not inhibit the EV71 3D polymerase activity in uridylylating VPg. These results indicate that DTriP-22 may inhibit EV71 3D polymerase activity associated with chain elongation but not that associated with VPg uridylylation. DTriP-22 exhibited broad-spectrum activity against other RNA viruses. Several viral polymerase inhibitors, such as ribavirin, exhibit broad-spectrum activity against various viruses (10, 23, 38). We next examined the activities of DTriP-22 against other viruses by using a neutralization test. DTriP-22 inhibited the cytopathic effects induced by all three genotypes

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TABLE 3. Antiviral activities of DTriP-22 against various viruses EC50 (␮M)a

Virus

EV71 BrCr (genotype A)...............................................................0.15 ⫾ 0.00 1743 (genotype B)................................................................0.44 ⫾ 0.00 2231 (genotype C)................................................................0.98 ⫾ 0.51 4643 (genotype C)................................................................0.13 ⫾ 0.00

Echovirus 9 ...............................................................................1.22 ⫾ 0.21 HRV-2 .......................................................................................1.69 ⫾ 0.95 Influenza A virus (WSN) ........................................................5.98 ⫾ 0.43 Influenza B virus ......................................................................5.90 ⫾ 2.22 HSV-1........................................................................................ ⬎25 HSV-2........................................................................................ ⬎25 a Data are presented as means ⫾ standard deviations of results from at least two independent experiments.

FIG. 4. Effect of DTriP-22 on in vitro EV71 3D polymerase activity. (A) Poly(U) polymerase activity was measured with 1 ␮M polymerase in the reaction buffer as described in Materials and Methods. The percent activity on the vertical axis represents the activity of the tested polymerase (in the presence of the compound) divided by that of the untreated polymerase. Black and white bars represent wild-type (wt) EV71 3D polymerase. Patterned bars represent the R163K mutant polymerase. Data are presented as means ⫾ SEM of results from three independent experiments. The activities of wild-type and mutant 3D polymerase observed in the presence of 250 and 500 ␮M DTriP-22, respectively, are compared. Difference among different groups was assessed by one-way analysis of variance followed by Tukey’s post hoc multiple-comparison test (ⴱⴱⴱ, P ⬍ 0.001 versus the control group; a, P ⬍ 0.05; b, P ⬍ 0.01 versus the same treatment with wild-type polymerase). (B) Effect of DTriP-22 on VPg uridylylation in vitro. Poly(A) RNA was used as a template in the presence of DTriP-22. The reaction conditions for the upper and lower panels are the same, except for the treatment with different compounds in lanes 4 to 6.

of EV71. These three genotypes were those associated with prototype BrCr (genotype A), TW/1743/98 (genotype B), and Tainan/4643/98 (genotype C), with EC50s from 0.13 to 0.44 ␮M (Table 3). DTriP-22 also had strong antiviral activity against other enteroviruses, including coxsackieviruses A and B and echovirus 9 (EC50s of 0.07 to 1.22 ␮M). Additionally, this compound exhibited antiviral activity against HRV-2 (EC50 ⫽ 1.69 ␮M) and influenza viruses A and B, whereas the prototype compound (compound 1) in this series of compounds did not (9). However, DTriP-22 failed to inhibit two DNA viruses, HSV-1 and HSV-2 (EC50s of ⬎25 ␮M). The CC50 values of DTriP-22 for Vero, MDCK, and HeLa cells were all greater than 100 ␮M, according to MTS assays. The results show that DTriP-22 has a broader spectrum than the prototype pyrazolo[3,4-d]pyrimidine-containing compound (compound 1).

DTriP-22, targeting the EV71 3D polymerase, exhibits a broader spectrum of activity against RNA viruses, especially picornaviruses. However, the antiviral mechanisms of DTriP-22 activity against the aforementioned non-EV71 viruses, especially the enveloped viruses, need to be further studied. DISCUSSION In this study, we demonstrated that R163K mutations in EV71 polymerase render the virus resistant to DTriP-22. In the polymerase elongation assay, DTriP-22 affected the R163K mutant polymerase activity significantly less than it affected the wild-type polymerase activity. The sequence alignment of the EV71 polymerase region with other enteroviruses shows that the Arg-163 residue is highly conserved within the Enterovirus genus (present in 99.3% of 136 isolates). The Arg-163 residue is also found in HRV 3D polymerase, which may explain the activity of DTriP-22 against HRV 2. The crystal structure of the poliovirus 3D polymerase revealed that the Arg-163 residue is located in the ring finger domain of the right-hand structure, which contains conserved basic residues (Arg-163, Lys-167, and Arg-174) that interact with the incoming nucleoside triphosphate for chain elongation (43). Therefore, DTriP-22 may interfere with 3D activity by obstructing the nucleoside triphosphate entry cavity of 3D polymerase but not by incorporation into the growing RNA chains. DTriP-22 efficiently reduced the amount of EV71 RNA accumulation in the cell base system. However, DTriP-22 was less effective in inhibiting the in vitro polymerase activity of EV71 3D (Fig. 4A). The amounts of DTrip-22 required to inhibit purified EV71 3D polymerase in vitro were more than 100 times larger than those required to inhibit virus replication in RD cells. One possibility is that DTriP-22 may accumulate

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Coxsackieviruses A9...........................................................................................0.99 ⫾ 0.10 A10.........................................................................................1.06 ⫾ 0.59 A16.........................................................................................0.08 ⫾ 0.01 A24.........................................................................................0.08 ⫾ 0.00 B1 ...........................................................................................0.24 ⫾ 0.15 B2 ...........................................................................................0.07 ⫾ 0.00 B3 ...........................................................................................0.26 ⫾ 0.19 B4 ...........................................................................................0.23 ⫾ 0.02 B5 ...........................................................................................0.13 ⫾ 0.00 B6 ...........................................................................................0.33 ⫾ 0.03

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CHEN ET AL.

rapine and efavirenz (nonnucleoside reverse transcriptase inhibitors) have been reported to combine with other anti-HIV agents in treatment against HIV-1 (36, 40). Several nonnucleoside inhibitors of hepatitis C virus polymerase have also been shown, including benzothiadiazines (12, 42, 44, 45). Although many polymerase inhibitors have been shown to exhibit antienterovirus activity, most are nucleoside analogues (8, 11, 18– 20, 24). Here, we report on a novel nonnucleoside analogue which targets 3D polymerase and may have great potential in the development of a broad-spectrum antienteroviral agent. ACKNOWLEDGMENTS This research was supported by the National Science Council of the Republic of China, Taiwan, under contract no. NSC-94IDP002-1. We thank Yi-Yu Ke for helpful discussions. REFERENCES 1. AbuBakar, S., Y. F. Chan, and S. K. Lam. 2000. Outbreaks of enterovirus 71 infection. N. Engl. J. Med. 342:355–356. 2. AbuBakar, S., H. Y. Chee, M. F. Al-Kobaisi, J. Xiaoshan, K. B. Chua, and S. K. Lam. 1999. Identification of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalomyelitis in Malaysia. Virus Res. 61:1–9. 3. Alexander, J. P., Jr., L. Baden, M. A. Pallansch, and L. J. Anderson. 1994. Enterovirus 71 infections and neurologic disease—United States, 1977–1991. J. Infect. Dis. 169:905–908. 4. Arita, M., T. Wakita, and H. Shimizu. 2008. Characterization of pharmacologically active compounds that inhibit poliovirus and enterovirus 71 infectivity. J. Gen. Virol. 89:2518–2530. 5. Chan, K. P., K. T. Goh, C. Y. Chong, E. S. Teo, G. Lau, and A. E. Ling. 2003. Epidemic hand, foot and mouth disease caused by human enterovirus 71, Singapore. Emerg. Infect. Dis. 9:78–85. 6. Chang, L. Y., L. M. Huang, S. S. Gau, Y. Y. Wu, S. H. Hsia, T. Y. Fan, K. L. Lin, Y. C. Huang, C. Y. Lu, and T. Y. Lin. 2007. Neurodevelopment and cognition in children after enterovirus 71 infection. N. Engl. J. Med. 356: 1226–1234. 7. Chang, L. Y., T. Y. Lin, K. H. Hsu, Y. C. Huang, K. L. Lin, C. Hsueh, S. R. Shih, H. C. Ning, M. S. Hwang, H. S. Wang, and C. Y. Lee. 1999. Clinical features and risk factors of pulmonary oedema after enterovirus-71-related hand, foot, and mouth disease. Lancet 354:1682–1686. 8. Chen, T. C., K. F. Weng, S. C. Chang, J. Y. Lin, P. N. Huang, and S. R. Shih. 2008. Development of antiviral agents for enteroviruses. J. Antimicrob. Chemother. 62:1169–1173. 9. Chern, J. H., K. S. Shia, T. A. Hsu, C. L. Tai, C. C. Lee, Y. C. Lee, C. S. Chang, S. N. Tseng, and S. R. Shih. 2004. Design, synthesis, and structureactivity relationships of pyrazolo[3,4-d]pyrimidines: a novel class of potent enterovirus inhibitors. Bioorg. Med. Chem. Lett. 14:2519–2525. 10. De Clercq, E. 1993. Antiviral agents: characteristic activity spectrum depending on the molecular target with which they interact. Adv. Virus Res. 42:1–55. 11. De Palma, A. M., G. Purstinger, E. Wimmer, A. K. Patick, K. Andries, B. Rombaut, E. De Clercq, and J. Neyts. 2008. Potential use of antiviral agents in polio eradication. Emerg. Infect. Dis. 14:545–551. 12. Di Marco, S., C. Volpari, L. Tomei, S. Altamura, S. Harper, F. Narjes, U. Koch, M. Rowley, R. De Francesco, G. Migliaccio, and A. Carfi. 2005. Interdomain communication in hepatitis C virus polymerase abolished by small molecule inhibitors bound to a novel allosteric site. J. Biol. Chem. 280:29765–29770. 13. Fujimoto, T., M. Chikahira, S. Yoshida, H. Ebira, A. Hasegawa, A. Totsuka, and O. Nishio. 2002. Outbreak of central nervous system disease associated with hand, foot, and mouth disease in Japan during the summer of 2000: detection and molecular epidemiology of enterovirus 71. Microbiol. Immunol. 46:621–627. 14. Furuta, Y., K. Takahashi, M. Kuno-Maekawa, H. Sangawa, S. Uehara, K. Kozaki, N. Nomura, H. Egawa, and K. Shiraki. 2005. Mechanism of action of T-705 against influenza virus. Antimicrob. Agents Chemother. 49:981– 986. 15. Gerber, K., E. Wimmer, and A. V. Paul. 2001. Biochemical and genetic studies of the initiation of human rhinovirus 2 RNA replication: identification of a cis-replicating element in the coding sequence of 2Apro. J. Virol. 75:10979–10990. 16. Gohara, D. W., C. S. Ha, S. Kumar, B. Ghosh, J. J. Arnold, T. J. Wisniewski, and C. E. Cameron. 1999. Production of “authentic” poliovirus RNA-dependent RNA polymerase (3D(pol)) by ubiquitin-protease-mediated cleavage in Escherichia coli. Protein Expr. Purif. 17:128–138. 17. Goodfellow, I., Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W.

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to higher concentrations in cells than in the extracellular environment. Another explanation is that DTriP-22 needs to be metabolized in cells to exhibit full efficacy. A previous report described a similar scenario in which the anti-influenza compound T-705 needed to be modified to T-705RTP (T-705-4ribofuranosyl-5-triphosphate) by cellular kinases, subsequently inhibiting influenza RNA polymerase activity (14). Another possibility is that DTriP-22 may not only inhibit 3D polymerase elongation activity but also interfere with other 3D polymerase-involving cellular functions during viral replication in infected cells. DTriP-22 inhibited EV71 3D polymerase activity associated with chain elongation but not that associated with VPg uridylylation. The possibility that the inability of DTriP-22 to inhibit VPg uridylylation is due to reactions that are not similar to the physiological conditions cannot be excluded. The VPg uridylylation assay employed poly(A) as the template and Mn2⫹ as a cofactor of EV71 3D polymerase. Mn2⫹ in the VPg uridylylation reaction mixture allows for enhanced/exaggerated poliovirus 3D polymerase activity, relative to the levels in reaction mixtures containing Mg2⫹, when poly(A) is used as the template (35). However, Mn2⫹-based conditions allow 3D polymerase-catalyzed VPg uridylylation, which would not occur naturally. Another consideration is that appropriate RNA templates (cis-acting replication elements [CREs]) in the in vitro VPg uridylylation reaction are closer than a poly(A) template to the physiological conditions. The CRE structures are located in the 2C-encoding region of poliovirus, the capsidencoding region of HRV-14 and cardiovirus, the 2A-encoding region of HRV-2, the 5⬘ noncoding region of foot-and-mouthdisease virus, and the 3D-encoding region of hepatitis A virus (15, 17, 30–32, 47). However, the CRE of EV71 has not yet been identified in any biochemical experiment. A stem-loop structure was observed in the 2C region of EV71, with a typical AAACA/G CRE motif in its top loop, with the MFOLD program (data not shown). Biochemical analysis needs to be performed to map the CRE of EV71. When the appropriate RNA template is used under Mg2⫹-based conditions, DTriP-22 may be able to inhibit the VPg uridylylation of EV71 3D polymerase in vitro. The high mutation rate of enteroviruses could result in the emergence of drug-resistant viruses. The present study and our earlier studies have shown that two EV71 inhibitors, DTriP-22 and pyridyl imidazolidinone, target different molecules and may be effective in drug combinations for delaying or preventing the generation of drug-resistant viruses. The results of the time-of-addition assay show that unlike pyridyl imidazolidinone, DTriP-22 acted after virus absorption. Moreover, DTriP-22 efficiently inhibited the viral replication of the VP1 V192M mutant, a pyridyl imidazolidinone-resistant virus (Table 2). However, pyridyl imidazolidinone exhibited great activity in the inhibition of DTriP-22-resistant viruses (Table 2). DTriP-22 had a broader spectrum of antiviral activity than pyridyl imidazolidinone against enteroviruses, which result is consistent with the fact that 3D polymerase is a more conserved target than is that of the capsid protein VP1. Viral polymerases have been considered to be potent targets for drug development. Successful clinical studies of nonnucleoside reverse transcriptase inhibitor-based human immunodeficiency virus (HIV)/AIDS therapies have been reported. Nevi-

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DTriP-22 TARGETS EV71 RNA POLYMERASE