Discovery of a novel antiviral agent targeting the nonstructural protein 4 (nsP4) of chikungunya virus

Virology 505 (2017) 102–112

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Discovery of a novel antiviral agent targeting the nonstructural protein 4 (nsP4) of chikungunya virus

MARK

Yuji Wadaa, Yasuko Orbaa, Michihito Sasakia, Shintaro Kobayashia,1, Michael J. Carrb,c, ⁎ Haruaki Noboria,d, Akihiko Satoa,d, William W. Hallb,c,e, Hirofumi Sawaa,b,e, a

Division of Molecular Pathobiology, Research Center for Zoonosis Control, Hokkaido University, Sapporo, Hokkaido, Japan Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University, Sapporo, Hokkaido, Japan c National Virus Reference Laboratory, University College Dublin, Belfield, Ireland d Discovery Research Laboratory for Core Therapeutic Areas, Shionogi & Co., Ltd., Toyonaka, Osaka, Japan e Global Virus Network, 801 W. Baltimore, MD, United States b

A R T I C L E I N F O

A BS T RAC T

Keywords: Chikungunya virus Antiviral compound RdRp inhibitor

Chikungunya fever (CHIKF) is caused by chikungunya virus (CHIKV) infection which is a re-emerging mosquito-borne zoonosis. At present, there are no approved therapeutics for CHIKF. Herein, we have investigated candidate compounds which can inhibit CHIKV infection. Screening of chemical compound libraries were performed and one candidate, a benzimidazole-related compound designated Compound-A was found to inhibit infection by several CHIKV strains and a Sindbis virus strain at nanomolar concentrations. To investigate the inhibitory mechanism of action, a Compound-A resistant CHIKV (res-CHIKV) was isolated and a key mutation associated with resistance was identified by reverse-genetic recombinant CHIKVs containing amino acid substitutions present in res-CHIKV. These results demonstrated that the target site of Compound-A was the M2295 residue in the nonstructural protein 4 (nsP4), which is located in one of the functional domains of RNA-dependent RNA-polymerase (RdRp). We also confirmed that Compound-A inhibits RdRp function of CHIKV by using CHIKV replicons.

1. Introduction Chikungunya virus (CHIKV) is a plus sense, single-stranded RNA virus belonging to the genus Alphavirus, family Togaviridae. The genome is composed of two open reading flames; one encoding a nonstructural polyprotein which is post-translationally processed to four nonstructural proteins (nsP1-4) that are involved in genome replication, and a second region encoding a structural polyprotein which is also processed and forms viral particles which are composed of a capsid and two envelope proteins (Rupp et al., 2015; Abdelnabi et al., 2015; Thiberville et al., 2013). Chikungunya fever (CHIKF) is a re-emerging mosquito-borne zoonosis caused by CHIKV infection (Pialoux et al., 2007). The major symptoms of CHIKF are an acute febrile illness with arthralgia, myalgia, rash, lymphopenia, thrombocytopenia and gastrointestinal symptoms (Pialoux et al., 2007; Couderc and Lecuit, 2015; Borgherini et al., 2007; Chusri et al., 2014). In severe cases of CHIKF, encephalitis has been reported in neonates and fatal disease can occur although, rare in both the young and the elderly (Couderc and Lecuit, 2015; ⁎

1

Gérardin et al., 2016). The arthralgia is characterized by severe joint pain and can become chronic in significant numbers of patients persisting over years as a rheumatoid-like disorder (Schilte et al., 2013; Javelle et al., 2015). This painful, debilitating arthralgia is a major factor affecting the quality of life in convalescent CHIKF patients (de Andrade et al., 2010). Outbreaks of CHIKF have occurred worldwide since 2004, in Africa, Asia and North, Central and South America (Javelle et al., 2015; Weaver and Forrester, 2015; Nasci, 2014; Raghavendhar et al., 2015; Nagpal et al., 2012; Grandadam et al., 2011; Nunes et al., 2015). It is thought that one factor in the reemergence of CHIKF could be related to viral genome mutations which have resulted in an increased efficiency of viral transmission in different mosquito vectors and/or possibly an increased virulence in humans (Kumar et al., 2014; Tsetsarkin et al., 2014). Currently there are no licensed prophylactic vaccines or chemotherapeutic options available for prevention or treatment (Cardona-Ospina et al., 2015; Krishnamoorthy et al., 2009). Recently, two vaccine candidates have showed promising results in phase I clinical studies; however, the development of antiviral therapeutics is still in early

Corresponding author at: Division of Molecular Pathobiology, Research Center for Zoonosis Control, Hokkaido University, Sapporo, Hokkaido, Japan. E-mail address: [email protected] (H. Sawa). Current address: Laboratory of Public Health, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido, Japan.

http://dx.doi.org/10.1016/j.virol.2017.02.014 Received 9 November 2016; Received in revised form 9 February 2017; Accepted 15 February 2017 Available online 23 February 2017 0042-6822/ © 2017 Elsevier Inc. All rights reserved.

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diluted in DMEM containing 2% FCS in 96-well cell culture plates (50 μl /well). Vero cells containing 3×104 cells (100 μl) were added to each well containing diluted compound solutions and incubated for 1 h. Thereafter, 10 TCID50 (median tissue culture infective dose) of CHIKV or SINV was added into each well and incubated for 3 days (finally 200 μl /well). When the rCHIKV-M2295I which has low growth efficiency was evaluated, the longer incubation period was needed (4 days). The MTT reagent (Sigma-Aldrich, St. Louis, MO, USA, 5 mg/ml) was then added to each well and incubated for 1 h. Culture supernatants (150 μl) were then discarded and 150 μl of formazan dissolution reagent (2-propanol with 10% Triton-X100 and 0.28% HCl) added to each well and incubated overnight at room temperature. The absorbance intensity was measured at a 570 nm wavelength with a 630 nm reference wavelength using a Model 680 microplate reader (Bio-Rad, Hercules, CA, USA). T-705 was used as positive control, as it has been previously reported to inhibit CHIKV infection (Delang et al., 2014). Each assay was performed in duplicate and independently at least 3 times. The 50% effective concentration (EC50), 30% effective concentration (EC30) and 50% cytotoxicity concentration (CC50) were calculated from the results of MTT assay. In the case of the DENVs assays, compounds were serially diluted in DMEM containing 5% FCS in 96-well cell culture plates (50 μl /well). BHK-21 cells containing 2×104 cells (100 μl) were added to each well containing diluted compound solutions and incubated for 1 h. Thereafter, 10 TCID50 of DENVs was added into each well and incubated for 4 days (finally 200 μl/well).

development (Abdelnabi et al., 2015; Chang et al., 2014; Ramsauer et al., 2015). Several studies have suggested that the development of chronic arthralgia may be related to the severity of the symptoms during the acute phase (Chow et al., 2011; Yaseen et al., 2014; Reddy et al., 2014). Therefore, effective antiviral treatment during the acute phase could potentially reduce symptoms not only in the acute but also the chronic phases. Recently, several candidates which can inhibit CHIKV infection have been reported; however, the basis of their inhibitory effects have not been fully elucidated (Abdelnabi et al., 2015). In the present study, we have identified an anti-CHIKV candidate which is effective at nanomolar concentrations. Using whole genome sequencing of resistant clones, reverse genetics and CHIKV replicons, the viral target region was identified in the nsP4 and the anti-CHIKV candidate inhibits the RNA-dependent RNA-polymerase (RdRp) function of CHIKV. 2. Methods 2.1. Cells and viruses Vero cells were obtained from the Japan Health Sciences Foundation (Tokyo, Japan). BHK-21 cells were obtained from the Shionogi & Co., LTD. (Osaka, Japan). HEK-293T cells were obtained from the RIKEN BRC (Tsukuba, Japan). The cells were maintained in Dulbecco's Modified Eagle Medium (DMEM), containing 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 unit/ml penicillin and 100 μg/ml streptomycin. The media for HEK-293T cells was additionally supplied 4,500 mg/L of glucose. Cells were cultured at 37 °C in 5% CO2. CHIKV-SL10571 (AB455494), CHIKV-BaH306, Dengue virus serotype 1 (DENV-1)-hu/PHL/10-07 and DENV-2-hu/INDIA/09–74 strains were kindly provided by Dr. Tomohiko Takasaki (National Institute of Infectious Diseases, Tokyo, Japan) (Lim et al., 2009; Nakao and Hotta, 1973). The both of DENV strains were clinically isolated in Japan. CHIKV-S27 strain (NC004162) was kindly provided by Dr. Kouichi Morita (Institute of Tropical Medicine, Nagasaki, Japan) (Khan et al., 2002). Sindbis virus (SINV)-Ar339 strain was obtained from the American Type Culture Collection (Manassas, VA, USA). All viruses were initially amplified in Vero cells after inoculation at a multiplicity of infection (MOI) of 0.01 until cytopathic effects (CPE) were observed. Supernatants from the CHIKV-infected cells were harvested, cell debris was removed by brief centrifugation and the supernatants stored at −80 °C.

2.5. Evaluation of viral replication efficiency in the presence or absence of Compound-A CHIKV-SL10571 (MOI =0.001) was inoculated onto Vero cells in 24-well cell culture plates and incubated for 1 h to allow virus attachment. After removing virus-containing supernatants, DMEM containing 2% FCS (500 μl) with or without 1 μM of Compound-A was added in each well and incubated for 12, 24 or 36 h. At each time point, the supernatants from the CHIKV-inoculated cells were harvested, cell debris was removed by brief centrifugation and the supernatants stored at −80 °C. Viral titers of the supernatants were calculated by plaque assay. Each assay was performed in duplicate and independently at least 3 times. 2.6. Isolation of Compound-A resistant CHIKV clones Vero cells (3×105 cells) in 12-well cell culture plates were inoculated with CHIKV-SL10571 (MOI =1) in the presence of Compound-A (2 μM). When CPEs were detected by microscopy, the supernatants from the culture (500 μl) were transferred to fresh Vero cells in the presence of Compound-A. Passages of the cells under the same conditions were repeated 12 times in duplicate. After twelve passages, the CHIKVs were cloned by limiting dilution. Briefly, the passaged viruses were serially diluted 10-fold to 10−6 and inoculated onto Vero cells (3×104 cells) in 96-well cell culture plates in the presence of Compound-A (2 μM). The supernatants from the cells with CPE were inoculated onto fresh Vero cells in the presence of Compound-A (2 μM). Finally, Compound-A-resistant viruses were isolated from supernatants of the cell cultures after a total of 14 passages in the presence of Compound-A. The supernatants from CHIKV-SL10571-inoculated cells after 20 passages in the absence of Compound-A were used as controls (passaged-CHIKV).

2.2. Compound libraries Chemical compound libraries were synthesized and provided by the Shionogi & Co., LTD. 2.3. Plaque assay Viral dilutions were inoculated onto Vero cells in 24-well cell culture plates and incubated for 1 h to allow virus attachment. After removal of virus-containing supernatants, the cells were overlaid with culture media containing 1.25% methyl cellulose and incubated for 2 days. Cells were then fixed by buffered-formalin, stained by crystal violet and plaque numbers counted. All of the plaque assays were performed in duplicate. 2.4. Cell viability assays The MTT [3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay was employed to examine cell viability following virus infection using methods as previously described (Delang et al., 2014). In the case of the CHIKVs and SINV, compounds were serially

2.7. Genome sequence analysis Full genome sequence analysis was performed using the Ion PGM system (Life Technologies, Carlsbad, CA, USA) as previously described (Sasaki et al., 2015). Briefly, viral genomic RNA was extracted using the 103

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Table 1 Efficacy and cell toxicity of the chemical compounds against alphaviruses. EC50 (μM)

Compound-A T-705

CC50 (μM)

CHIKV-SL10571

CHIKV-S27

CHIKV-BaH306

SINV-Ar339

0.54 ± 0.08 23.86 ± 1.28

0.98 ± 0.08 67.27 ± 0.94

0.77 ± 0.02 34.38 ± 1.36

0.38 ± 0.29 23.98 ± 3.63

Compound-A

3.70 ± 0.30 > 100

Table 2 Efficacy and cell toxicity of the chemical compounds against flaviviruses. EC50 (μM)

CC50 (μM)

DENV-1 hu/PHL/10-07 Compound-A T-705

DENV-2 hu/INDIA/09–74 > CC50 22.10 ± 4.02

> CC50 18.52 ± 2.02

8.02 ± 0.17 > 100

rCHIKV-SL10571

log10 (PFU / ml)

Fig. 1. Chemical structure of Compound-A. Compound-A possesses a benzimidazole structure.

High Pure Viral RNA kit (Roche Diagnostics, Basel, Switzerland) and reverse transcribed to double-stranded cDNA (ds-cDNA) using random hexamers with tag-sequences using a cDNA Synthesis kit (Takara Bio, Shiga, Japan). Synthesized ds-cDNA was purified by Agencourt AMPure XP (Beckman Coulter, Pasadena, CA, USA) and amplified by the PrimeSTAR Max (Takara Bio) using tag-sequence primers. PCR products were fragmented using the Covaris S2 focused-ultrasonicator (Covaris, Woburn, MA, USA) and used to prepare a 400-base-read library using the Ion Plus Fragment Library kit (Life Technologies) and E-Gel SizeSelect 2% agarose gels (Life Technologies). Emulsion PCR was performed by the Ion PGM Template Hi-Q OT2 400 kit (Life Technologies). Sequencing was performed using the Ion PGM sequencer with the Ion PGM Sequencing 400 kit and Ion 318 Chip V2 (Life Technologies). Data analysis was performed with the CLC Genomics Workbench ver 7.5.1 (CLC bio Japan, Tokyo, Japan). Sanger sequencing was performed using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the Applied Biosystems 3130 and 3130xl Genetic Analyzers (Applied Biosystems). Data analysis was carried out with the GENETYX software v.10 (Genetyx Corporation, Tokyo, Japan).

**

8 ** 6

**

4 2 0 0

12

24

36

h.p.i Fig. 2. Compound-A significantly inhibits CHIKV infection. The replication efficiency of rCHIKV-SL10571 in the presence (black bars) or absence (white bars) of Compound-A was evaluated by plaque assay at 12, 24 and 36 h post infection (h.p.i.). rCHIKV-SL10571 was inoculated at an MOI of 0.01. Growth media containing 1 μM of Compound-A was added after virus attachment. The viral titers are shown (logarithmic scale) on the y-axis. Data represent mean values ± SD of at least three independent experiments. Significance was analysed by the Student's t-test and indicated by asterisks (** p < 0.01). Table 3 Amino acid mutations in res-CHIKV.

2.8. Generation of recombinant CHIKVs (rCHIKVs) by reverse genetics Recombinant CHIKVs (rCHIKVs) were synthesized as previously described (Kümmerer et al., 2012). Briefly, CHIKV-SL10571 genomic RNA was extracted as described above and cDNAs generated with random hexamers and SuperScript III reverse transcriptase (Life Technologies). The viral genome was amplified by PrimeSTAR Max, and the T7 promoter with Not I restriction sites being added at the 5′ and 3′ ends of the viral sequence, respectively. The viral genome was inserted into the pSMART-LCKan vector (Lucigen, Middleton, WI, USA) using the In-Fusion HD cloning kit (Takara Bio). The viral cDNA plasmid was transformed in E. coli HST08 Premium competent cells (Takara Bio), cloned and amplified. Plasmid extraction was performed using the QIAprep Spin Miniprep kit (Qiagen, Valencia, CA, USA). Non-synonymous mutation(s) associated with resistance for Compound-A were introduced by the In-Fusion HD cloning kit (Takara Bio). Extracted plasmids were linearized with Not I and in vitro transcribed with the mMESSAGE mMACHINE T7 transcription kit (Life Technologies). Transcribed RNA was transfected to Vero cells with the TransIT-mRNA transfection kit (Mirus Bio LLC, Madison, WI,

Viral protein

nsP2

nsP3

Nucleotide Residue CHIKV-SL10571 passaged-CHIKV res-CHIKV

3550 1158 Arg – Ser

5347 1757 Met – Ile

nsP4 5486 1804 Ser – Pro

6961 2295 Met – Ile

USA). The supernatants from the RNA-transfected cells were harvested when CPE was observed; thereafter cell debris was removed by brief centrifugation and supernatants stored at −80 °C. 2.9. Competition assays The viral mixtures were prepared to contain equal MOI of rCHIKVs (rCHIKV-SL10571 and rCHIKV-M2295I) and inoculated into Vero cells (1.5×105 cells) in 24-well cell culture plates in the presence or absence of Compound-A (2 μM). When CPE was detected by microscopy, the supernatants were collected. Viral RNAs were extracted from the inoculated viral mixtures and the passaged supernatants and cDNAs generated as described above. Partial genome sequences of CHIKV around M2295 residue were amplified by TaKaRa EX-Taq Hot Start Version (Takara Bio). The sequence at M2295 residue (rCHIKV-SL10571: ATG, rCHIKVM2295I: ATA) was bidirectionally analysed by Sanger sequencing and 104

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Fig. 3. The M2295I mutation in rCHIKVs is related to a decreased susceptibility to Compound-A. The susceptibility of rCHIKVs with amino acid mutation(s) against Compound-A were assessed. The rCHIKVs were inoculated at MOI of 0.01 and cultivated for 12, 24 and 36 h.p.i. in the presence (black bars) or absence (white bars) of Compound-A. Growth media containing 1 μM of Compound-A was added after virus attachment. (A) The rCHIKV-R1158S-M1751I-S1804P-M2295I was generated with all four amino acid mutations detected in the res-CHIKV (Table 3). (B-E) The rCHIKVs with three of four amino acid mutations (rCHIKV-R1158S-M1751I-S1804P, rCHIKV-R1158S-M1751I-M2295I, rCHIKVR1158S-S1804P-M2295I, rCHIKV-M1751I-S1804P-M2295I) were also assessed. (F-H) The rCHIKVs with M2295I and other single amino acid mutations (rCHIKV-R1158S-M2295I, rCHIKV-M1751I-M2295I, rCHIKV-S1804P-M2295I) were assessed. (I-L) The rCHIKVs with single amino acid mutations (rCHIKV-R1158S, rCHIKV-M1751I, rCHIKV-S1804P, rCHIKV-M2295I) were assessed. The viral titers are shown (logarithmic scale) on the y-axis. Data represent mean values ± SD of at least three independent experiments. Significance was analysed by the Student's t-test and indicated by asterisks (* p < 0.05, ** p < 0.01).

(100 μl) with or without 1 μM of Compound-A was added in each well and incubated for 6, 12, 24 or 36 h. At each time point, NanoLuc activities were measured using the Nano-Glo luciferase assay kit (Promega) and GloMax 96 microplate luminometer (Promega). Each assay was performed in duplicate and independently at least 3 times.

mean value of nucleotide proportions from bidirectional sequencing were calculated by the ab1 Peak Reporter (Life Thechnologies: https://apps. lifetechnologies.com/ab1peakreporter). Each assay was performed in duplicate and independently at least 3 times. 2.10. Luciferase assays using CHIKV replicon

2.11. Sequence alignment analysis A CHIKV replicon was constructed as previously described (Gläsker et al., 2013). Genome recombination was performed as described above. Briefly, the sequences encoding structural polyprotein in CHIKV cDNA plasmid, which had been constructed to produce rCHIKV, were replaced by a NanoLuc luciferase sequence derived from pNL2.1 vector (Promega, Madison, WI, USA) using the In-Fusion HD cloning kit. Transformation, extraction of plasmids, in vitro mutagenesis (GDD to GAA and M2295I) and in vitro transcription were performed as described in the generation of rCHIKVs. The transcribed RNA (100 ng) was transfected into HEK-293T cells (5×104 cells), presheeted in Poly-L-Lysine coated-96-well cell culture plates, by the Lipofectamine Messenger MAX (Life Technologies) and incubated for 1 h. After removing replicon RNA-containing supernatants, DMEM containing 10% FCS and 4500 ml/L of glucose

The amino acid sequences of CHIKV-SL10571, CHIKV-S27, CHIKV-BaH306, O'nyong-nyong virus (ONNV: AF079456), Mayaro virus (MAYV: AF237947), Semliki Forest virus (SFV: X04129), Ross River virus (RRV: M20162), Getah virus (GETV: EU015061), Barmah Forest virus (BFV: U73745), Western Equine Encephalitis virus (WEEV: GQ287640), Eastern Equine Encephalitis virus (EEEV: EF151503), Venezuelan Equine Encephalitis virus (VEEV: L01442), Rubella virus (RV: AAB81187), DENV-1 (AAB40695), Hepatitis C virus (HCV: AAA45676), Bovine viral diarrhea virus 1(BVDV: AAA42854) and Polio virus (PV: AAA46910) were downloaded from GenBank for multiple sequence alignments. Multiple alignment analysis was performed by GENETYX software v.10. Since the genomic sequence of CHIKV-BaH306 has not been 105

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Fig. 3. (continued) Table 4 Summary of the relationship between amino acid mutations and susceptibility against Compound-A. Number of mutations

4

3

Viral regions

nsP2 nsP3 nsP4 R1158S M1757I S1804P M2295I

nsP2 nsP3

nsP2 nsP3 nsP4

R1158S M1757I S1804P

R1158S M1757I M2295I

R

S

R

Amino acid mutations

Susceptibility against Compound-A

2

1

nsP3

nsP2

nsP3

nsP4

nsP4

nsP4

R1158S S1804P M2295I

M1757I S1804P M2295I

R1158S M2295I

M1757I M2295I

R

R

R

R

nsP2

nsP3

nsP4

S1804P M2295I

R1158S

M1757I

S1804P

M2295I

R

S

S

S

R

R; resistance, S; sensitive.

3. Results

reported, the partial amino acid sequence of the viral strain was estimated from the genome sequence obtained in our laboratory.

3.1. MTT screening of chemical compound libraries and characterization of the inhibitory effect of an identified chemical compound To identify novel chemical compounds which could inhibit CHIKV infection, we performed MTT screening assays with chemical compound libraries. We optimized the conditions of the MTT assay and confirmed that the EC50 of T-705 was comparable to that previously reported (Table 1) (Delang et al., 2014). The CHIKV-SL10571 strain

2.12. Statistical analysis Statistical significance was calculated by Student's t-test or TukeyKramer test based on One-way ANOVA analysis from at least three independent experiments (*: p < 0.05, **: p < 0.01). 106

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A)

Replication efficiency of rCHIKVs

log10 (PFU / ml)

8

**

6

**

4

**

2 0 0

12

24

36

h.p.i rCHIKV-SL10571 rCHIKV-M1757I rCHIKV-M2295I

Viral growth competition in the absence of Compound-A

C)

Viral growth competition in the presence of Compound-A

100%

100%

80%

80%

Viral population

Viral population

B)

rCHIKV-R1158S rCHIKV-S1804P

60% 40% 20%

60% 40% 20%

0%

0% 0

1

0

Passage time CHIKV-SL10571

1 Passage time

CHIKV-M2295I

CHIKV-SL10571

CHIKV-M2295I

Fig. 4. Selective pressure of Compound-A against rCHIKVs. (A) rCHIKVs were inoculated at an MOI of 0.01 and cultivated for 12, 24 and 36 h.p.i. in the absence of CompoundA. The viral titers are shown (logarithmic scale) on the y-axis. Data represent mean values ± SD of at least three independent experiments. Significance was analysed by the Student's ttest and indicated by asterisks (** p < 0.01). (B and C) The mixture of rCHIKV-SL10571 (lines) and rCHIKV-M2295I (dotted lines) was inoculated into Vero cells in the presence or absence of Compound-A (1 μM). After one time passage, the supernatants were collected and analysed change of proportions of inoculated rCHIKVs by Sanger sequencing. Data represent mean values of bidirectional sequence analysis from three independent experiments.

assessed to evaluate the inhibitory effect of Compound-A using recombinant CHIKV-SL10571 (rCHIKV-SL10571) generated by reverse genetics. Supernatants from the rCHIKV-SL10571-inoculated cells were collected at 12, 24 and 36 h post infection (h.p.i.) and the viral titers were assessed by plaque assay. Compound-A was found to significantly inhibit CHIKV replication at each time point with reductions of viral titers (plaque forming unit: PFU/ml) by 14.5, 81.6 and 75.2 times compared to the untreated controls (Fig. 2).

belonging to the Indian Ocean Lineage (IOL) was used in this assay. We evaluated 63 compounds from the chemical compound library and identified a group of compounds which possessed a benzimidazole structure that inhibited CHIKV infection at low concentrations. Based on this result, we then evaluated 103 compounds which also possessed a benzimidazole central structure with different chemical side chains. We subsequently identified a compound (designated Compound-A) which inhibited CHIKV infection at nanomolar concentrations (Fig. 1 and Table 1). As far as we are aware there are no reports that a compound with a benzimidazole structure would inhibit CHIKV infection at nanomolar concentrations, and as such we focused on Compound-A in further experiments. We performed the MTT assay with two additional CHIKV strains and another alphavirus, Sindbis virus (SINV). CHIKV-S27 and CHIKVBaH306 strains belong to the East/Central/South African (ECSA) linage and the Asian linage, respectively. SINV is a member of the genus Alphavirus, but is classified in a different complex (WEEV complex) from that of CHIKV (SFV complex) (Forrester et al., 2012). Consistent with the prior experiments with CHIKV-SL10571, Compound-A inhibited infection by all three viruses at nanomolar concentrations (Table 1). These findings suggest that Compound-A has the potential to inhibit not only different lineages of CHIKV strains but potentially other alphavirus infections. We also evaluated the inhibitory effect of Compound-A against DENV-1 and -2 belonging to genus Flavivirus, family of Flaviviridae; however, Compound-A did not inhibit infection by these viruses (Table 2). Viral growth efficiency in the presence of Compound-A was

3.2. Viral genome sequence analysis of Compound-A resistant CHIKV clones To investigate the inhibitory effect of Compound-A on CHIKV infection, we attempted to isolate CHIKV clones with reduced susceptibility to Compound-A. In this study, we performed an isolation of Compound-A-resistant CHIKV clones in duplicate, and a total of 12 Compound-A-resistant CHIKV clones (9 clones from one well and 3 clones from another) were analysed by full genome sequence analysis. The complete genome sequence of a control, passaged-CHIKV (CHIKV passaged 20 times in the absence of Compound-A) and the resistant CHIKV clones were analysed. Amino acid mutations which were detected in both the passaged-CHIKV and the resistant CHIKV clones were omitted from the analysis, as it was assumed these were unrelated to resistance to Compound-A. Furthermore, mutations in the resistant CHIKV clones whose frequency was < 20% by deep sequencing were also omitted from the analysis. For further investigation, we focused on 3 resistant CHIKV clones 107

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A)

B)

Genome replication efficiency in wild type CHIKV replicon

7

7

** Log10(Luminescence)

** Log10 (Luminescence)

Genome replication efficiency in M2295I mutant CHIKV replicon

6

5

4

**

6

**

5

4

3

3 6

12

24

6

36

12

24

36

h.p.t.

h.p.t. rep-CHIKV-SL10571 rep-CHIKV-SL10571 with Compound-A rep-CHIKV-SL10571-GAA rep-CHIKV-SL10571-GAA with Compound-A

rep-CHIKV-M2295I rep-CHIKV-M2295I with Compound-A rep-CHIKV-M2295I-GAA rep-CHIKV-M2295I-GAA with Compound-A

Fig. 5. Genome replication efficiency of CHIKV replicon encoding NanoLuc luciferase in the presence or absence of Compound-A. The CHIKV replicons were transfected into HEK-293T cells for 1 h and cultivated for 6, 12, 24 and 36 h.p.t. in the presence or absence of Compound-A. Growth media containing 1 μM of Compound-A was added after RNA transfection and the activity of NanoLuc luciferase produced from CHIKV replicons was measured at each time point. (A and B) The RNAs of rep-CHIKV-SL10571, repCHIKV-SL10571-GAA, rep-CHIKV-M2295I and rep-CHIKV-M2295I-GAA were transfected and cultivated in the presence (lines) or absence (dotted lines) of Compound-A (1 μM). The RdRp function of rep-CHIKV-SL10571-GAA and rep-CHIKV-M2295I-GAA was inactivated by introducing mutations in the Motif C (GDD motif) from GDD to GAA. Data represent mean values ± SD of at least three independent experiments. Significance was analysed by the Student's t-test and indicated by asterisk (* p < 0.05, ** p < 0.01).

Based on these results, we then evaluated the rCHIKVs containing the M2295I in combination with the other amino acid mutations (rCHIKV-R1158S-M2295I, rCHIKV-M1751I-M2295I, rCHIKVS1804P-M2295I) (Fig. 3F–H) and single mutations (rCHIKVR1158S, rCHIKV-M1751I, rCHIKV-S1804P, rCHIKV-M2295I) (Fig. 3I–L). Consistently, only the rCHIKVs which had the M2295I mutation in nsP4 showed no significant difference in viral replication efficiency in the presence or absence of Compound-A. In contrast the rCHIKVs not harboring the M2295I showed significant differences in viral replication efficiency in the presence or absence of Compound-A (Fig. 3F–L). On the basis of these findings, it can be concluded that the M2295I mutation was critical for the inhibitory effect of Compound-A and the target of Compound-A is the M2295 in the viral nonstructural protein, nsP4 (Fig. 3 and Table 4). To further understand the relevance of M2295I mutation in CHIKV and resistance to Compound-A, we additionally analysed the phenotype of rCHIKV-M2295I. The rCHIKV-M2295I exhibited a significant decrease in viral growth efficiency compared to rCHIKV-SL10571, rCHIKV-R1158S, rCHIKV-M1751I and rCHIKV-S1804P in the absence of Compound-A (Fig. 4A). The decrease of viral growth efficiency is certainly a disadvantage for viral survival; however, the res-CHIKV possessing the mutation of the M2295I became dominant in the presence of Compound-A. We also performed competition assays with rCHIKV-SL10571 and rCHIKV-M2295I. In these, rCHIKV-SL10571 became dominant in the absence of Compound-A; on the other hand, the rCHIKV-M2295I became dominant in the presence of CompoundA (Fig. 4B-C). These results suggest that the M2295I mutation is an advantage to overcome the selective pressure of Compound-A and also supports our conclusion that Compound-A targets M2295.

which were isolated from the same single well. These 3 resistant CHIKV clones possessed same set of four amino acid mutations (R1158S in nsP2, M1751I and S1804P in nsP3, and M2295I in nsP4 with frequencies > 98% for each mutation), and we designated the mutant CHIKVs as res-CHIKV (Table 3). These four amino acid mutations in the res-CHIKV were also confirmed by Sanger sequence analysis and were not detected in the untreated passaged-CHIKV control or other 9 resistant CHIKV clones. We next evaluated the replication efficiency of the res-CHIKV in the presence of Compound-A, which revealed that the res-CHIKV showed no significant difference of viral replication efficiency either in the presence or absence of Compound-A (data not shown). Based on the findings, we hypothesized Compound-A targeted a single or a combination of these four amino acid mutations. 3.3. Susceptibility of CHIKV against Compound-A evaluated by reverse-engineered CHIKV To specifically identify the target region(s) in CHIKV of CompoundA, we generated recombinant CHIKVs (rCHIKVs) using reverse genetics containing the amino acid mutation(s) detected in the resCHIKV. Firstly, we examined the susceptibility to Compound-A of a rCHIKV containing all four amino acid mutations in the nsPs (rCHIKVR1158S-M1751I-S1804P-M2295I). Consistent with the analysis using the res-CHIKV, the rCHIKV-R1158S-M1751I-S1804P-M2295I showed no significant decrease in viral replication efficiency in the presence of Compound-A (Fig. 3A). Next, we evaluated the susceptibility of rCHIKVs which had three of four introduced mutations (rCHIKV-R1158S-M1751I-S1804P, rCHIKV-R1158S-M1751I-M2295I, rCHIKV-R1158S-S1804P-M2295I and rCHIKV-M1751I-S1804P-M2295I) associated with reduced susceptibility to Compound-A. Notably, each rCHIKV which had the M2295I mutation introduced showed no significant difference in replication efficiency in the presence or absence of Compound-A, although the rCHIKV without the M2295I mutation had significant inhibition of viral replication in the presence of Compound-A (Fig. 3B– E).

3.4. CHIKV genome replication efficiency in the presence of Compound-A evaluated by a CHIKV replicon Generally, it is well known that the main role of the alphavirus nsP4 is RdRp which regulates viral genome replication. Therefore, we assessed whether Compound-A can inhibit viral genome replication 108

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A)

Motif B (SGxxxT)

CHIKV-SL10571 CHIKV-S27 CHIKV-BaH306 ONNV MAYV SFV RRV GETV BFV SINV WEEV EEEV VEEV

2245 2245 2245 2284 2208 2199 2251 2238 2181 2284 2242 2254 2267

SLALTALMLLEDLGVDHSLL .................... .................... ......M.........QPI. .....G..I.......NQ.. .....G..I.......QY.. ................QE.. ................QE.. .M......I.......QN.M AM...G..I.......QP.. AI.IS...I.......QP.. AI..S...I.......QA.. AM......I.......AE..

CHIKV-SL10571 CHIKV-S27 CHIKV-BaH306 ONNV MAYV SFV RRV GETV BFV SINV WEEV EEEV VEEV

2305 2305 2305 2344 2268 2259 2311 2298 2241 2344 2302 2314 2327

LNITIASRVLEDRLTKSACA .................... .................... ...........E...T.... ...........A...N.... ...........Q...D.... ...V..C...REK..N.I.. ...V..C...R.K.SS.... ..VV..C.....Q.AQ.PWP ..VV.......E..KT.R.. V..M......RE...T.... V..M......RE...N.P.. I..V......RE...G.P..

DLIEAAFGEISSCHLPTGTR .................... .................... .................... ........Q.T......... .................... ....E.....T.V....... ..........T.V....... N.........V.T....... ....C............... ........N.T.V....... ........D.T.V....... T...........I....K.K

FKFGAMMKSGMFLTLFVNTL .................... .................... .................... ................I..V ................I..V ................I... ................I... ...................I ...................V .................... ................I..V ...................V

2304 2304 2304 2343 2267 2258 2310 2297 2240 2343 2301 2313 2326

RCATWMNMEVKIIDAVVSLK .................... .................... ..................E. .....V..........MCI. ...S.V..........MGE. ...S.V.........TMCE. ...S.V.........TMCE. ............L.SI.GIR .....L..........IGER .....L..........IGI. .....L...........G.. .....L...........GE.

2364 2364 2364 2403 2327 2318 2370 2357 2300 2403 2361 2373 2386

Motif C (GDD)

AFIGDDNIIHGVVSDELMAA .................... .................... ...............A.... .......VV......K...D ........V...I..K...E ........V...R..P...E ........V...R..P...E ...........II..K...D ...............KE..E ........V......T...E ........VK..K..A...D ........VK..K..K...D

B)

Motif B (SGxxxT)

CHIKV RV DENV-1 HCV BVDV PV

2245 1885 3045 2654 3626 1988

SLALTALMLLEDLGVDHSLL TLATRDVELEISAALLGLPC ITDIMEPEHALLATSIFKLT RTEEAIYQCCDLDPQARVAI DLQLIGEIQKYYYKKEWHKF SLSPAWFEALKMVLEKIGFG

DLIEAAFGEISSCHLPTGTR AEDYRALRAGSYCTLRELGS YQNKVVRVQRPAKNGTVMDV KSLTERLYVGGPLTNSRGEN IDTITDHMTEVPVITADGEV DRVDYIDYLNHSHHLYKNKT

CHIKV RV DENV-1 HCV BVDV PV

2305 1945 3105 2714 3686 2048

LNITIASRVLEDRLTKSACA VAMCMAMRMVPKGVRWAGIF TNMEAQLIRQMESEGIFSPS TCYIKARAACRAAGLQDCTM LNVLTMMYGFCESTGVPYKS INNLIIRTLLLKTYKGIDLD

AFIGDDNIIHGVVSDELMAA QGDDMVIFLPEGARSAALKW ELETPNLAERVLDWLKKHGT LVCGDDLVVICESAGVQEDA FNRVARIHVCGDDGFLITEK HLKMIAYGDDVIASYPHEVD

FKFGAMMKSGMFLTLFVNTL TETGCERTSGEPATLLHNTT ISRRDQRGSGQVGTYGLNTF CGYRRCRASGVLTTSCGNTL YIRNGQRGSGQPDTSAGNSM YCVKGGMPSGCSGTSIFNSM

2304 1944 3104 2713 3685 2047

RCATWMNMEVKIIDAVVSLK TPAEVGLFGFHIPVKHVSTP ERLKRMAISGDDCVVKPIDD ASLRAFTEAMTRYSAPPGDP GLGLKFANKGMQILHEAGKP ASLLAQSGKDYGLTMTPADK

2364 2004 3164 2773 3745 2107

Motif C (GDD)

Fig. 6. Sequence alignment analyses of viral nsP4. (A) Multiple alignment analysis among the genus Alphavirus. The conserved sequences of motif B and C in the nsP4 of CHIKV strains and other representative alphavirus members are shown as bold and underlined characters. The M2295 in CHIKV and its corresponding amino acid residues in other alphaviruses are highlighted in gray. Each dot of other viruses represents identical amino acid residues as those of CHIKV-SL10571. ONNV; O'nyong-nyong virus. MAYV; Mayaro virus. SFV; Semliki Forest virus. RRV; Ross River virus. GETV; Getah virus. BFV; Barmah Forest virus. SINV; Sindbis virus. WEEV; Western Equine Encephalitis virus. EEEV; Eastern Equine Encephalitis virus. VEEV; Venezuelan Equine Encephalitis virus. (B) Sequence alignment comparing residues adjacent to motif B in the nsP4 RdRp with other plus/positive-strand RNA viruses (rubivirus, flavivirus, hepacivirus, pestivirus and enterovirus). The conserved sequences of motif B and C are shown as bold and underlined characters. The M2295 in CHIKV and its corresponding amino acid residues of other positive-sense RNA viruses are highlighted in gray. RV; Rubella virus. DENV-1; Dengue virus serotype 1. HCV; Hepatitis C virus. BVDV; Bovine viral diarrhea virus 1. PV; Polio virus.

as the GDD motif (rep-CHIKV-SL10571-GAA). At 6, 12, 24 and 36 h post transfection (h.p.t.) of these replicons, NanoLuc activities were measured using the cell lysates. Genome replication of rep-CHIKVSL10571 was observed from 12 h.p.t. and found to be significantly inhibited in the presence of Compound-A at 24 and 36 h.p.t. with reductions of luminescence by 106.0 and 216.7 times compared to that in the absence of Compound-A (Fig. 5A). The NanoLuc activity of the rep-CHIKV-SL10571-GAA was stable from 6 to 36 h.p.t., therefore, this demonstrated that the RdRp function of rep-CHIKV-SL10571GAA was precisely inactivated and the RNAs of rep-CHIKV-SL10571-

by using CHIKV encoding NanoLuc luciferase whose activity represents replication of the virus. To evaluate the replication efficiency of CHIKV in the presence of Compound-A, we constructed a wild type replicon and a RdRpinactivated replicon. The sequence alignment of the wild type replicon is based on CHIKV-SL10571, but sequences encoding structural polyprotein were replaced by the sequences encoding NanoLuc luciferase (rep-CHIKV-SL10571). The RdRp-inactivated replicon had introduced amino acid mutations from GDD to GAA in the motif C of repCHIKV-SL10571, which is the catalytic core of RdRp and also known

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other RNA viruses (rubivirus, flavivirus, hepacivirus, pestivirus and enterovirus). This analysis revealed that M2295 is located in motif B (SGxxxT), which is one of the functional domains of the viral RdRp coding region (Fig. 6) (Shatskaya and Dmitrieva, 2013). The M2295 and its adjacent amino acids were highly conserved throughout the genus alphavirus; however, these were not conserved in other RNA viruses except for motif B (Fig. 6). The R1158, M1751, S1804 and their surrounding sequences were also not seen to be conserved in the alphaviruses (data not shown). These multiple sequence alignments further support our conclusion that Compound-A inhibits RdRp function of CHIKV in vitro by targeting the M2295 residue in the nsP4.

Compound-B

Compound-C

3.6. Sensitivity of rCHIKV-M2295I against benzimidazole compounds To investigate whether anti-CHIKV activity targeting M2295 of Compound-A was specific or not, we evaluated sensitivity of rCHIKVM2295I against the other compounds which have similar benzimidazole structure as Compound-A. Two of the benzimidazole compounds that inhibited CHIKV-SL10571 infection at a relatively low concentration (designated Compound-B and -C), and a third benzimidazole compound that has almost the same structure as Compound-A (designated Compound-D) were evaluated by the MTT assay (Fig. 7 and Table 5). Firstly, we evaluated inhibitory effect of Compound-A and T-705 (as an assay control) against rCHIKV-M2295I (Table 6). The EC50 value against rCHIKV-M2295I for Compound-A was 2.39 times higher than that against CHIKV-SL10571 even though that for T-705 was slightly (0.80 times) decreased. In this condition, inhibitory effects of Compound-B, -C, and -D against rCHIKV-M2295I were evaluated (Fig. 7 and Table 5). As a result, the EC50 or EC30 values (depending on the compound) for Compound-B, -C, and -D against rCHIKV-M2295I were similar to or lower than the corresponding EC50 or EC30 values against CHIKV-SL10571 (0.99, 0.82 and 0.43 times, respectively). From these observations, we conclude that the anti-CHIKV activity targeting M2295 is specific for Compound-A rather than broadly present in compounds containing a benzimidazole structure similar to that of Compound-A, such as Compound-B, -C, and -D. In addition, these results also support our conclusion that the M2295I mutation is critical to the anti-CHIKV activity of Compound-A.

Compound-D

*

Fig. 7. Chemical structure of Compound-B, -C, and-D. Compound-B, -C, and -D each possess a benzimidazole structure. An asterisk indicates a different structure from a 2-Pyrrolidone of Compound-A at the terminal region.

GAA were translated until 6 h.p.t.. In addition, genome translation efficiency of rep-CHIKV-SL10571-GAA was not affected by CompoundA. From these observations, it was confirmed that Compound-A significantly inhibited CHIKV genome replication, but did not inhibit genome translation. We also analysed the replicons with the introduced M2295I mutation in rep-CHIKV-SL10571 or rep-CHIKV-SL10571-GAA (repCHIKV-M2295I and rep-CHIKV-M2295I-GAA). Notably, the genome replication of rep-CHIKV-M2295I was significantly inhibited at 24 and 36 h.p.t. in the presence of Compound-A; however, the reductions of luminescence were quite different from that of rep-CHIKV-SL10571, being only 4.5 and 5.1 times at each time point compared to the luminescence in the absence of Compound-A (Fig. 5B). From this analysis, we confirmed that the M2295I mutation is critical for resistance to the inhibitory effect of Compound-A. Taken together, we conclude that Compound-A inhibits the RdRp function of CHIKV targeting to M2295 at nsP4 but does not inhibit genome translation.

4. Discussion CHIKV is associated with significant acute and chronic morbidity in endemic areas, and as such the development of CHIKV antiviral therapeutics is of great importance. In the present study, we performed screening of chemical compound libraries and discovered a novel antiCHIKV candidate which we refer to as Compound-A. This is a benzimidazole-related molecule and can inhibit CHIKV infection in vitro at nanomolar concentrations. We also specifically identified the viral target of Compound-A by using Compound-A resistant CHIKV clones and rCHIKVs synthesized by reverse engineering and also demonstrated the mechanism of action of Compound-A as a RdRp inhibitor by using CHIKV replicons. Compound-A inhibited CHIKV-SL10571, CHIKV-S27 and CHIKVBaH306 strains (Table 1). CHIKV-SL10571 strain is classified as IOL

3.5. nsP4 amino acid sequence analysis The consensus of amino acid sequence frequencies around M2295 in nsP4 was assessed by aligning alphaviral nsP4 proteins together with Table 5 Efficacy and cell toxicity of the chemical compounds against CHIKV-SL10571.

EC50 (μM) EC30 (μM) CC50 (μM)

Compound-A

Compound-B

Compound-C

Compound-D

T-705

0.54 ± 0.08 NT 3.70 ± 0.32

2.38 ± 0.80 NT > 10

2.11 ± 1.08 NT 5.52 ± 1.48

ND 2.18 ± 0.75 > 10

23.86 ± 1.28 NT > 100

ND: Not Detected, NT: Not Tested.

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Table 6 Efficacy and cell toxicity of the chemical compounds against rCHIKV-M2295I.

EC50 (μM) EC30 (μM) CC50 (μM)

Compound-A

Compound-B

Compound-C

Compound-D

T-705

1.29 ± 0.51 NT 5.84 ± 0.17

2.38 ± 0.29 NT > 10

1.73 ± 0.39 NT 4.31 ± 0.30

1.37 ± 0.18 0.93 ± 0.01 > 10

19.32 ± 2.80 NT > 100

NT: Not Tested.

all alphavirus infections; therefore, the broad spectrum of antialphaviral activity exhibited by Compound-A is important. In this study, we identified that the inhibitory mechanism of action of Compound-A is a RdRp inhibitor targeting M2295 in motif B. To our knowledge, this is the first report of a compound which targets CHIKV RdRp with the exception of certain nucleotide analogues (Delang et al., 2014; Briolant et al., 2004). It is unlikely that Compound-A operates as nucleotide analog, because the chemical structure of Compound-A is completely different (Delang et al., 2014; Rada and Dragún, 1977). Notably, Compound-A did not inhibit infections by Dengue virus. It is known that ribo-nucleotide analogues inhibit infection by a broad range of RNA viruses (Briolant et al., 2004; Crance et al., 2003; Gowen et al., 2007; Furuta et al., 2009), and that the RdRp fundamentally forms similar structures in other RNA viruses even if the sequence alignment is very different (Shatskaya and Dmitrieva, 2013; Koonin, 1991; Rupp et al., 2011). In other RNA virus analyses motif B plays a role in ribo- or deoxyribo-nucleotide selection as synthetic substrates and RNA synthesis cooperating with motif C (known as the GDD motif) which is the catalytic core of RdRp and is located near to the motif B (Shatskaya and Dmitrieva, 2013; Gohara et al., 2000; Sesmero and Thorpe, 2015). Accordingly, it could be possible that the M2295 residue might be located in the active site of the CHIKV RdRp and Compound-A might disrupt the vital function of RdRp by inhibiting ribo-nucleotide selection. However, we could not demonstrate that Compound-A directly binds M2295 and inhibits the function of motif B due to a lack of information on the RdRp structure in the genus Alphavirus. We believe that Compound-A could contribute to development of novel candidates targeting alphaviral RdRp with a mechanism of action which is different from nucleotide analogues. In summary, CompoundA could contribute to progress in the development of therapeutic strategies for CHIKF or potentially other diseases caused by pathogenic alphaviruses, and such studies would also contribute to basic research on the replication of CHIKV.

and is derived from the ECSA lineage and representative of recently circulating CHIKV strains endemic in Asia and Africa (Weaver and Forrester, 2015; Casal et al., 2015). CHIKV-S27 and CHIKV-BaH306 strains are classified as classical ECSA lineage and Asian lineages, respectively (Weaver and Forrester, 2015; Nakao and Hotta, 1973; Khan et al., 2002). The ECSA and Asian lineages of CHIKV are now endemic in North, Central and South America (Nasci, 2014; Nunes et al., 2015; Stapleford et al., 2016). Therefore, it can be expected that Compound-A should inhibit CHIKV strains worldwide. In our study, the results also suggest that the inhibitory effect of Compound-A was specific for CHIKV as this failed to inhibit DENV infection. Unfortunately, the safety index (CC50 / EC50) of Compound-A was not as high when compared to other approved antivirals (Table 1) and as such it may be necessary for this to be chemically modified to address this (Delang et al., 2014; Briolant et al., 2004). Our assessment that analysed sensitivity of rCHIKV-M2295I would be useful to consider the strategy for chemical modification of Compound-A (Tables 5 and 6). Especially, the structure of Compound-D is the same as that of Compound-A except for a 2Pyrrolidone at the terminal region (Fig. 7: asterisk). However, the antiCHIKV effect of Compound-D against CHIKV-SL10571 was much weaker than that of Compound-A, and we were unable to calculate EC50 value against CHIKV-SL10571 for Compound-D, even though the EC50 values of both Compound-A and -D against rCHIKV-M2295I were similar (Tables 5 and 6). These results suggest that the 2Pyrrolidone at the terminal region of the benzimidazole structure is important for anti-CHIKV activity. Furthermore, the EC50 values against CHIKV-SL10571 of Compound-B and -C, which have side branches that differ from those in Compound-A, were different from that of Compound-A. Based on these data, we suppose that modification of the structure of the branches in Compound-A may improve its selectivity index. The present studies suggest that Compound-A has the potential to contribute to future outbreaks of CHIKF as a preemptive measure. Previous studies have suggested that CHIKV genome mutations are associated with adaptation to new mosquito vector species and/or to an increase in viral pathogenicity (Kumar et al., 2014; Tsetsarkin et al., 2014; Stapleford et al., 2016). As CHIKV evolves and adapts further, it is possible that additional mutations will arise which may cause an increase efficiency of viral replication and/or viral transmission. Importantly, the nsP4 residue M2295 is located in the highly conserved functional motif B (Fig. 6) and therefore, it is less likely that mutations might be introduced at this site in a natural situation. This is supported by our findings which assessed the viral growth efficiency and competition of rCHIKVs. The rCHIKV-M2295I showed significant decrease of the viral growth efficiency and became minor population compared to rCHIKV-SL10571 in the absence of Compound-A (Fig. 4). If M2295I was accidentally introduced in the CHIKV genome, this virus would probably be eliminated due to its low replication ability. Compound-A also inhibited SINV infection at nanomolar concentrations (Table 1). SINV is classified within the WEEV complex in the genus Alphavirus and is distinct from the SFV complex to which CHIKV belongs (Forrester et al., 2012). The M2295 residue is conserved in the genus Alphavirus and as such Compound-A may have the potential to inhibit other pathogenic alphaviruses. There are currently no approved chemotherapeutic options for the treatment of

Acknowledgments This work was supported in part by grants for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Science, Sports and Technology (MEXT) of Japan (16H06429, 16H06431, 16K21723); grants from the Ministry of Education, Culture, Sports, Science and Technology; the Ministry of Health, Labour and Welfare, Japan (MEXT)/JSPS KAKENHI (16H05805); and the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID) from Japan Agency for Medical Research and Development, AMED. We greatly appreciate Dr. Tomohiko Takasaki (National Institute of Infectious Diseases, Tokyo, Japan) and Dr. Kouichi Morita (Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan) for giving CHIKV strains to us. References Abdelnabi, R., Neyts, J., Delang, L., 2015. Towards antivirals against chikungunya virus. Antivir. Res. 121, 59–68. http://dx.doi.org/10.1016/j.antiviral.2015.06.017, (PubMed PMID: 26119058). de Andrade, D.C., Jean, S., Clavelou, P., Dallel, R., Bouhassira, D., 2010. Chronic pain associated with the Chikungunya Fever: long lasting burden of an acute illness. BMC Infect. Dis. 10, 31. http://dx.doi.org/10.1186/1471-2334-10-31, (PubMed PMID:

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