A novel flavanone derivative inhibits dengue virus fusion and infectivity

Antiviral Research 151 (2018) 27–38

Contents lists available at ScienceDirect

Antiviral Research journal homepage: www.elsevier.com/locate/antiviral

A novel flavanone derivative inhibits dengue virus fusion and infectivity a,1

a,1

b

T

a

Pimsiri Srivarangkul , Wanchalerm Yuttithamnon , Aphinya Suroengrit , Saran Pankaew , Kowit Hengphasatpornc, Thanyada Rungrotmongkolc,d, Preecha Phuwapriasirisane, Kiat Ruxrungthamf, Siwaporn Boonyasuppayakornf,g,∗ a

Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand Graduate Program, Faculty of Medicine, Chulalongkorn University, Bangkok, 10330, Thailand c Bioinformatics and Computational Biology Program, Graduated School, Chulalongkorn University, Bangkok, 10330, Thailand d Structural and Computational Biology Research Group, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand e Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand f Chula Vaccine Research Center (Chula VRC), Faculty of Medicine, Chulalongkorn University, Bangkok, 10330, Thailand g Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, 10330, Thailand b

A R T I C L E I N F O

A B S T R A C T

Keywords: Flavanone Dengue Envelope Drug discovery Flavivirus

Dengue infection is a global burden affecting millions of world population. Previous studies indicated that flavanones were potential dengue virus inhibitors. We discovered that a novel flavanone derivative, 5-hydroxy7-methoxy-6-methylflavanone (FN5Y), inhibited DENV2 pH-dependent fusion in cell-based system with strong binding efficiency to DENV envelope protein at K (P83, L107, K128, L198), K' (T48, E49, A50, L198, Q200, L277), X' (Y138, V354, I357), and Y' (V97, R99, N103, K246) by molecular dynamic simulation. FN5Y inhibited DENV2 infectivity with EC50s (and selectivity index) of 15.99 ± 5.38 (> 6.25), and 12.31 ± 1.64 (2.23) μM in LLC/MK2 and Vero cell lines, respectively, and inhibited DENV4 at 11.70 ± 6.04 (> 8.55) μM. CC50s in LLC/ MK2, HEK-293, and HepG2 cell lines at 72 h were higher than 100 μM. Time-of-addition study revealed that the maximal efficacy was achieved at early after infection corresponded with pH-dependent fusion. Inactivating the viral particle, interfering with cellular receptors, inhibiting viral protease, or the virus replication complex were not major targets of this compound. FN5Y could become a potent anti-flaviviral drug and can be structurally modified for higher potency using simulation to DENV envelope as a molecular target.

1. Introduction Dengue virus causes a global burden with 390 million infections per year (Bhatt et al., 2013). This viral hemorrhagic fever was first emerged in South East Asia in 1970s and has become a regional public health burdens (Ooi and Gubler, 2009). Moreover, a rapid increase by 30-fold to all tropical regions within the recent decade was statistically shown (WHO, 2012). Dengue virus is a member of the family Flaviviridae consisting of 4 serotypes (DENV1-4). The genome is a single stranded positive sense RNA with a guanosine cap but no poly-A tail (Lindenbach et al., 2007). The transmission occurred by infected Aedes aegypti and Aedes albopictus mosquitoes injecting the virus to human while taking blood meal. The virus infected dendritic cells and was carried into circulatory system causing systemic infection. Severe dengue is a pathological condition with plasma leakage, internal bleeding, or multiple organ failure, hypovolemic shock, and death. Those serious

∗

1

pathological manifestations are caused by secondary heterotypic infection that the immune system misdirectedly responds to the previously infected serotype (WHO, 2009). Cellular and humoral immunemediated responses create excessive cytokine production, endothelial damage, and coagulopathy which leads to vascular leakage, hemorrhage and shock. A vaccine (Dengvaxia, CYD-TDV) is currently available but the efficacy against DENV1 and 2 were still limited (Vannice et al., 2017). This vaccine is recommended only in endemic population at the age group of 9–45 years old (WHO, 2016). Besides preventive vaccines, therapeutic drugs were extensively developed in order to alleviate the clinical severity of infected individuals. Cumulative evidences suggested the level of viral load was associated with progression to severe dengue (Libraty et al., 2002; Pozo-Aguilar et al., 2014; Wang et al., 2003); therefore, a small molecule that inhibits viral replication should reduce the viral load, and eventually prevent the disease progression

Corresponding author. Department of Microbiology, Faculty of Medicine, Chulalongkorn University,1873 Rama 4 Road, Pathumwan, Bangkok, 10330, Thailand. E-mail address: [email protected] (S. Boonyasuppayakorn). These authors contributed equally to this work.

https://doi.org/10.1016/j.antiviral.2018.01.010 Received 13 June 2017; Received in revised form 10 October 2017; Accepted 17 January 2018 0166-3542/ © 2018 Elsevier B.V. All rights reserved.

Antiviral Research 151 (2018) 27–38

P. Srivarangkul et al.

prepared to 6–10 different concentrations in filter-sterilized dimethylsulfoxide (Merck®, Darmstadt, Germany) before addition to the cells. Plates were incubated for 48, 72, or 120 h as indicated in the experiment. MTS reagent (Promega®, Madison, USA) was added to cells according to manufacturer's protocol and incubated for 4 h before analysis by spectrophotometry at A450nm. Each compound was tested in triplicate. Cytotoxic concentrations (CC50) were calculated using nonlinear regression analysis and the results were reported as means and standard deviation of three independent experiments.

(reviewed by Lim et al. (2013)). Dengue drug discovery was extensively studied in recent decades since it has acquired technological advancements of target identification, screening, and validation (Lim et al., 2013). Flavonoid derivatives are potential inhibitors to flaviviral replications (Frabasile et al., 2017) (Allard et al., 2011; Du et al., 2016; Johari et al., 2012; Moghaddam et al., 2014; Sanchez et al., 2000; Zandi et al., 2011; Zhang et al., 2012). Previous reports suggested potential molecular targets of the compounds were at flaviviral protease (de Sousa et al., 2015) (Senthilvel et al., 2013), RdRP (Coulerie et al., 2012, 2013) and envelope (Ismail and Jusoh, 2016). Moreover, our preliminary results showed that flavanone derivatives were potentially inhibited DENV2 infectivity analyzed by plaque assay (Boonyasuppayakorn et al., 2016). In this study, we verified the previous findings and characterized a flavanone derivative, 5-hydroxy-7-methoxy-6-methylflavanone, as a dengue virus fusion inhibitor blocking DENV envelope protein to perform dimer to trimer conformational change.

2.4. Effective concentration (EC50) test LLC/MK2 at 5 × 104 cells per well were seeded into 24-well plate in growth medium and incubated overnight at 37 °C under 5% CO2. Cells were infected with DENV2 at the multiplicity of infection (MOI) of 0.1 for 1 h with gentle rocking every 15 min. Cells were washed with PBS and incubated with MEM supplemented with 1% fetal bovine serum, 100 I.U./ml penicillin, and 100 μg/ml streptomycin. The compound was added to the virus-infected cells during and after infection. Cells were incubated for 72 h, unless otherwise indicated, at 37 °C under 5% CO2. Supernatants were collected and the viral infectivity were analyzed by 96-well plaque titration (Boonyasuppayakorn et al., 2016). Data were plotted and the EC50 values were calculated by nonlinear regression analysis. Each concentration was tested in duplicate and the results were reported as means and standard deviation of three independent experiments. Selectivity index was calculated from the ratio of CC50 and EC50.

2. Materials and methods 2.1. Cells and viruses LLC/MK2 (ATCC® CCL-7), and C6/36 (ATCC® CRL-1660) cell lines were maintained in minimal essential medium (Gibco®, Langley, USA) supplemented with 10% fetal bovine serum (Gibco®, Langley, USA), 100 I.U./ml penicillin (Bio Basic Canada®, Ontario, Canada), and 100 μg/ml streptomycin (Bio Basic Canada®, Ontario, Canada), and 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (Sigma Aldrich®, St. Louis, USA) at 37 °C under 5% CO2 for LLC/MK2 and 28 °C for C6/ 36 cell line. Vero (ATCC® CCL-81) was maintained in Medium 199 (Gibco®, Langley, USA) supplemented with 10% fetal bovine serum, 100 I.U./ml penicillin, and 100 μg/ml streptomycin, and 10 mM HEPES at 37 °C under 5% CO2. HEK-293 (ATCC® CRL-1573) and HepG2 (ATCC® HB-8065) were maintained in DMEM (Gibco®, Langley, USA) supplemented with 10% fetal bovine serum, 100 I.U./ml penicillin, and 100 μg/ml streptomycin, and 10 mM HEPES at 37 °C under 5% CO2. Reference strains of DENV2 (New Guinea C strain, NGC), and DENV4 (c0036) were propagated in Vero cell line with minimal essential medium supplemented with 1% fetal bovine serum, 100 I.U./ml penicillin, and 100 μg/ml streptomycin at 37 °C under 5% CO2.

2.5. Time of drug addition study LLC/MK2 cells were seeded into 24-well plates and incubated as previously described. Cells were then infected with DENV2 at the M.O.I. of 0.1 for 1 h with gentle rocking every 15 min. FN5Y at 25 μM was added at the following conditions; (i) 1 h prior to DENV infection; (ii) during DENV infection; or (iii) after DENV infection at 1, 2, 4, 8, 12, 24, 48, and 72 h. Supernatants and cells were collected at 72 h post infection to determine the viral titers by plaque titration and RT-qPCR, respectively. RT-qPCR was performed by total intracellular RNAs extraction using TRIzol reagent (Invitrogen™ Carlsbad, USA) and purification using Direct-zol™ RNA MiniPrep Kit (Zymo Research, Irvine, USA). The RT-qPCR was performed with a Step-One Plus RealTime PCR System (Applied Biosystem, Foster City, CA, USA) with 1 × Power SYBRGreen PCR Master Mix (Applied Biosystem, Foster City, CA, USA), 50 μM each of DN-F and DN-R primers (Shu et al., 2003) under the following condition. Each reaction mixture at 20 μl contains 5 μl of sample RNA, 50 nM of each primer, and 1 μg of total RNA. The reactions were then cycled at 48 °C for 30 min and 95 °C for 10 min, followed by 45 cycles of 95 °C for 20s (denaturation), 55 °C for 30s (annealing), 72 °C for 30s (extension). Each sample was analyzed in triplicated and results were confirmed by three independent experiments.

2.2. Isolation of natural flavonoids Natural flavonoids in this experiment were isolated and purified from the leaves of rose apple, Syzygium samarangense, collected from Nakhonratchasima, Thailand, in October 2011. The dried leaves (214 g) were extracted (2 × 2L) at 80 °C for 3 h. the aqueous extract was partitioned with EtOAc to afford the organic extract. The EtOAc extract was fractionated with silica gel flash column chromatography eluted with CH2Cl2, MeOH-CH2Cl2 (5:95, 15:85 and 3:7) MeOH-CH2Cl2 and MeOH, thus yielding 5 combined fractions. Fraction 1 was triturated with hot hexane to afford hexane soluble fraction and insoluble solid. The hexane soluble fraction was further purified using silica gel (3:2 CH2Cl2-hexane) to obtain 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (F5Y), 5-hydroxy-7-methoxy-6-methylflavone (FN5Y), pinostrobin (F7Y) and aurentiacin (F8Y). The hexane insoluble solid was crystallized in 9:1 hexane-CH2Cl2 to obtain demthoxymatteucinol (F9Y). Fraction 2 was purified by Sephadex LH20 (5:4:1 CH2Cl2hexane-MeOH) followed by silica gel column chromatography (1:1 CH2Cl2-hexane) to yield pinocembrin (F12Y). The purity and identity of the isolated flavonoids were verified by NMR.

2.6. Pre-incubation study LLC/MK2 cells were seeded in 24-well plates and incubated as previously described. FN5Y at 25 μM was added to DENV2 for 1 or 2 h before adsorption (pre-incubation). Cells were then infected with the FN5Y-treated DENV2 (M.O.I. of 1) at 4 °C for 1 h with continuous rocking, followed by washing three times with cold PBS. Cells were incubated at 37 °C, under 5% CO2 for 2 days before supernatant collection. The level of virus production was quantified by plaque titration. DMSO-treated samples were used as a no-inhibition control. Results were confirmed by three independent experiments.

2.3. Cytotoxicity test LLC/MK2, Vero, HEK-293, or HepG2 cells were seeded at 104 cells per well of 96-well plate and incubated overnight. Compounds were 28

Antiviral Research 151 (2018) 27–38

P. Srivarangkul et al.

2.7. Pre-treatment study

2.10. In vitro protease assay

LLC/MK2 cells were seeded in 24-well plates and incubated as previously described. FN5Y at 25 μM was added to LLC/MK2 cells for 1 or 2 h before adsorption (pre-treatment). Cells were then adsorbed by DENV2 (M.O.I. of 1) at 37 °C for 1 h with continuous rocking, followed by a PBS wash. Cells were incubated at 37 °C, under 5% CO2 for 2 days before supernatant collection. The level of virus production was quantified using plaque titration. DMSO-treated samples were used as a no-inhibition control. Results were confirmed by three independent experiments.

Assays were performed in triplicate in a 96-well half area black plate (Greiner Bio-One, Monroe, USA). The reaction mixture (100 μl) contained 200 mM Tris-HCl, pH 9.5, 30% glycerol, 0.1% CHAPS, 1% DMSO, 50 nM DENV2 NS2BH-(QR)-NS3pro enzyme (Yon et al., 2005), 10 μM fluorogenic tetrapeptide substrate, Bz-Nle-Lys-Arg-Arg-AMC, and designated concentrations of compounds in DMSO. The compoundenzyme mixture was pre-incubated for 15 min at room temperature before addition of the substrate. The reaction was continued at 37 οC for 30 min. The release of AMC from the substrate was recorded every 1.5 min at 380 nm excitation and 460 nm emission in a SpectraMax Gemini EM spectrofluorometer (Molecular Devices, Sunnyvale, CA, USA). DMSO alone (concentration of 1%) was used as the no-inhibitor control (100% protease activity) and the bovine pancreatic trypsin inhibitor (BPTI, also known as aprotinin), which has a Ki of 26 nM against the DENV2 protease, was used at 5 μM as a positive control (0% protease activity). Data were plotted and reported as percent inhibition of each compound to protease activity.

2.8. Fusion inhibition assay The protocol was adapted from Poh et al. (2009). C6/36 were seeded at 105 cells per well into 24-well plate and incubated overnight at 28 °C. Cells were infected with DENV2 NGC at the M.O.I. of 0.02 for 1 h with gentle rocking every 15 min. Cells were washed with PBS and incubated with MEM supplemented with 1% fetal bovine serum, 100 I.U./ml penicillin, and 100 μg/ml streptomycin. FN5Y at 25 μM was added to the virus-infected cells. DMSO-treated cells and 4G2 antibodytreated cells were used as controls in the experiment. Cells were incubated for two days before addition of 0.5 mM 2-morpholinoethanesulfonic acid (MES), pH 5, to induce pH-dependent fusion. Cells were closely monitored under microscope until fused cells were observed. Pictures were taken using Elipse TS100 inverted routine microscope (Nikon, USA). Results were confirmed by three independent experiments.

2.11. Molecular docking of NS2B/NS3 protease FN5Y structure was drawn by HyperChemTM (Hypercube, Inc., Gainsville, USA) and optimized with molecular mechanics method for geometry optimization. The crystallized DENV3 NS2B-NS3 in complex with aldehyde inhibitor Bz-nKRRR-H (pdb: 3U1I) was used in the study. The protein was visualized by Visual Molecular Dynamics software (VMD) and molecular docking was performed using Autodock4 (The Scripps Research Institute, La Jolla, CA, USA). The results were obtained from 20 snapshots and analyzed for estimated binding free energy.

2.9. Molecular dynamics simulation on FN5Y/E complex 2.12. Replicon inhibition assay The crystal structure of DENV2 E protein with n-octyl-beta-D-glucoside (β-OG) (pdb: 1OKE) (Modis et al., 2003) was used to construct the FN5Y/E complex using molecular docking technique. The blind docking approach was used to search all possible binding sites for FN5Y using 400 independent runs by AutodockVina version 1.2.1 (Trott and Olson, 2010). At each site, the conformer with preferential binding, or the lowest binding affinity, was adopted as the starting structure for molecular dynamic simulations. All-atom molecular dynamics (MD) simulation of FN5Y/E complex was performed with periodic boundary condition at the 8 different sites using AMBER 14 package program (Case et al., 2014), similar to previous studies (Kaiyawet et al., 2013; Meeprasert et al., 2012, 2014). The partial atomic charges for FN5Y were developed in accordance with the standard procedure (Nutho et al., 2014; Sangpheak et al., 2014), whereas AMBER ff03.r1 (Duan et al., 2003) and general AMBER (Wang et al., 2004) force fields were adopted for the protein and FN5Y, respectively. The protonation state of all amino acids was assigned by PROPKA 3.1 (Olsson et al., 2011). The system was fully solvated by the TIP3P water model with 10 Å distance dimension from protein surface and the counter ions were used to electrical charge neutralization. The hydrogen and water atoms were minimized by the 1500 steps of steepest descents (SD) followed by 1500 steps of conjugated gradients (CG) using the SANDER module implemented in Amber14. Then, the system was heated up to 300 K for 200 ps, and was subsequently simulated at the same temperature for 100 ns. The MD trajectories were collected every 0.2 ps from the production phase. Using the CPPTRAJ module, the convergences of energies, temperature, and global root meansquare displacement were used to verify the stability of the FN5Y/E complex. The MD snapshots extracted from the last 50 ns were used for total binding free energy analysis based on MM/GBSA method (Kollman et al., 2000) and its per-residue decomposition energy contribution.

BHK-21 cells expressing DENV2 replicon (BHK-21/DENV2) were maintained in minimal essential medium (MEM) supplemented with 10% FBS, and 0.3 mg/ml G418 (Bio Basic Canada®, Ontario, Canada). Cells were seeded at 5 × 104 cells per well into 24-well plate and incubated overnight at 37 °C under 5% CO2. FN5Y, or ribavirin (TargetMol, Boston, MA, USA), a known flaviviral replication inhibitor, at 1, 10, 25 μM were added to the cells. DMSO at the concentration of 1% was used as a no-inhibition control. Cells were incubated at 37 °C for 48 h and lysed to quantified DENV2 replicon by RT-qPCR (Manzano et al., 2011). Data were reported as percent inhibition of DMSO control and results were confirmed by three independent experiments. 3. Results 3.1. Cytotoxicity and efficacy study of selected flavanones A previous screening was done with two different concentrations (10 or 25 μM) of flavanone derivatives with DENV2 at the M.O.I. of 0.1 for 120 h, and the results suggested that the compounds were potential flaviviral inhibitors (Boonyasuppayakorn et al., 2016). In this study, we explored CC50 and EC50 of the flavanones and chalcones (Table 1). Results suggested that FN5Y, F8Y, and F12Y cytotoxicities were not observed to at least 100 μM, whereas cytotoxicities (CC50) of F5Y and F7Y were 48.87 ± 3.69 and 78.58 ± 3.29 μM, respectively. F9Y was not properly dissolved in DMSO therefore its cytotoxicity was undetermined. Effective concentration (EC50) against DENV2 infectivity was also performed and analyzed by plaque titration of 120 h supernatants. FN5Y showed high potency with EC50 of 4.21 μM (Table 1), similar to those of F5Y and F7Y with EC50s of 5.09 and 6.11 μM, respectively. F5Y was previously characterized with anti-tumor activity (Ye et al., 2004) and protective effects against metabolic syndrome (Choi et al., 2016; Hu et al., 2014). F7Y was also characterized as one of 29

Antiviral Research 151 (2018) 27–38

P. Srivarangkul et al.

Table 1 Screening of LLC/MK2 cytotoxicity (CC50), and DENV2 efficacy (EC50) at 120 h of selected flavanones. Abbreviation

Name

CC50 (μM)

EC50 (μM)

FN5Y F5Y F7Y F8Y F9Y F12Y

5-hydroxy-7-methoxy-6-methylflavanone 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone Pinostrobin (5-hydroxy-7-methoxyflavanone) Aurentiacin (2′-hydroxy-4′,6′-dimethoxy-3′-methylchalcone) Demethoxymatteucinol ((2S)-5,7-Dihydroxy-6,8-dimethylflavanone) Pinocembrin (5,7-dihydroxyflavanone) Naringenin (5,7-Dihydroxy-2-(4-hydroxyphenyl) chroman-4-one) (Frabasile et al., 2017)

> 100 48.87 ± 3.69 78.58 ± 3.29 > 100 Not dissolved > 100 311.3 (Huh7.5 cells)

4.21 5.09 6.11 31.00 15.45 35.81 17.97 117.1 177.5

(DENV1/FGA) (DENV2/ICC) (DENV3/5532) (DENV4/TVP360)

showed at least two major inhibitions of viral production, one at the early after infection (1 hpi) and the other at the late steps of infection (12–48 hpi). In contrast, RT-qPCR analysis revealed only a single genome inhibition at the early steps. Note that FN5Y did not inhibit the virus if the compound was introduced prior to (−1 hpi) or simultaneous with (0 hpi) the infection (Fig. 3B–C). In addition, the pre-incubation (Fig. 4A) and pre-treatment (Fig. 4B) studies were done to confirm the TOA findings and no inhibition was observed in either study. Therefore, FN5Y neither inactivated the virus nor interfered with the attachment and receptor-binding steps. Also, the penetration efficacy was explored using molecular calculation (swissADME.ch) and parameters including lipophilicity, water solubility, pharmacokinetics, and druglikeness were determined (Supplementary 1). From these parameter, FN5Y should efficiently diffuse across lipid bilayer including plasma membrane and endosomal membrane without specific binding receptor. The major inhibitory effect would rather be the step immediately after internalization (e.g. fusion, initial translation) suggested from the TOA viral loads (Fig. 3B–C). Moreover, the time of drug addition assay showed that the maximal efficiency of the drug was at 1 hpi (Fig. 3), or exactly when fusion occurred. The results suggested that the drug was rapidly absorbed and diffused freely to every compartment in the cell. Adding the drug prior to fusion (e.g. −1 or 0 hpi) did not increase the efficacy. Therefore, the pretreatment method did not provide any advantage to the compound inhibition. Previous studies suggested that flavonoid compounds inhibited particular viral targets such as envelope (Ismail and Jusoh, 2016), protease (de Sousa et al., 2015), or RNA-dependent RNA polymerase (Anusuya and Gromiha, 2016). In this section, we studied FN5Y against pH-dependent fusion (Poh et al., 2009). Cell-cell fusion was observed in DENV2-infected cells under acidic pH (Fig. 5A, arrow) after MES treatment, whereas no fusion was found in DMSO-treated, no-virus control (Fig. 5B). Apparently, FN5Y-treated cells did not exhibit virusinduced cell-cell fusion (Fig. 5C) suggesting that FN5Y potently inhibited virus-induced fusion. Similar finding was also found in that of 4G2 antibody control (Fig. 5D), a macromolecular inhibitor that targeted DENV envelope (E) protein and blocked the fusion. Therefore, the results suggested that FN5Y actively inhibited DENV fusion under acidic environment. Note that the compound was inactive during preincubation to the virus (Fig. 4A) or pre-treatment to the cells (Fig. 4B), therefore the proposed mechanism of FN5Y was to inhibit the E protein during conformational change at pH-dependent fusion. In addition to FN5Y, F7Y and F12Y were tested and virus-induced fusion was obviously noticed under light microscope (Supplementary 2). Therefore, both F7Y and F12Y failed to inhibit the virus-induced fusion. It is possible that the additional 6-methyl group of FN5Y plays an important role towards this fusion activity. Molecular dynamic (MD) simulation was chosen to verify the FN5Y/ E binding sites and their interaction energy. Briefly, the eight possible binding regions on DENV2 E homodimer were demonstrated for FN5Y binding as follows (Fig. 6A); 1) K and K' (20% for kl hairpin, domain I/II

the breast cancer resistant proteins (BRCP) (Pick et al., 2011). FN5Y, however, was newly discovered and it showed a promising CC50 and EC50 results. Therefore, FN5Y was chosen for further investigation as a potential DENV inhibitor. 3.2. FN5Y detailed cytotoxic study Next, we examined FN5Y CC50s to LLC/MK2, Vero, HEK-293, and HepG2 cells (Fig. 1) at various conditions. LLC/MK2 cells were treated with FN5Y for 48, 72, and 120 h (Fig. 1A–C) and the CC50s were at > 100, > 100, and 33.20 ± 4.33 μM, respectively. Similarly, Vero cells were treated with FN5Y for 48, and 72 h and the CC50s were at 78.11 ± 12.10, and 26.61 ± 7.20 μM, respectively. Note that the prolonged treatment of FN5Y increases cytotoxicity in both cell lines but LLC/MK2 was more tolerant than Vero towards FN5Y-induced toxicity. In addition, HEK-293 and HepG2 cell lines were treated with FN5Y for 72 h and no toxicity was observed upto 100 μM (Fig. 1F–G). 3.3. FN5Y efficacy study in LLC/MK2 and Vero cells The efficacy of FN5Y to DENV2 was studied in LLC/MK2 and Vero cells. DENV2 NGC was used to infect LLC/MK2 cells and FN5Y was added during and after infection. The efficacy (EC50) of DENV2 inhibition obtained from plaque titration of supernatants at 72, and 120 h were 15.99 ± 5.38, and 3.72 ± 1.03 μM (Fig. 2A–B), respectively. Similarly, Vero cells was also treated with FN5Y and the EC50 was 12.31 ± 1.64 μM at 72 h (Fig. 2C). Although EC50s obtained from DENV2 infected LLC/MK2 (Fig. 2A) and Vero (Fig. 2C) cells were similar at 72 h, the selectivity indices (SI, CC50/EC50) of LLC/MK2 was more than two-fold higher than that of Vero cells. Since CC50 of LLC/ MK2 and Vero at 72 h were > 100 and 26.61 (Fig. 1B and E), SI of DENV2 inhibition to the respective cell lines were > 6.25 and 2.23 (Fig. 2A and C). Interestingly, SI of FN5Y at 120 h was 8.93 with the EC50 of 3.72 ± 1.03 μM (Fig. 2B) suggesting that the compound was stable for at least five days of incubation period. In addition, we studied FN5Y-treated DENV4 in LLC/MK2 cells (Fig. 2D) and results were 11.70 ± 6.04 (> 8.55) μM, similar to those of DENV2 in LLC/MK2 (Fig. 2A) and Vero (Fig. 2C) cells. This data suggested that FN5Y similarly inhibited both DENV2 and DENV4 in cell-based assay system and FN5Y could potentially be a broad-spectrum anti-dengue inhibitor. 3.4. FN5Y inhibited fusion by preventing DENV E protein conformational change Time of drug addition (TOA) assay was the first mechanism of action study aiming to reveal if the primary target would either locate at early or late steps of DENV life cycle. FN5Y (25 μM) was added to LLC/ MK2 cells at different time points before (−1 hpi), during (0 hpi), and after DENV2 infection (1–72 hpi). Supernatants and cells were collected for studying viral production by plaque titration (Fig. 3A–B), and intracellular RNA level by RT-qPCR (Fig. 3C), respectively. Results 30

Antiviral Research 151 (2018) 27–38

P. Srivarangkul et al.

Fig. 1. Cytotoxicity of FN5Y to LLC/MK2 cells (A–C) incubated at 48, 72, and 120 h, respectively. Vero cells (D–E) were incubated at 48, and 72 h. HEK-293 (F) and HepG2 cells (G) were incubated at 72 h.

31

Antiviral Research 151 (2018) 27–38

P. Srivarangkul et al.

Fig. 2. Efficacy of FN5Y to DENV2 infecting LLC/MK2 (A–B) cells, Vero (C) cells, and DENV4 infecting LLC/MK2 (D) cells.

hinge region, or β-OG pocket) (Modis et al., 2003), 2) X and X' (10% for domain I/III hinge region) (Yennamalli et al., 2009), 3) Y and Y' (25% for conserved region among flaviviruses) (Ismail and Jusoh, 2016) and 4) Z and Z' (12% for interface between the two monomers) (HernándezSantoyo et al., 2013). Other sites were surrounded by lipid membrane such that they were unsuitable for proper ligand binding (Kuhn et al., 2002); (Oliveira et al., 2017). MD simulation suggested that FN5Y bound stably to the K, K′, X′ and Y′ sites under 100-ns simulation but inconsistently bound to X, Y, Z and Z′ pockets. Moreover, MM/GBSA binding free energy suggested that FN5Y preferentially bound to K, K′, X′, and Y′ sites at −28.4, −23.5, −16.5, and −21.0 kcal/mol, respectively (Fig. 6B). The important residues associated with FN5Y binding were calculated from per-residue decomposition energy (Fig. 6C). The following residues, K (P83, L107, K128 and L198), K' (T48, E49, A50, L198, Q200 and L277), X' (Y138, V354 and I357), and Y' (V97, R99, N103 and K246), were reported to interact with FN5Y. Taken together, the MD simulation suggested the maximum binding affinity of FN5Y to K and K′ sites, thereby interfering the hinge from opening and re-orienting to trimer conformation triggered by acidic environment. In addition, we studied the role of 6-methyl group of FN5Y towards the binding efficiency of DENV envelope proteins. Briefly, we chose F7Y (5-hydroxy-7-methoxyflavanone) for a parallel MD simulation study because F7Y lacked the 6-methyl group and failed to inhibit fusion in cell-based assay. MD study under 100-ns simulations of FN5Y/E and F7Y/E at the K, K′, X′ and Y′ sites were run and the binding efficiencies were compared (Table 2). Results showed that FN5Y bound more stably to K, X′, and Y′ sites of DENV envelope, whereas F7Y bound slightly better at K′ site. Overall, the presence of a 6-methyl group in FN5Y

could be the major reason that strengthened the binding efficiencies towards DENV E protein. 3.5. FN5Y did not inhibit NS2B/3 DENV2 protease or DENV2 replicon replication Despite the E protein, we studied if flaviviral protease was inhibited by the flavanones since it was previously suggested as a target of certain flavanone derivatives (Kiat et al., 2006; Othman et al., 2008). The compounds, FN5Y, F7Y, F8Y, F9Y, and F12Y, at 5, 10, and 25 μM were tested for percent inhibition of DENV2 NS2BH-(QR)-NS3PRO protease activity using in vitro protease assay (Fig. 7A). Results, however, showed that all compounds at their highest concentration (25 μM) inhibited less than 50% of the protease activities. We then concluded that the protease was unlikely the major target of our tested flavanone compounds. In addition, the molecular docking between FN5Y and DENV3 NS2B-NS3 showed merely a weak hydrogen bond with an estimated binding energy at −7.23 kcal/mol (Fig. 7B). From the results, we concluded that the FN5Y, as well as other flavanones, did not primarily target flaviviral NS2B-NS3 protease. Moreover, we tested whether FN5Y inhibited DENV2 replication in the replicon system (Fig. 8). Results showed that FN5Y did not inhibit replicon replication at 1, 10, or 25 μM. Ribavirin, a flaviviral replication inhibitor, was assayed in parallel and a dose-dependent inhibition was found. The fact that FN5Y did not inhibit replicon replication can be implied that the drug target was not a self-sustaining RNA replication machinery. This result corresponded to the previous TOA finding that the genome titer was not reduced in late time points (Fig. 3C). The plaque titer inhibition at late time points (Fig. 3B) could possibly result 32

Antiviral Research 151 (2018) 27–38

P. Srivarangkul et al.

Fig. 3. Time-of-addition assay. LLC/MK2 cells (5 × 104 in 0.5 ml of 24-well plate) were seeded, incubated overnight, and infected with DENV2 at the MOI of 0.1 for 1 h with gentle rocking. FN5Y at 25 μM in 1% DMSO were added at 1 h before, during, and after infection at various time points as shown. Cells were washed and maintained in culture medium containing FN5Y for 72 h. (A–B) Supernatants were collected to analysis by plaque titration and (C) cell lysates were collected to analysis by RT-qPCR.

33

Antiviral Research 151 (2018) 27–38

P. Srivarangkul et al.

Fig. 4. Pre-incubation and pre-treatment assays LLC/MK2 cells(5 × 104 in 0.5 ml of 24-well plate) were seeded, incubated overnight.(A) FN5Y at 25 μM was added to DENV2 (M.O.I. of 1) for 1 or 2 h before adsorption (pre-incubation). (B) FN5Y at 25 μM was added to LLC/MK2 cells for 1 or 2 h before DENV2 (M.O.I. of 1) adsorption (pre-treatment). Cells were incubated for 48 h and the level of virus production was quantified by plaque titration of cultured supernatant. DMSO-treated samples were used as a no-inhibition control.

to bind to DENV envelope proteins at K, K′, X′ and Y′ positions, prevent the conformational change, and subsequently inhibit fusion (Fig. 6). Since DENV protease (de Sousa et al., 2015; Kiat et al., 2006; Othman et al., 2008) and RdRP (Coulerie et al., 2013) were previously reported as possible targets, we explored the activities using NS2B/3 protease and viral replicon replication assays. However, no significant inhibition was found suggesting that the viral protease and the self-replicating RNA machinery were not targets of FN5Y (Figs. 7–8). Flavanone, one of the flavonoid subclasses, has shown versatile biological activities (Patel et al., 2016; Rasul et al., 2013; Zandi et al., 2011). However, only naringenin was clearly shown to inhibit DENV1-4 viruses and DENV1 and DENV3 replicons in Huh7.5 cell line (Frabasile et al., 2017) (Table 1) and the drug was effective when introduced at post-infection (Frabasile et al., 2017). This report was contrast to an earlier study (Zandi et al., 2011) showing that naringenin and

from the drug inhibited post-replication steps such as maturation or budding.

4. Discussion This is the first report of a novel flavanone, 5-hydroxy-7-methoxy-6methylflavanone (FN5Y), as a potential inhibitor of dengue virus with the efficacy (and selectivity index) of 15.99 ± 5.38 (> 6.25), and 12.31 ± 1.64 (2.23) μM in LLC/MK2 and Vero cells, respectively (Fig. 2). The compound inhibited DENV4 at 11.70 ± 6.04 (> 8.55) μM. The maximal efficacy was achieved at early after infection (Fig. 3), similar to the DENV E inhibitor previously described (Wang et al., 2009). Also, FN5Y inhibited pH-dependent fusion (Fig. 5). The compound did not inactivate the viral particle, or interfere with receptormediated endocytosis (Fig. 4). The proposed mechanism of action was

Fig. 5. Fusion inhibition assay. C6/36 cells (105 in 0.5 ml of 24-well plate) were seeded, incubated overnight, and infected with (A) DENV2 at the MOI of 0.02 for 1 h with gentle rocking. DMSOtreated, (B) no virus infection was used as a mock treatment. (C) FN5Y at 25 μM and (D) 4G2 antibody were added to the DENV2 infected cells. Cells were washed and maintained in culture medium in 28 °C for 48 h before MES addition. Cells were washed at 4 h later and replaced by the culture medium. Pictures were taken when fused cells (A, arrow) were observed or a day after MES treatment. Results were confirmed by three independent experiments.

34

Antiviral Research 151 (2018) 27–38

P. Srivarangkul et al.

Fig. 6. Binding mechanism of FN5Y towards DENV2 envelope homodimer. (A) Eight possible binding sites of FN5Y on molecular surface of DENV2 E protein: kl hairpin (K), domain I/III hinge region (X), conserved region among flaviviruses (Y) and interface between the two monomers (Z) and their opposite sites (K′, X′, Y′ and Z′). Domains I, II and III are colored by red, yellow and blue, respectively. (B) Binding free energies of FN5Y/E complex (ΔGFN5Y binding) at the K, K′, X′, and Y′ sites. (C) Closed-up of FN5Y binding orientations at the K, K′, X′, and Y′ sites, where the interacting residues are colored according to energy values as shown in color scale. The residues with energy contribution lower than −0.5 kcal/mol are labeled. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

345 ± 70 μM (Kiat et al., 2006). We also investigated pinostrobin (F7Y) for inhibition of dengue virus infectivity (Table 1), fusion (Table 2, Supplementary 2), and protease (Fig. 7A) activities. Upon cytotoxicity and efficacy screening (Table 1), the compound inhibited DENV2 virus replication in LLC/MK2 cells suggesting that it was a potential dengue inhibitor. However, F7Y targeted neither fusion nor DENV2 protease and target identification will require further investigations such as chemical affinity-tag purification coupled with identification by liquid chromatography and mass spectrophotometry. Another extensively studied flavanone was pinocembrin, 5,7-dihydroxyflavanone, or F12Y. This compound exhibited a wide variety of biological activities (reviewed by Rasul et al. (2013)) including a modest anti-HIV inhibitor (Pasetto et al., 2014). Similar to F7Y, pinocembrin inhibited neither DENV protease (Fig. 6 (Kiat et al., 2006)), nor pH-dependent fusion (Supplementary 2). Moreover, both F7Y and F12Y were less potent than FN5Y with higher EC50 value (Table 1). However, both F7Y and F12Y provided instructive information towards developing a model for structure-activity relationship study. Our molecular dynamic study showed the strong binding efficacies of FN5Y to K, K′, X′, and Y′ sites of DENV envelope dimer. The K and K′ sites are hydrophobic pockets in the kl hairpins of DENV2 envelope proteins, which is one of the most promising targets in designing antidengue drugs (Jadav et al., 2015; Tambunan et al., 2015; Wang et al., 2009). FN5Y well occupied into this pocket by sharing intermolecular interactions with the residues T48, E49 K128 and Q200, similar to a potent DENV2 entry inhibitor (EC50 of 119 nM) (Wang et al., 2009).

Table 2 Molecular dynamic study of FN5Y/E and F7Y/E under 100-ns simulations. DENV E Binding sites

ΔGbinding (kcal/mol) (means ± SD) FN5Y/E

K K′ X′ Y′

−28.4 −23.5 −21.0 −16.5

± ± ± ±

p-value

F7Y/E 0.24 0.10 0.15 0.26

−25.11 −24.35 −19.62 −14.00

± ± ± ±

0.20 0.25 0.40 0.50

< .001 < .001 < .001 < .001

hesperidin did not significantly inhibit DENV2 virus RNA level accessed by RT-qPCR. It is possible that naringenin inhibition was undetectable by RT-qPCR because its target was at replication or post-replication steps. In TOA experiment, we explored both virion production and genome replication levels by plaque titration and RT-qPCR methods, respectively (Fig. 3B–C). We noticed that plaque titration detected the compound inhibition at both early and late time points, whereas RTqPCR detected only at the early time points. In other words, plaque titration can detect the compound inhibition at any step in viral life cycle but RT-qPCR can detect only the steps preceding the genome replication. We concluded from our results that there were two possible FN5Y targets located at fusion and post-replication steps. In addition to cell-based assays, in vitro DENV protease was used to characterize flavanone inhibitors. For example, pinostrobin was reported to non-competitively inhibit DENV2 protease with the Ki of 35

Antiviral Research 151 (2018) 27–38

P. Srivarangkul et al.

Fig. 8. Replicon inhibition assay BHK-21 cells expressing DENV2 replicon (BHK-21/ DENV2) cells (5 × 104/well) were seeded into 24-well plate and were incubated overnight at 37 °C under 5% CO2 followed by compounds addition at indicated final concentrations. Cells were incubated at 37 °C for 48 h and lysed to quantified DENV2 replicon by RT-qPCR. Data were reported as percent inhibition of DMSO control.

simulations and functional studies reinstated that the additional 6methyl group potentiated FN5Y to bind to DENV envelope proteins and inhibited fusion. In conclusion, we reported a novel compound, 5-hydroxy-7methoxy-6-methylflavanone (FN5Y), inhibiting dengue virus with the efficacy (and selectivity index) of 15.99 ± 5.38 (> 6.25), and 12.31 ± 1.64 (2.23) μM in LLC/MK2 and Vero cells, respectively. It also inhibited DENV4 suggesting a possible pan-dengue inhibitor. The drug elicited strong bindings to K, K′, X′, and Y′ sites of DENV envelope protein and prevented the pH-dependent fusion. Conflicts of interest The authors declare no conflict of interest. Acknowledgement This research is supported by the Thailand Research Fund and Office of Higher Education Commission (OHEC) Thailand (MRG6080017), and Chulalongkorn University; Grants for Development of New Faculty Staff and Part of the ‘Research Grant for New Scholar CU Researcher's Project’ (RGN_2558_008_02_30), Ratchadaphiseksomphot Endowment Fund. Cell lines, reference dengue viruses, and 4G2 antibody were gifts from Drs. Chutitorn Ketloy, Padet Siriyasatien, Parvapan Bhattarakosol, and Nattiya Hirankarn, Faculty of Medicine, Chulalongkorn University. We thank Drs. Anuradha Balasubramanian and Radhakrishnan Padmanabhan, Georgetown University for in vitro protease assay and DENV2/BHK-21 replicon cells. We thank Dr. Pornthep Sompornpisut, Faculty of Science, Chulalongkorn University for molecular docking of DENV2 protease.

Fig. 7. (A) In vitro NS2BH-(QR)-NS3PRO protease assay The reaction contains 200 mM Tris-HCl, pH 9.5, 30% glycerol, 0.1% CHAPS, 1% DMSO, 50 nM DENV2 NS2BH-(QR)NS3pro enzyme, 10 μM Bz-Nle-Lys-Arg-Arg-AMC, and 5, 10, and 25 μM of compounds in 1% DMSO. The reaction was continued at 37 οC for 30 min. The release of AMC was recorded every 1.5 min at 380 nm excitation and 460 nm emission. DMSO alone (concentration of 1%) was used as the no-inhibitor control (100% protease activity) and triplicate results were plotted and reported as percent inhibition of each compound to protease activity. (B) Binding of FN5Y to DENV3 NS2B-NS3 (3U1I). A dash line represents a hydrogen bond of interacting residues.

Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.antiviral.2018.01.010.

The X′ site is a domain I/III hinge region involved in the DENV vaccine development (Dubey et al., 2017; Yennamalli et al., 2009). And the Y′ site is one of the conserved region among flaviviruses that were targeted by particular flavonoids such as baicalein, quercetin, and epigallocatechin gallate (Ismail and Jusoh, 2016). Therefore, the strong binding efficiencies towards these four sites recapitulated that pH-dependent fusion performed by DENV envelope proteins should be the true target of FN5Y. Also, F7Y/E showed the less potent binding efficiencies than FN5Y/E at three out of the four sites, which corresponded to the results obtained from cell-based fusion study. Therefore, MD

References Allard, P.M., Dau, E.T., Eydoux, C., Guillemot, J.C., Dumontet, V., Poullain, C., Canard, B., Gueritte, F., Litaudon, M., 2011. Alkylated flavanones from the bark of Cryptocarya chartacea as dengue virus NS5 polymerase inhibitors. J. Nat. Prod. 74, 2446–2453. Anusuya, S., Gromiha, M.M., 2016. Quercetin derivatives as non-nucleoside inhibitors for dengue polymerase: molecular docking, molecular dynamics simulation, and binding free energy calculation. J. Biomol. Struct. Dynam. 1–15. Bhatt, S., Gething, P.W., Brady, O.J., Messina, J.P., Farlow, A.W., Moyes, C.L., Drake, J.M., Brownstein, J.S., Hoen, A.G., Sankoh, O., Myers, M.F., George, D.B., Jaenisch, T., Wint, G.R., Simmons, C.P., Scott, T.W., Farrar, J.J., Hay, S.I., 2013. The global

36

Antiviral Research 151 (2018) 27–38

P. Srivarangkul et al.

Meeprasert, A., Hannongbua, S., Rungrotmongkol, T., 2014. Key binding and susceptibility of NS3/4A serine protease inhibitors against hepatitis C virus. J. Chem. Inf. Model. 54, 1208–1217. Meeprasert, A., Khuntawee, W., Kamlungsua, K., Nunthaboot, N., Rungrotmongkol, T., Hannongbua, S., 2012. Binding pattern of the long acting neuraminidase inhibitor laninamivir towards influenza A subtypes H5N1 and pandemic H1N1. J. Mol. Graph. Model. 38, 148–154. Modis, Y., Ogata, S., Clements, D., Harrison, S.C., 2003. A ligand-binding pocket in the dengue virus envelope glycoprotein. Proc. Natl. Acad. Sci. U. S. A. 100, 6986–6991. Moghaddam, E., Teoh, B.T., Sam, S.S., Lani, R., Hassandarvish, P., Chik, Z., Yueh, A., Abubakar, S., Zandi, K., 2014. Baicalin, a metabolite of baicalein with antiviral activity against dengue virus. Sci. Rep. 4, 5452. Nutho, B., Khuntawee, W., Rungnim, C., Pongsawasdi, P., Wolschann, P., Karpfen, A., Kungwan, N., Rungrotmongkol, T., 2014. Binding mode and free energy prediction of fisetin/β-cyclodextrin inclusion complexes. Beilstein J. Org. Chem. 10, 2789–2799. Oliveira, E.R.A., Mohana-Borges, R., de Alencastro, R.B., Horta, B.A.C., 2017. The flavivirus capsid protein: structure, function and perspectives towards drug design. Virus Res. 227, 115–123. Olsson, M.H.M., Søndergaard, C.R., Rostkowski, M., Jensen, J.H., 2011. PROPKA3: consistent treatment of internal and surface residues in empirical pKa predictions. J. Chem. Theor. Comput. 7, 525–537. Ooi, E.E., Gubler, D.J., 2009. Dengue in Southeast Asia: epidemiological characteristics and strategic challenges in disease prevention. Cad. Saúde Pública 25 (Suppl. 1), S115–S124. Othman, R., Kiat, T.S., Khalid, N., Yusof, R., Newhouse, E.I., Newhouse, J.S., Alam, M., Rahman, N.A., 2008. Docking of noncompetitive inhibitors into dengue virus type 2 protease: understanding the interactions with allosteric binding sites. J. Chem. Inf. Model. 48, 1582–1591. Pasetto, S., Pardi, V., Murata, R.M., 2014. Anti-HIV-1 activity of flavonoid myricetin on HIV-1 infection in a dual-chamber in vitro model. PLoS One 9, e115323. Patel, N.K., Jaiswal, G., Bhutani, K.K., 2016. A review on biological sources, chemistry and pharmacological activities of pinostrobin. Nat. Prod. Res. 30, 2017–2027. Pick, A., Muller, H., Mayer, R., Haenisch, B., Pajeva, I.K., Weigt, M., Bonisch, H., Muller, C.E., Wiese, M., 2011. Structure-activity relationships of flavonoids as inhibitors of breast cancer resistance protein (BCRP). Bioorg. Med. Chem. 19, 2090–2102. Poh, M.K., Yip, A., Zhang, S., Priestle, J.P., Ma, N.L., Smit, J.M., Wilschut, J., Shi, P.Y., Wenk, M.R., Schul, W., 2009. A small molecule fusion inhibitor of dengue virus. Antivir. Res. 84, 260–266. Pozo-Aguilar, J.O., Monroy-Martínez, V., Díaz, D., Barrios-Palacios, J., Ramos, C., UlloaGarcía, A., García-Pillado, J., Ruiz-Ordaz, B.H., 2014. Evaluation of host and viral factors associated with severe dengue based on the 2009 WHO classification. Parasites Vectors 7. Rasul, A., Millimouno, F.M., Ali Eltayb, W., Ali, M., Li, J., Li, X., 2013. Pinocembrin: a novel natural compound with versatile pharmacological and biological activities. BioMed Res. Int. Sanchez, I., Gomez-Garibay, F., Taboada, J., Ruiz, B.H., 2000. Antiviral effect of flavonoids on the dengue virus. Phytother Res. : PT 14, 89–92. Sangpheak, W., Khuntawee, W., Wolschann, P., Pongsawasdi, P., Rungrotmongkol, T., 2014. Enhanced stability of a naringenin/2,6-dimethyl β-cyclodextrin inclusion complex: molecular dynamics and free energy calculations based on MM- and QMPBSA/GBSA. J. Mol. Graph. Model. 50, 10–15. Senthilvel, P., Lavanya, P., Kumar, K.M., Swetha, R., Anitha, P., Bag, S., Sarveswari, S., Vijayakumar, V., Ramaiah, S., Anbarasu, A., 2013. Flavonoid from Carica papaya inhibits NS2B-NS3 protease and prevents Dengue 2 viral assembly. Bioinformation 9, 889–895. Shu, P.Y., Chang, S.F., Kuo, Y.C., Yueh, Y.Y., Chien, L.J., Sue, C.L., Lin, T.H., Huang, J.H., 2003. Development of group- and serotype-specific one-step SYBR green I-based realtime reverse transcription-PCR assay for dengue virus. J. Clin. Microbiol. 41, 2408–2416. Tambunan, U.S.F., Zahroh, H., Parikesit, A.A., Idrus, S., Kerami, D., 2015. Screening analogs of β-OG pocket binder as fusion inhibitor of dengue virus 2. Drug Target Insights 9, 33–49. Trott, O., Olson, A.J., 2010. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. J. Comput. Chem. 31, 455–461. Vannice, K., Chocarro, L., Pfleiderer, M., Bellah, A., Ward, M., Yoon, I.K., Hombach, J., 2017. The value of multi-country joint regulatory reviews: the experience of a WHO joint technical consultation on the CYD-TDV (Dengvaxia(R)) dossier. Vaccine. Wang, J., Wolf, R.M., Caldwell, J.W., Kollman, P.A., Case, D.A., 2004. Development and testing of a general amber force field. J. Comput. Chem. 25, 1157–1174. Wang, Q.-Y., Patel, S.J., Vangrevelinghe, E., Xu, H.Y., Rao, R., Jaber, D., Schul, W., Gu, F., Heudi, O., Ma, N.L., Poh, M.K., Phong, W.Y., Keller, T.H., Jacoby, E., Vasudevan, S.G., 2009. A small-molecule dengue virus entry inhibitor. Antimicrob. Agents Chemother. 53, 1823–1831. Wang, W.K., Chao, D.Y., Kao, C.L., Wu, H.C., Liu, Y.C., Li, C.M., Lin, S.C., Ho, S.T., Huang, J.H., King, C.C., 2003. High levels of plasma dengue viral load during defervescence in patients with dengue hemorrhagic fever: implications for pathogenesis. Virology 305, 330–338. WHO, 2009. Dengue Guideline for Diagnosis, Treatment, Prevention, and Control, 2009 ed. WHO, Geneva, Switzerland. WHO, 2012. Global Strategy for Dengue Prevention and Control 2012-2020. World Health Organization. WHO, 2016. Dengue Vaccine Research. The World Health Organization. Ye, C.L., Liu, J.W., Wei, D.Z., Lu, Y.H., Qian, F., 2004. In vitro anti-tumor activity of 2',4'dihydroxy-6'-methoxy-3',5'-dimethylchalcone against six established human cancer cell lines. Pharmacol. Res. 50, 505–510.

distribution and burden of dengue. Nature 496, 504–507. Boonyasuppayakorn, S., Suroengrit, A., Srivarangkul, P., Yuttithamnon, W., Pankaew, S., Saelee, T., Prompetchara, E., Salakij, S., Bhattarakosol, P., 2016. Simplified dengue virus microwell plaque assay using an automated quantification program. J. Virol. Methods 237, 25–31. Case, D.A., Babin, V., Berryman, J.m.U.o.L.F.o.S., Technology and Communication (FSTC) > Physics and Materials Science Research Unit > ,Betz, R.M., Cai, Q., Cerutti, D.S., Cheatham III, T.E., Darden, T.A., Duke, R.E., Gohlke, H., Goetz, A.W., Gusarov, S., Homeyer, N., Janowski, P., Kaus, J., Kolossváry, I., Kovalenko, A., Lee, T.S., LeGrand, S., Luchko, T., Luo, R., Madej, B., Merz, K.M., Paesani, F., Roe, D.R., Roitberg, A., Sagui, C., Salomon-Ferrer, R., Seabra, G., Simmerling, C.L., Smith, W., Swails, J., Walker, R.C., Wang, J., Wolf, R.M., Wu, X., Kollman, P.A., 2014. Amber, vol. 14 University of California, San Francisco. Choi, J.W., Kim, M., Song, H., Lee, C.S., Oh, W.K., Mook-Jung, I., Chung, S.S., Park, K.S., 2016. DMC (2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone) improves glucose tolerance as a potent AMPK activator. Metab. Clin. Exp. 65, 533–542. Coulerie, P., Eydoux, C., Hnawia, E., Stuhl, L., Maciuk, A., Lebouvier, N., Canard, B., Figadere, B., Guillemot, J.C., Nour, M., 2012. Biflavonoids of Dacrydium balansae with potent inhibitory activity on dengue 2 NS5 polymerase. Planta Med. 78, 672–677. Coulerie, P., Nour, M., Maciuk, A., Eydoux, C., Guillemot, J.C., Lebouvier, N., Hnawia, E., Leblanc, K., Lewin, G., Canard, B., Figadere, B., 2013. Structure-activity relationship study of biflavonoids on the Dengue virus polymerase DENV-NS5 RdRp. Planta Med. 79, 1313–1318. de Sousa, L.R., Wu, H., Nebo, L., Fernandes, J.B., da Silva, M.F., Kiefer, W., Kanitz, M., Bodem, J., Diederich, W.E., Schirmeister, T., Vieira, P.C., 2015. Flavonoids as noncompetitive inhibitors of Dengue virus NS2B-NS3 protease: inhibition kinetics and docking studies. Bioorg. Med. Chem. 23, 466–470. Du, J., Chen, Z., Zhang, T., Wang, J., Jin, Q., 2016. Inhibition of dengue virus replication by diisopropyl chrysin-7-yl phosphate. Science China. Life Sci. 59, 832–838. Duan, Y., Wu, C., Chowdhury, S., Lee, M.C., Xiong, G., Zhang, W., Yang, R., Cieplak, P., Luo, R., Lee, T., Caldwell, J., Wang, J., Kollman, P., 2003. A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations. J. Comput. Chem. 24, 1999–2012. Dubey, K.D., Tiwari, G., Ojha, R.P., 2017. Targeting domain-III hinging of dengue envelope (DENV-2) protein by MD simulations, docking and free energy calculations. J. Mol. Model. 23, 102. Frabasile, S., Koishi, A.C., Kuczera, D., Silveira, G.F., Verri Jr., W.A., Duarte Dos Santos, C.N., Bordignon, J., 2017. The citrus flavanone naringenin impairs dengue virus replication in human cells. Sci. Rep. 7, 41864. Hernández-Santoyo, A., Tenorio-Barajas, A.Y., Altuzar, V., Vivanco-Cid, H., MendozaBarrera, C., 2013. Protein-protein and protein-ligand docking. In: Ogawa, T. (Ed.), Protein Engineering - Technology and Application. InTech, Rijeka Ch. 03. Hu, Y.C., Hao, D.M., Zhou, L.X., Zhang, Z., Huang, N., Hoptroff, M., Lu, Y.H., 2014. 2',4'Dihydroxy-6'-methoxy-3',5'-dimethylchalcone protects the impaired insulin secretion induced by glucotoxicity in pancreatic beta-cells. J. Agric. Food Chem. 62, 1602–1608. Ismail, N.A., Jusoh, S.A., 2016. Molecular docking and molecular dynamics simulation studies to predict flavonoid binding on the surface of DENV2 E protein. Interdiscipl. Sci. Comput. Life Sci. Jadav, S.S., Kaptein, S., Timiri, A., De Burghgraeve, T., Badavath, V.N., Ganesan, R., Sinha, B.N., Neyts, J., Leyssen, P., Jayaprakash, V., 2015. Design, synthesis, optimization and antiviral activity of a class of hybrid dengue virus E protein inhibitors. Bioorg. Med. Chem. Lett 25, 1747–1752. Johari, J., Kianmehr, A., Mustafa, M.R., Abubakar, S., Zandi, K., 2012. Antiviral activity of baicalein and quercetin against the Japanese encephalitis virus. Int. J. Mol. Sci. 13, 16785–16795. Kaiyawet, N., Rungrotmongkol, T., Hannongbua, S., 2013. Effect of halogen substitutions on dUMP to stability of thymidylate synthase/dUMP/mTHF ternary complex using molecular dynamics simulation. J. Chem. Inf. Model. 53, 1315–1323. Kiat, T.S., Pippen, R., Yusof, R., Ibrahim, H., Khalid, N., Rahman, N.A., 2006. Inhibitory activity of cyclohexenyl chalcone derivatives and flavonoids of fingerroot, Boesenbergia rotunda (L.), towards dengue-2 virus NS3 protease. Bioorg. Med. Chem. Lett 16, 3337–3340. Kollman, P.A., Massova, I., Reyes, C., Kuhn, B., Huo, S., Chong, L., Lee, M., Lee, T., Duan, Y., Wang, W., Donini, O., Cieplak, P., Srinivasan, J., Case, D.A., Cheatham, T.E., 2000. Calculating structures and free energies of complex Molecules: combining molecular mechanics and continuum models. Acc. Chem. Res. 33, 889–897. Kuhn, R.J., Zhang, W., Rossmann, M.G., Pletnev, S.V., Corver, J., Lenches, E., Jones, C.T., Mukhopadhyay, S., Chipman, P.R., Strauss, E.G., Baker, T.S., Strauss, J.H., 2002. Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell 108, 717–725. Libraty, D.H., Endy, T.P., Houng, H.S., Green, S., Kalayanarooj, S., Suntayakorn, S., Chansiriwongs, W., Vaughn, D.W., Nisalak, A., Ennis, F.A., Rothman, A.L., 2002. Differing influences of virus burden and immune activation on disease severity in secondary dengue-3 virus infections. J. Infect. Dis. 185, 1213–1221. Lim, S.P., Wang, Q.Y., Noble, C.G., Chen, Y.L., Dong, H., Zou, B., Yokokawa, F., Nilar, S., Smith, P., Beer, D., Lescar, J., Shi, P.Y., 2013. Ten years of dengue drug discovery: progress and prospects. Antivir. Res. 100, 500–519. Lindenbach, D., Thiel, H.J., Rice, C., 2007. Flaviviridae: the viruses and their replication. In: Knipe, D.M., Howley, P.M. (Eds.), Field's Virology, 5 ed. Lippincott-Raven Publishers, Philadelphia, pp. 1101–1152. Manzano, M., Reichert, E.D., Polo, S., Falgout, B., Kasprzak, W., Shapiro, B.A., Padmanabhan, R., 2011. Identification of cis-acting elements in the 3'-untranslated region of the dengue virus type 2 RNA that modulate translation and replication. J. Biol. Chem. 286, 22521–22534.

37

Antiviral Research 151 (2018) 27–38

P. Srivarangkul et al.

Yennamalli, R., Subbarao, N., Kampmann, T., McGeary, R.P., Young, P.R., Kobe, B., 2009. Identification of novel target sites and an inhibitor of the dengue virus E protein. J. Comput. Aided Mol. Des. 23, 333. Yon, C., Teramoto, T., Mueller, N., Phelan, J., Ganesh, V.K., Murthy, K.H., Padmanabhan, R., 2005. Modulation of the nucleoside triphosphatase/RNA helicase and 5'-RNA triphosphatase activities of Dengue virus type 2 nonstructural protein 3 (NS3) by interaction with NS5, the RNA-dependent RNA polymerase. J. Biol. Chem. 280, 27412–27419.

Zandi, K., Teoh, B.T., Sam, S.S., Wong, P.F., Mustafa, M.R., Abubakar, S., 2011. Antiviral activity of four types of bioflavonoid against dengue virus type-2. Virol. J. 8, 560. Zhang, T., Wu, Z., Du, J., Hu, Y., Liu, L., Yang, F., Jin, Q., 2012. Anti-Japanese-encephalitis-viral effects of kaempferol and daidzin and their RNA-binding characteristics. PLoS One 7, e30259.

38