Inhibition of dengue virus replication by novel inhibitors of RNA-dependent RNA polymerase and protease activities

Article abstract of DOI:10.1080/14756366.2017.1355791

Dengue virus (DENV) is the leading mosquito-transmitted viral infection in the world. With more than 390 million new infections annually, and up to 1 million clinical cases with severe disease manifestations, there continues to be a need to develop new antiviral agents against dengue infection. In addition, there is no approved anti-DENV agents for treating DENV-infected patients. In the present study, we identified new compounds with anti-DENV replication activity by targeting viral replication enzymes–NS5, RNA-dependent RNA polymerase (RdRp) and NS3 protease, using cell-based reporter assay. Subsequently, we performed an enzyme-based assay to clarify the action of these compounds against DENV RdRp or NS3 protease activity. Moreover, these compounds exhibited anti-DENV activity in vivo in the ICR-suckling DENV-infected mouse model. Combination drug treatment exhibited a synergistic inhibition of DENV replication. These results describe novel prototypical small anti-DENV molecules for further development through compound modification and provide potential antivirals for treating DENV infection and DENV-related diseases.

Full text of DOI:10.1080/14756366.2017.1355791

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Inhibition of dengue virus replication by novel
inhibitors of RNA-dependent RNA polymerase and
protease activities
Sveva Pelliccia, Yu-Hsuan Wu, Antonio Coluccia, Giuseppe La Regina, Chin-
Kai Tseng, Valeria Famiglini, Domiziana Masci, John Hiscott, Jin-Ching Lee &
Romano Silvestri
To cite this article: Sveva Pelliccia, Yu-Hsuan Wu, Antonio Coluccia, Giuseppe La Regina, Chin-
Kai Tseng, Valeria Famiglini, Domiziana Masci, John Hiscott, Jin-Ching Lee & Romano Silvestri
(2017) Inhibition of dengue virus replication by novel inhibitors of RNA-dependent RNA polymerase
and protease activities, Journal of Enzyme Inhibition and Medicinal Chemistry, 32:1, 1091-1101,
DOI: 10.1080/14756366.2017.1355791
To link to this article: http://dx.doi.org/10.1080/14756366.2017.1355791
© 2017 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 04 Aug 2017.
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Date: 05 August 2017, At: 01:52

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 32, NO. 1, 1091–1101
https://doi.org/10.1080/14756366.2017.1355791
RESEARCH PAPER
Inhibition of dengue virus replication by novel inhibitors of RNA-dependent RNA
polymerase and protease activities
Sveva Pellicciaa, Yu-Hsuan Wub,c, Antonio Colucciaa, Giuseppe La Reginaa, Chin-Kai Tsengb,c, Valeria Famiglinia,
Domiziana Mascia, John Hiscottd, Jin-Ching Leee,f,g,h and Romano Silvestria
aDepartment of Drug Chemistry and Technologies, Sapienza University of Rome, Laboratory affiliated to Istituto Pasteur Italia – Fondazione
b
c
Cenci Bolognetti, Roma, Italy; Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan; Center
d
of Infectious Disease and Signaling Research, College of Medicine, National Cheng Kung University, Tainan, Taiwan; Istituto Pasteur Italia –
Fondazione Cenci Bolognetti, Roma, Italy; Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung,
Taiwan; Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan; Research Center for
Natural Products and Drug Development, Kaohsiung Medical University, Kaohsiung, Taiwan; Graduate Institute of Medicine, College of
e
f
g
h
Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
ABSTRACT
ARTICLE HISTORY
Received 30 April 2017
Revised 1 July 2017
Accepted 12 July 2017
Dengue virus (DENV) is the leading mosquito-transmitted viral infection in the world. With more than 390
million new infections annually, and up to 1 million clinical cases with severe disease manifestations, there
continues to be a need to develop new antiviral agents against dengue infection. In addition, there is no
approved anti-DENV agents for treating DENV-infected patients. In the present study, we identified new
compounds with anti-DENV replication activity by targeting viral replication enzymes – NS5, RNA-depend-
ent RNA polymerase (RdRp) and NS3 protease, using cell-based reporter assay. Subsequently, we per-
formed an enzyme-based assay to clarify the action of these compounds against DENV RdRp or NS3
protease activity. Moreover, these compounds exhibited anti-DENV activity in vivo in the ICR-suckling
DENV-infected mouse model. Combination drug treatment exhibited a synergistic inhibition of DENV repli-
cation. These results describe novel prototypical small anti-DENV molecules for further development
through compound modification and provide potential antivirals for treating DENV infection and DENV-
related diseases.
KEYWORDS
DENV inhibitors; RdRp; NS3
protease; ICR-suckling
mouse; synergy
Introduction
is a multifunctional protein, which contains protease, helicase and
triphosphatase domain. The N-terminal amino acids (residue
1–184) of NS3 is responsible for protease activity. NS2B serves as a
cofactor of NS3 protease and forms complex with NS3; the central
amino acid hydrophilic domain (residue 49–92) of NS2B is critical
for cofactor activity13. DENV replication also requires the non-
structural protein 5 (NS5) which is the essential RNA-dependent
RNA polymerase (RdRp) activity14. DENV NS5 RdRp has proven to
be a promising target for direct-acting antiviral (DAA) drug devel-
opment, because it is structurally conserved among the four DENV
serotypes, and NS5 RdRp has no enzymatic counterpart in mam-
malian cells15.
Currently, no licensed antiviral drugs are available to block
DENV infection, and while vector control efforts remain the only
means to stop the spread of the infection, they have not success-
fully inhibited annual epidemic outbreaks throughout the
tropics16. Recently, a live-attenuated DENV vaccine17 based on
the yellow fever virus 17 D backbone was licensed for use in the
Philippines, Brazil and Mexico. However, the serotype-specific effi-
cacy of the DENV vaccine is varying, and the long-term protection
and safety of this vaccine still need more investigation18. In the
present study, we have focused on the identification of potential
Dengue virus (DENV) is responsible of worldwide arthropodborne
viral infection, which globally represents a serious human health
concern. DENV is the etiological agent of disease in more than
hundred countries with up to 3 billion people exposed to the risk
of infection in the tropical regions1–3. DENV has expanded its glo-
bal range with sustained outbreaks in South America and Asia,
with these epidemics accompanied by increased disease severity.
DENV are single-stranded, positive sense RNA viruses belonging
to the Flaviviridae family. The DENV family can be viewed as falling
in four related, but antigenically distinct, DENV 1–4 serotypes. The
carriers of DENV to humans are the mosquitoes Aedes aegypti and
Aedes albopictus. The DENV infection causes a variety of illness,
including asymptomatic or subclinical disease, dengue fever (DF)
symptoms and the most severe dengue haemorrhagic fever (DHF)
and dengue shock syndrome (DSS) which cause millions of infec-
tions around the world4–8. DENV has a 10.7 kb, positive-sense RNA
genome with 50- and 30-untranslated regions flanking a polypro-
tein that encodes three structural (C, prM/M and E) and seven
non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) pro-
teins. prM and E structural proteins are the primary antigenic tar-
gets of the humoural immune response in humans9–12. DENV NS3 anti-DENV inhibitors by targeting the enzymatic activities of the
ꢀ
CONTACT Romano Silvestri
romano.silvestri@uniroma1.it
Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Universita di Roma, Piazzale Aldo Moro
5, I-00185 Roma, Italy; Jin-Ching Lee
jclee@kmu.edu.tw
Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung,
Taiwan
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

1092
S. PELLICCIA ET AL.
Table 1. Activity of Compounds 1–3 against DENV-2 Replication and DENV-2 NS5 RdRp.
DENV-2a
RNA
NS5 RdRp
SId
Cell-based EC50 SD (lM)
Enzyme-based EC50 SD (lM)
b
c
e
f
Structure
CC50 (lM)
EC50 SD (lM)
1
2
196
11.7 0.2
16.7
8.1 0.3
7.8 0.3
EtO
N
O
O
Cl
O S
N
O S
O
N
Cl
Me
>200
7.6 0.4
>26.3
>35.1
7.2 0.4
5.3 0.2
EtO
O
O
N
NH
N
N
S
O
O
Me
3
>200
5.7 0.3
6.0 0.3
4.9 0.2
EtO
O
O
NO2
O S
N
O S
O
N
N
NO2
aData are mean values of two to three independent experiments each one in triplicate.
bCC50: half maximal cytotoxicity concentration.
cEC50 (DENV-2 RNA): half maximal effective concentration. Huh-7 cells were infected with DENV-2 and followed by RdRp inhibitors treatment for 3 days. The cell
lysates were collected to analyse DENV RNA synthesis by qRT-PCR with specific primers targeting NS5.
dSI: selectivity index calculated as CC50/EC50 ratio.
eEC50 (cell-based DENV-2 NS5 RdRp): cell-based RdRp reporter assay. The Huh-7 cells were transiently expressed p(þ)RLuc-(–)DV-UTRDC-FLuc and DENV NS5 expres-
sion vector pcDNA-NS5-Myc.
fEC50 (enzyme-based DENV-2 NS5 RdRp): enzyme-based RdRp activity assay. The (ꢁ) 30UTR RNA was incubated with RdRp polymerase protein and CTP, GTP, UTP and
BBT-ATP. The fluorescence signal was measured at excitation wavelength of 422 nm and emission wavelength of 566 nm, respectively.
NS5 RdRp polymerase and NS3 protease in vitro and in vivo19–21
.
the mixture was cooled to 25 ꢀC via air jet cooling. Organic solu-
tions were dried over anhydrous sodium sulphate. Evaporation of
the solvents was carried out on a Bu€chi Rotavapor R-210 equipped
with a Bu€chi V-850 vacuum controller and a Bu€chi V-700 vacuum
pump. Column chromatography was performed on columns
packed with silica gel from Macherey-Nagel (70 ꢁ 230 mesh). Silica
gel thin-layer chromatography (TLC) cards from Macherey-Nagel
(silica gel pre-coated aluminium cards with fluorescent indicator
visualisable at 254 nm) were used for TLC. Developed plates were
visualised with a Spectroline ENF 260 C/FE UV apparatus. Melting
points (mp) were determined on a Stuart Scientific SMP1 appar-
atus and are uncorrected. Infrared spectra (IR) were run on a
PerkinElmer Spectrum 100 FT-IR spectrophotometer equipped with
universal attenuated total reflectance (ATR) accessory and IR data
acquired and processed by PerkinElmer Spectrum 10.03.00.0069
As part of a continuation of our studies22–24, we developed new
pyrazole derivatives25 as potential DENV NS5 RdRp inhibitors
(Table 1). In addition, we carried out virtual screening (VS) studies
on the NS2B/NS3 protease to design and synthesise new DENV
NS3 protease inhibitors (Table 2). In sum, we identified five
compounds that exhibited anti-DENV replication activity without
cytotoxicity; two compounds exhibited anti-DENV activity in DENV-
infected ICR-suckling mouse model. Interestingly, combination
treatment with compounds, respectively, targeting NS5 RdRp poly-
merase and NS3 protease, demonstrated a synergistic inhibitory
effect on DENV replication.
Materials and methods
software. Band position and absorption ranges are given in cmꢁ1
.
Chemistry
Proton nuclear magnetic resonance (1H NMR) spectra were
recorded a Bruker Avance (400 MHz) spectrometer in the indicated
solvent and corresponding fid files were processed by MestreLab
Research SL MestreReNova 6.2.1–769 software (Santiago de
Compostela, Spain). Chemical shifts are expressed in d units (ppm)
from tetramethylsilane. Elemental analyses of biologically eval-
uated compounds were found to be within 0.4% of the theoret-
ical values and their purity was found to be >95% by high-
pressure liquid chromatography (HPLC). The HPLC system used
Thermo Fisher Scientific Dionex UltiMate 3000, consisted of a SR-
Microwave (MW)-assisted reactions were performed on a CEM
Discover SP single-mode reactor equipped with Explorer 72 auto-
sampler, controlling the instrument settings by PC-running CEM
Synergy 1.60 software (Matthews, NY, USA). Closed vessel experi-
ments were carried out in capped MW-dedicated vials (10 ml) with
cylindrical stirring bar (length 8 mm, diameter 3 mm). Stirring, tem-
perature, irradiation power, maximum pressure (Pmax), PowerMAX
(simultaneous cooling while heating), ActiVent (simultaneous vent-
ing while heating), and ramp and hold times were set as indi-
cated. Temperature of the reaction was monitored by an external 3000 solvent rack, a LPG-3400SD quaternary analytical pump, a
fibre optic temperature sensor. After completion of the reaction, TCC-3000SD column compartment, DAD-3000 diode array
a

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1093
Table 2. Activity of DENV inhibitors 4 and 5 against DENV-2 replication.
DENV-2a
RNA
NS3
SId
Cell-based EC50 SD (lM)
Enzyme-based EC50 SD (lM)
b
c
e
f
Structure
CC50 (lM)
EC50 SD (lM)
4
5
181
4.6 0.3
39.3
6.7 0.2
4.7 0.3
>200
7.3 0.3
27.4
7.9 0.6
6.9 0.4
aData are mean values of two to three independent experiments each one in triplicate.
bCC50: half maximal cytotoxicity concentration.
cEC50 (DENV-2 RNA): half maximal effective concentration. Huh-7 cells were infected with DENV-2 and followed by RdRp inhibitors treatment for 3 days. The cell
lysates were collected to analyse DENV RNA synthesis by qRT-PCR with specific primers targeting NS5.
dSI: selectivity index calculated as CC50/EC50 ratio.
eEC50 (cell-based DENV-2 NS3 protease): Huh-7 cells were transfected with pEG(MITA)SEAP and pcDNA-NS2B-GSG-NS3-Myc followed by incubation of each compound.
The SEAP activity was measured by Phodpha-Light assay kit after 3 days post incubation.
fEC50 (enzyme-based DENV-2 NS3 protease). The 7-amino-4-methylcoumarin (AMC) fluorophore-linked peptide substrate Boc-GRR-AMC (Bachem, USA) was incubated
with DENV NS2B/NS3pro protein and compound. The fluorescence signal was detected at excitation wavelength of 380 nm and emission wavelength of 465 nm,
respectively.
detector and an analytical manual injection valve with a 20 mL starting from 6 and 4-nitrobenzenesulphonyl chloride. Yield 33%,
loop. Samples were dissolved in acetonitrile (1 mg/mL). HPLC ana- mp 220–222 ꢀC (from ethanol). 1H NMR (DMSO-d6):
d 0.93
lysis was performed by using a Thermo Fisher Scientific Acclaim (t, J ¼ 7.1 Hz, 3H), 3.54 (s, 3H), 3.69–3.74 (m, 2H), 8.11 (s, 1H), 8.23
120 C18 column (5 mm, 4.6 mm ꢂ 250 mm) at 25 1 ꢀC with an (d, J ¼ 8.9 Hz, 4H), 8.52 ppm (d, J ¼ 8.9 Hz, 4H). FT-IR (ATR): ꢀ 1173,
appropriate solvent gradient (acetonitrile/water), flow rate of 1349, 1528, 1717 cmꢁ1. Anal. calcd. for C19H17N5O10S2 (539.49): C,
1.0 mL/min and signal detector at 206, 230, 254 and 365 nm. 42.30; H, 3.18; N, 12.98; S, 11.89. Found: C, 42.12; H, 3.14; N, 12.60;
Chromatographic data were acquired and processed by Thermo S, 11.70.
Fisher Scientific Chromeleon 6.80 SR15 Build 4656 software
(Waltham, MA, USA).
Ethyl 1-methyl-5–(4-nitrophenylsulphonamido)-1H-pyrazole-4-carb-
oxylate (9). A mixture of 8 (0.032 g, 0.06 mmol) and lithium hydrox-
General procedure for preparation of compounds 1, 3 and 8. ide monohydrate (0.01 g, 0.24 mmol) in THF/H2O (1:1, 5 mL) was
Ethyl 5–(4-chloro-N-((4-chlorophenyl)sulphonyl)phenylsulphonamido)- heated at 50 ꢀC for 15 min, cooled, made acid with 1 N HCl aqueous
1-methyl-1H-pyrazole-4-carboxylate (1). 4-Chlorobenzenesulphonyl solution (pH ꢃ 5) and extracted with ethyl acetate. The organic layer
chloride (0.15 g, 0.72 mmol) was added to solution of commercially was washed with brine, dried and filtered. Removal of the solvent
available 6 (0.10 g, 0.6 mmol) in pyridine (2 mL) while stirring. The gave 9 (0.02 g, 95%), mp 150–152 ꢀC (from ethanol). 1H NMR
reaction was stirred at 60 ꢀC overnight. The mixture was neutral- (DMSO-d6): d 0.98 (t, J ¼ 7.08, 3H), 3.71–3.76 (m, 5H), 7.78 (s, 1H),
ised with 1 N HCl and extracted with ethyl acetate. The organic 7.87 (d, J ¼ 8.9 Hz, 2H), 8.40 (d, J ¼ 8.9 Hz, 2H), 11.01 ppm (br s, dis-
layer was washed with brine, dried and filtered. Removal of the appeared on treatment with D2O, 1H). FT-IR (ATR): ꢀ 1170, 1347,
solvent gave a residue that was purified by column chromatog- 1532, 1709 cmꢁ1. Anal. calcd. for C13H14N4O6S (354.34): C, 44.07; H,
raphy (silica gel, n-hexane:ethyl acetate ¼ 2:1 as eluent) to furnish 3.98; N, 15.81; S, 9.05. Found: C, 43.84; H, 3.92; N, 15.62; S, 8.91.
1
1 (0.10 g, 33%), mp 132–135 ꢀC (from ethanol). H NMR (DMSO-d6):
Ethyl
5–(4-aminophenylsulphonamido)-1-methyl-1H-pyrazole-4-
d 0.96 (t, J ¼ 6.2 Hz, 3H), 3.52 (s, 3H), 3.71–3.77 (m, 2H), 7.81 (d, carboxylate (10). A mixture of 9 (0.14 g, 0.4 mmol) and tin(II) chlor-
J ¼ 8.0 Hz, 4H), 7.94 (d, J ¼ 7.9 Hz, 4H), 8.06 ppm (s, 1H). FT-IR (ATR): ide dihydrate (0.27 g, 1.2 mmol) in ethyl acetate (15 ml) was heated
ꢀ 1176, 1391, 1709 cmꢁ1. Anal. calcd. for C19H17Cl2N3O6S2 (518.38): at reflux for 3 h. After cooling, the mixture was made basic
C, 44.02; H, 3.31; N, 8.11; Cl, 13.68; S, 12.37. Found: C, 43.78; H, (pH ꢃ 8) with a saturated aqueous solution of NaHCO3 and filtered.
3.26; N, 7.84; Cl, 13.36; S, 12.02.
The layers were separated and the organic one was washed with
Ethyl 5–(4-nitro-N-((4-nitrophenyl)sulphonyl)phenylsulphonamido)- brine, dried and filtered. Removal of the solvent gave a residue
1-phenyl-1H-pyrazole-4-carboxylate (3). Was synthesised as 1 starting that was purified by column chromatography (silica gel,
from 726 and 4-nitrobenzensulphonyl chloride. Yield 79%, mp dichloromethane:ethanol ¼ 9.8:0.2 as eluent) to furnish 10 (0.06 g,
1
230–232 ꢀC (from ethanol). 1H NMR (DMSO-d6): d 1.01 (t, J ¼ 7.0, 3H), 50%), mp 118–120 ꢀC (from ethanol). H NMR (DMSO-d6): d 1.09 (t,
3.70–3.76 (m, 2H), 7.32–7.38 (m, 3H), 7.48 (d, J ¼ 6.9 Hz, 2H), 8.04 (d, J ¼ 7.1 Hz, 3H), 3.66 (s, 3H), 3.83–3.88 (m, 2H), 6.02 (s, 2H), 6.52 (d,
J ¼ 8.9 Hz, 4H), 8.33–8.36 ppm (m, 5H). FT-IR (ATR): ꢀ 1167, 1336, J ¼ 8.7 Hz, 2H), 7.20 (d, J ¼ 8.7 Hz, 2H), 7.72 (s, 1H), 9.71 ppm (br s,
1597, 1706 cmꢁ1. Anal. calcd. for C24H19N5O10S2 (601.06): C, 47.92; H, disappeared on treatment with D2O, 1H). FT-IR (ATR): ꢀ 1152,
3.18; N, 11.64; S, 10.66. Found: C, 47.78; H, 3.11; N, 11.39; S, 10.42.
Ethyl 1-methyl-5–(4-nitro-N-((4-nitrophenyl)sulphonyl)phenylsul-
phonamido)-1H-pyrazole-4-carboxylate (8). Was synthesised as 1 zole-4-carboxylate (11). A mixture of 10 (0.05 g, 0.15 mmol) and
1378, 1698, 3397 cmꢁ1
Ethyl 5–(4-(1H-pyrrol-1-yl)phenylsulphonamido)-1-methyl-1H-pyra-
.

1094
S. PELLICCIA ET AL.
2,5-dimethoxytetrahydrofuran (0.02 g, 0.02 mL, 0.15 mmol) in gla- aqueous solution of NaHCO3 and extracted with ethyl acetate. The
cial acetic acid (1 mL) was heated at reflux for 30 min. The solvent
was removed in vacuo, and after cooling water and ethyl acetate
were added. Layers were separated and the organic one was
washed with a saturated aqueous solution of NaHCO3, brine and
dried. Removal of the solvent gave a residue that was purified by
column chromatography (silica gel, n-hexane:ethyl acetate ¼ 1:1 as
eluent) to furnish 11 (0.035 g, 66%), mp 200–203 ꢀC (from ethanol).
1H NMR (DMSO-d6): d 0.97 (t, J ¼ 7.1 Hz, 3H), 3.73–3.77 (m, 5H),
6.32 (t, J ¼ 2.2 Hz, 2H), 7.49 (t, J ¼ 2.2 Hz, 2H), 7.62 (d, J ¼ 8.8 Hz,
2H), 7.76–7.78 (m, 3H), 10.48 ppm (br s, disappeared on treatment
with D2O, 1H). FT-IR (ATR): ꢀ 1132, 1336, 1702, 3139 cmꢁ1. Anal.
calcd. for C17H18N4O4S (374.41): C, 54.53; H, 4.85; N, 14.96; S, 8.56.
Found: C, 54.28; H, 4.79; N, 14.69; S, 8.22.
organic layer was washed with brine, dried and filtered. Removal
of the solvent gave a residue that was purified by column chroma-
tography (silica gel, n-hexane/ethyl acetate ¼ 3:1 as eluent) to fur-
nish 5 (0.05 g, 50%), mp 182–185 ꢀC (from ethanol). 1H NMR
(CDCl3): d 3.71 (s, 6H), 3.92 (s, 3H), 4.43 (s, 2H), 6.30–6.31 (m, 1H),
6.41–6.44 (m, 2H), 7.25–7.35 (m, 2H), 7.58–7.60 (m, 1H), 8.80 ppm
(br s, disappeared on treatment with D2O, 1H). FT-IR (ATR): ꢀ 1690,
3323 cmꢁ1. Anal. calcd. for C19H18ClNO4 (359.81): C, 63.43; H, 5.04;
Cl, 9.85; N, 3.89. Found: C, 63.27; H, 4.97; Cl, 9.59; N, 3.70.
Molecular modelling studies
All molecular modelling studies were performed on a MacPro dual
2.66 GHz Xeon running Ubuntu 12. The DENV protease structure
(PDB ID: 2FOM)27 was downloaded from the protein data bank. An
in-house compound library matching Lipinski’s rule of five28 was
used as a training set. No pre-filter was applied. The docking simu-
lations were performed with PLANTS29 using a 13 Å radius grid
sphere. The centre of the binding site was settled according to
Tamiri30. Molecules were scored by ChemPLP scoring function.29
Figure 1 was generated by PyMOL31.
Ethyl
5–(4-(2-benzoyl-1H-pyrrol-1-yl)phenylsulphonamido)-1-
mixture of 11 (0.06 g,
methyl-1H-pyrazole-4-carboxylate (2).
A
0.14 mmol), benzoyl chloride (0.02 g, 0.016 mL, 0.14 mmol) and
anhydrous aluminium chloride (0.02 g; 0.14 mol) in anhydrous 1,2-
dichloroethane (3 mL) was placed into the MW cavity (closed ves-
sel mode, Pmax ¼ 250 psi). A starting MW irradiation of 70 W was
used, the temperature being ramped from 25 to 110 ꢀC, while stir-
ring. Once 110 ꢀC was reached, taking about 1 min, the reaction
mixture was held at this temperature for 4 min. The reaction mix-
ture was quenched on 1 N HCl aqueous solution/crushed ice and
extracted with dichloromethane. The organic layer was washed
with brine, dried and filtered. Removal of the solvent gave a resi-
due that was purified by column chromatography (silica gel,
n-hexane:acetone ¼ 2:1 as eluent) to furnish 2 (0.04 g, 71%), mp
Ethics statement and experimental animals
Six-day-old ICR strain-suckling mice were obtained from BioLasco
Taiwan Co. Ltd (Taipei, Taiwan). All animal studies were conducted
in specific pathogen-free conditions and carried out in accordance
with the Guide for the Care and Use of Laboratory Animals. The
experimental protocol was approved by the Animal Research
1
136–140 ꢀC (from ethanol). H NMR (DMSO-d6): d 1.07 (t, J ¼ 7.1 Hz,
3H), 3.67 (s, 3H), 3.87–3.93 (m, 2H), 6.43–6.45 (m, 1H), 6.87–6.89
(m, 1H), 7.45–7.46 (m, 1H), 7.52–7.56 (m, 4H), 7.63–7.69 (m, 3H),
7.78 (s, 1H), 7.82–7.84 (m, 2H) 10.60 ppm (br s, disappeared on Committee of Kaohsiung Medical University of Taiwan (IACUC,
treatment with D2O, 1H). FT-IR (ATR): ꢀ 1166, 1347, 1595, 1713, protocol number 102177) under the guidance of the Public Health
3102 cmꢁ1. Anal. calcd. for C24H22N4O5S (478.52): C, 60.24; H, 4.63;
Service (PHS) Policy on Humane Care and Use of Laboratory
N, 11.71; S, 6.70. Found: C, 59.92; H, 4.58; N, 11.46; S, 6.46. Further
elution with the same eluent gave ethyl 5–(4-(3-benzoyl-1H-pyrrol-
1-yl)phenylsulphonamido)-1-methyl-1H-pyrazole-4-carboxylate (0.02 g,
28%), mp 216–218 ꢀC (from ethanol). 1H NMR (DMSO-d6): d 0.97
(t, J ¼ 7.0 Hz, 3H), 3.73–3.79 (m, 5H), 6.80–6.82 (m, 1H), 7.53–7.57
(m, 2H), 7.62–7.68 (m, 4H), 7.76 (s, 1H), 7.84–7.86 (d, J ¼ 8.4 Hz, 2H),
7.93–7.96 (d, J ¼ 9.0 Hz, 2H), 8.04 (s, 1H), 10.58 ppm (br s, disap-
peared on treatment with D2O, 1H). FT-IR (ATR): ꢀ 1166, 1280,
1642, 1706, 3132 cmꢁ1. Anal. calcd. C24H22N4O5S (478.52): C, 60.24;
H, 4.63; N, 11.71; S, 6.70. Found: C, 59.88; H, 4.55; N, 11.65; S, 6.60.
Methyl 5-chloro-3–(4-hydroxy-3,5-dimethoxybenzoyl)-1H-indole-2-
carboxylate (4). Was synthesised as 2 starting from 12 and 4-
hydroxy-3,5-dimethoxybenzoyl chloride. Yield 11%, mp 118–120 ꢀC
Animals.
Cells and virus
Human hepatoma Huh-7 cells were cultured in Dulbecco’s modi-
fied Eagle’s medium (DMEM) containing 10% foetal bovine serum,
1% non-essential amino acids, and 1% antibiotic–antimycotic
within 5% CO2 supplement at 37 ꢀC. Aedes albopictus C6/36 mos-
quito cells were cultured in RPMI1640 medium containing 10%
fetal bovine serum, 1% non-essential amino acids, 1% antibioti-
c–antimycotic and 1% sodium pyruvate within 5% CO2 supple-
ment at 37 ꢀC. DENV-2 (DENV type-2 strain 16681) was amplified
in C6/36 mosquito cells. Spodoptera frugiperda (Sf9) insect cells
were cultured in Grace’s medium with 10% hear-inactivated foetal
bovine serum (FBS) and 1% antibiotic–antimycotic at 26 ꢀC.
1
(from ethanol). H NMR (CDCl3): d 3.70 (s, 3H), 3.86 (s, 6H), 6.01 (br
s, disappeared on treatment with D2O, 1H), 7.17 (s, 2H), 7.32–7.35
(m, 1H), 7.41–7.43 (m, 1H), 7.60–7.61 (m, 1H), 9.31 ppm (br s, disap-
peared on treatment with D2O, 1H). FT-IR (ATR):
ꢀ 1715,
3312 cmꢁ1. Anal. calcd. for C19H16ClNO6 (389.79): C, 58.55; H, 4.14;
N, 3.59; Cl, 9.09. Found: C, 58.36; H, 4.09; N, 3.23; Cl, 8.81.
Evaluation of anti-DENV RNA activity
Huh-7 cells were seeded in 24-well plate and infected DENV at an
MOI of 0.2 for 2 h and followed by test compound treatment at
concentration of 0, 1, 5, 10, 25, 50 and 100 mM for 3 days. The total
cellular RNA was harvested by RNA extraction kit32 following man-
ufacturer’s instrument. DENV RNA and cellular mRNA levels were
determined by quantitative real-time reverse-transcription poly-
merase chain reaction (RT-qPCR) with specific primers33. The DENV
RNA level was normalised by cellular glyceraldehydes-3-phosphate
dehydrogenase (GAPDH) mRNA level. The relative DENV RNA level
was calculated by StepOneTM Software v2.2.2 (Applied Biosystems,
Methyl 5-chloro-3–(3,5-dimethoxybenzoyl)-1H-indole-2-carboxylate
(13). Was synthesised as 2 starting from 12 and 3,5-dimethoxyben-
zoyl chloride. Yield 21%, mp 185–188 ꢀC (from ethanol). 1H NMR
(CDCl3): d 3.70 (s, 3H), 3.82 (s, 6H), 6.07 (t, J ¼ 2.3 Hz, 1H), 7.0 (d,
J ¼ 4.7 Hz, 2H), 7.33–7.36 (m, 1H), 7.41–7.44 (m, 1H), 7.65–7.66 (m,
1H), 9.31 ppm (br s, disappeared on treatment with D2O, 1H). FT-IR
(ATR): ꢀ 1698, 3287 cmꢁ1
.
Methyl 5-chloro-3–(3,5-dimethoxybenzyl)-1H-indole-2-carboxylate
(5). A mixture of 13 (0.1 g, 0.3 mmol), triethylsilane (0.072 g, 0.1 mL,
0.6 mmol) and trifluoroacetic acid (0.31 g, 0.21 mL, 3 mmol) was
stirred at 25 ꢀC for 48 h. The mixture was diluted with a saturated Foster City, CA, USA) following normalisation of cellular

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1095
Figure 1. Proposed binding for derivatives 4 (green) and 5 (magenta). Residues involved in interactions are reported as cyan stick. H-bonds are shown as yellow dot
lines. Protease is reported as cartoon: grey for NS3 and red for NS2 subunits.
glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA level. was subjected to metal affinity chromatography employing the
AKTA prime protein purification system35. The eluted DENV-2 NS5
The zero dose was defined as 100%. The EC50 values were calcu-
protein was concentrated by an Amicon Ultra-15 30 k centrifugal
filter device36 and then dialysed against PBS overnight at 4 ꢀC.
lated from non-linear regression curve fitting using GraphPad
Prism7 software (San Diego, CA, USA). Results were obtained from
three independent experiments.
Evaluation of anti-DENV RdRp activity
The cell-based experiments were performed as previously
described33. Briefly, the Huh-7 cells were transfected with 0.5 mg of
p(þ)RLuc-(–)DV-UTRDC-FLuc and DENV NS5 expression vector
pcDNA-NS5-Myc followed by compound treatment for 3 days. The
RLuc and FLuc activities were analysed by Dual-Glo Luciferase
Assay System37. The enzyme-based fluorescence-based alkaline
phosphatase-coupled polymerase assay (FAPA) was performed as
previously described33. Briefly, the template was amplified from
the cDNA of DENV-2 minus strand 30-UTR and its RNA was syn-
thesised by the T7 Megascript kit38. The 100 nM DENV NS5 protein
was incubated with 1, 5 or 10 mM test compound, 100 nM (ꢁ) 30-
UTR RNA, 20 mM CTP, GTP, UTP and 20 mM BBT-ATP (Jena
Bioscience) in a total volume of 30 mL in assay buffer (50 mM Tris-
HCl, pH 7.5, 10 mM KCl, 1 mM MgCl2, 0.3 mM MnCl2, 0.001% Triton
X-100 and 10 mM cysteine) at 25 ꢀC for 60 min. The 30 mL of 2.5ꢂ
stop buffer (200 mM NaCl, 25 mM MgCl2, 1.5 M diethanolamine,
pH 10) with 25 nM calf intestinal alkaline phosphatase (Promega)
was added to the reaction after 60-min incubation. The fluores-
cence signal was measured at excitation wavelength of 422 nm
and emission wavelength of 566 nm, respectively.
Cell cytotoxicity assay
The Huh-7 cells were seeded in 96-well and treated with test com-
pound at concentration of 0, 10, 25, 50, 100, 150 and 200 mM.
After 3 days incubation, the cell viability was determined by
CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS
assay) as previously reported34. Briefly, the MTS assay buffer was
added to the cultured plate following removal of the culture
medium. After 2 h of incubation at 37 ꢀC, the absorbance at
492 nm was measured. The cell viability was calculated from non-
linear regression curve fitting using GraphPad Prism7. The zero
dose was defined as 100%. Results were obtained from three inde-
pendent experiments.
Purification of DENV NS5 protein
The DENV-2 NS5 protein purification was performed as previously
described33. Briefly, Sf9 cells were infected by the recombinant
baculovirus vAc-DENV-NS5 at an MOI of 10 for 3 days. The cells
were pelleted by centrifugation at 1000ꢂg for 5 min at 4 ꢀC and
washed twice with phosphate-buffered saline (PBS). The cells were
suspended in 5 mL of binding buffer (20 mM sodium phosphate,
pH 7.5, 300 mM NaCl, 20 mM imidazole) containing protease
inhibitor cocktail, and then disrupted by sonication. After centrifu-
Purification of DENV NS2B/NS3pro protein
The DENV NS2B/NS3pro purification was performed as previously
gation, the supernatant containing His-tagged DENV-2 NS5 protein described39. Briefly, the pET24b-NS2B-NS3pro plasmid was

1096
S. PELLICCIA ET AL.
transformed into competent Escherichia coli BL21 (DE3) and grew
in kanamycin (50 mg/mL)-containing LB at 26 ꢀC until OD600 nm
Results and discussion
Chemistry
reached
0.6.
The
isopropyl-b-D-thiogalactopyranose
was
added into the bacterial culture at 0.5 mM for protein induction.
After 12-h incubation, cells were collected and subjected to pro-
tein purification following previous description.
Compounds 1–3 were synthesised as shown in Scheme 1. Ethyl
5-amino-1-methyl-1H-pyrazole-4-carboxylate (6) was purchased
from Maybridge. Treatment of ethyl 2-cyano-3-ethoxyacrylate with
phenylhydrazine hydrochloride yielded ethyl 5-amino-1-phenylpyr-
azole-4-carboxylate (7)26. Pyrazoles 6 and 7 were converted into
final derivatives 1, 3 or intermediate 8 by reaction with 4-chloro-
or 4-nitrobenzenesulphonyl chloride, respectively, in pyridine.
Treatment of 8 with lithium hydroxide in aqueous tetrahydrofuran
for 15 min gave the sulphonamide 9. Tin (II) chloride dihydrate
reduction of 9 for 3 h in boiling ethyl acetate afforded amino
derivative 10 that was converted into pyrrole derivative 11 with
2,5-dimethoxytetrahydrofuran in glacial acetic acid at 80 ꢀC for 1 h.
Derivative 2 was prepared from 11 by MW-assisted reaction with
benzoyl chloride in the presence of anhydrous aluminium chloride
in 1,2-dichloroethane at 110 ꢀC (70 W) for 3 min.
Indole derivatives 4 and 5 were synthesised as shown in
Scheme 2. Final compound 4 and the intermediate 13 were pre-
pared by MW-assisted aroylation of methyl 5-chloro-1H-indole-2-
carboxylate (12) with 4-hydroxy-3,5-dimethoxybenzoyl chloride or
3,5-dimethoxybenzoyl chloride, respectively, in the presence of
anhydrous aluminium chloride in 1,2-dichloroethane at 110 ꢀC
(70 W) for 2 min. Treatment of 13 with triethylsilane in trifluoro-
acetic acid for 48 h afforded the corresponding methylene com-
pound 5.
Evaluation of anti-DENV protease activity
For cell-based activity assay, the Huh-7 cells were cotransfected
with 0.5 mg of pEG(MITA)SEAP reporter vector and DENV protease
expression vector pcDNA-NS2B-GSG-NS3-Myc followed by com-
pound treatment for 3 days. The SEAP activity was analysed by
Phospha-Light SEAP Reporter Gene Assay System40. The enzyme-
based experiment was performed as previously described39
.
Briefly, the 7-amino-4-methylcoumarin (AMC) fluorophore-linked
peptide substrate Boc-GRR-AMC41 and purified DENV NS2B/
NS3pro protein were used to analyse DENV NS2B/NS3 protease
activity. The 100 nM DENV NS2B/NS3pro protein was incubated
with 1, 5 or 10 mM tested compound and 10 mM Boc-GRR-AMC in
cleavage buffer (200 mM Tris [pH 9.5], 20% glycerol) for 30 min at
25 ꢀC. The fluorescence signal was detected at excitation wave-
length of 380 nm and emission wavelength of 465 nm,
respectively.
Evaluation of anti-DENV activity by ICR-suckling mice
Breeder ICR mice were purchased from BioLasco Taiwan Co.
Ltd. The experimental protocol was approved by the Animal
Research Committee of Kaohsiung Medical University of Taiwan
(IACUC, protocol number 102177). All mice received humane
care and fed with standard rodent chew and water ad libitum.
Mice were kept under a standard laboratory condition following
the Animal Use Protocol of Kaohsiung Medical University. The
6-day-old ICR-suckling mice were intracerebrally injected with
DENV-2 followed by intracerebrally injecting saline or test com-
pound at 1, 3, 5 days postinfection (dpi). Mice intracerebrally
injected with 65 ꢀC heat-inactive DENV-2 followed with saline
treatment served as healthy control. The body weight, clinical
scores and survival rate were recorded every day. The mice
were sacrificed at 6 dpi.
Virtual screening studies
In search for allosteric inhibitors of the NS2B/NS3 protease, we car-
ried out virtual screening (VS) and docking studies. We focused
our VS studies against the allosteric site, of the enzyme42,43. To
our knowledge, only few protease allosteric inhibitors have been
reported30, and many of them are natural compounds that do not
possess drug-like properties30. We performed structure-based
screening studies on an open conformation of the NS2B/NS3 pro-
tease structure44 (pdb code: 2FOM)27 available at the protein data
bank webserver45. This structure of the NS2B/NS3 protease was
previously used in VS campaign in search for new allosteric
inhibitors43.
EtO
EtO
N
EtO
O
NHNH2
.
O
O
NC
COOEt
O2
S
b
c
a
HCl
N
+
N
N
NH
S
NH2
O
N
N
N
R1 = Ph
R2
NO2
R1
O2S
O
R1
Me
O
6: R1 = Me
7: R1 = Ph
R2
1: R1 = Me, R2 = Cl;
9
3: R1 = Ph; R2 = NO2
8: R1 = Me, R2 = NO2;
EtO
EtO
EtO
O
O
O
d
e
f
O
N
NH
S
N
NH
S
N
NH
S
N
N
N
NH2
N
N
O
O
O
Me
Me
Me
O
O
O
2
10
11
Scheme 1. Synthesis of the final compounds 1–3. (a) TEA, reflux, 7 h. (b) 4-Cl- or 4-NO2-C6H4SO2Cl, pyridine, 60 ꢀC, overnight. (c) LiOH, 50 ꢀC, 15 min. (d) SnCl2, 80 ꢀC,
3 h. (e) 2,5-(OMe)2-THF, 100ꢀC, 1 h. (f) benzoyl chloride, AlCl3, closed vessel, 110ꢀC, 70 W, 4 min.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1097
OMe
OMe
R
O
Cl
OMe
COOMe
Cl
OMe
COOMe
Cl
a
b
CCOMe
N
H
(13)
N
N
H
H
12
4: R = OH; 13: R = H;
5
Scheme 2. Synthesis of the final compounds 4 and 5. (a) 4-OH-3,5-OMe2- or 3,5-OMe2-C6H4COCl, AlCl3, closed vessel, 110 ꢀC, 70 W, 2 min. (b) TES, TFA, 48 h, rt.
Figure 2. Inhibition of DENV RNA replication and RdRp activity by compounds 1–3. Panel A. Huh-7 cells were infected with DENV-2 at a multiplicity of infection (M.O.I)
of 0.2 and followed by treatment with the DENV polymerase inhibitor for 3 days. The DENV RNA level was analysed by RT-qPCR with specific primer targeting viral NS5
gene, and relative viral RNA levels were normalised against cellular GADPH mRNA levels. Treatment of 50 lM 20-C-methylcytidine (2CMC) direct against DENV RdRp
served as positive control. 0.1% DMSO (Mock) served as negative control. Panel B. Huh-7 cells were transfected with pEG(MITA)SEAP and pcDNA-NS2B-GSG-NS3-Myc
followed by incubation with each compound. The luciferase activity was analysed after 3 days treatment. Error bars denote the means SD of three independent experi-
ꢄ
ꢄꢄ
ments. p < .05; p < .01.
Compounds 4 and 5 shared virtually superimposable binding the inhibitory specificity of compounds 1–3 against NS5 RdRp
modes: (a) the 2-methoxycarbonyl ester occupied the inner part of
the binding pocket and was stabilised by an H-bond with Asp152
side chain; (b) the indole NH formed an H-bond with Asn167; (c)
the indole ring arranged a series of hydrophobic contacts with
Val146, Leu149 and Leu76; (d) the methoxylphenyl moiety of both
inhibitors formed hydrophobic interactions with Trp83 and a pi-
cation interaction with Lys74. (Figure 1).
activity, we performed an enzyme-based FAPA assay. Our data
showed that compounds 1–3 exhibited direct-action against DENV
RdRp activity (Table 1) with EC50 of 7.78 0.31, 5.34 0.17 and
4.87 0.24 mM, respectively, whose values correlated with results
of cell-based assay. Taken together, these results demonstrated
that compounds 1–3 attenuate DENV replication by directly inhib-
iting DENV RdRp activity.
Biological activity
DENV NS3 protease inhibitors
DENV NS5 RdRp inhibitors
Huh-7 cells were infected with DENV-2 and then treated with pro-
tease inhibitors for 3 days and DENV RNA synthesis was evaluated
with specific qRT-PCR primers targeting viral NS5 gene.
Compounds 4 and 5 strongly inhibited the DENV-2 DNA replica-
tion with EC50s of 4.60 0.03 mM and 7.28 0.13 mM, respectively
(Table 2 and Figure 3, panel A). In addition, a cell-based DENV
protease reporter assay was used to characterise the protease spe-
cificity of compounds 4 and 5. Huh-7 cells were transfected
with pEG(MITA)SEAP and DENV protease expression vector
pcDNA-NS2B-GSG-NS3-Myc and then treated with 4 or 5. Both
compounds showed strong inhibitory activity against the DENV
protease with EC50 of 6.71 0.20 mM and 7.92 0.62 mM, respect-
ively (Table 2 and Figure 3, panel B). We further performed an
Compounds 1–3 were evaluated in DENV-2 infected Huh-7 cells
by determining DENV RNA levels using specific qRT-PCR primer
targeting viral NS5 levels, normalised to cellular GADPH mRNA lev-
els. Compound
1
inhibited the DENV-2 with EC50 of
11.09 0.21 mM and selectivity index (SI ¼ CC50/EC50 ratio) of 17.7.
Compound 2 bearing a benzoyl tail at position 2 of the pyrrole
potently inhibited DENV-2 with EC50 of 7.61 0.36 mM and SI
>26.3. 1-Phenylpyrazole derivative compound 3 with the 4-nitro-
phenyl group showed potent anti-DENV-2 activity with EC50 of
5.66 0.25 mM and SI > 35.3. Collectively, compounds 1–3 strongly
reduced DENV RNA level (Table 1 and Figure 2, panel A).
To confirm the anti-RdRp activity of 1–3, we performed a cell-
based RdRp reporter assay. The Huh-7 cells were transiently enzyme-based assay to evaluate the inhibitory specificity of com-
pounds 4–5. The data showed that compounds 4–5 specifically
expressed p(þ)RLuc-(ꢁ)DV-UTRDC-FLuc and DENV NS5 expression
suppressed DENV protease activity with EC50 of 4.72 0.3 mM and
6.90 0.11 mM, respectively (Table 2). In conclusion, the results
indicated that both compound 4 and 5 suppress DENV replication
by directly inhibiting DENV protease activity.
vector pcDNA-NS5-Myc and then treated with the tested com-
pound. Our data showed that compounds 1–3 efficiently inhibited
DENV NS5 RdRp activity with EC50 of 8.07 0.26, 7.22 0.38 and
5.99 0.30 mM (Table 1 and Figure 2, panel B). To further confirm

1098
S. PELLICCIA ET AL.
Figure. 3. Inhibition of DENV RNA and NS3-protease activity by compounds 4 and 5. Panel A. Huh-7 cells were infected with DENV-2 at a multiplicity of infection
(M.O.I) of 0.2 and followed by the treatment of each DENV protease inhibitors for 3 days. The DENV RNA level was analysed by RT-qPCR with specific primer targeting
viral NS5 gene, and relative viral RNA levels were normalised against cellular GADPH mRNA levels. Treatment of 50 lM 20-C-methylcytidine (2CMC) direct against DENV
RdRp served as positive control. 0.1% DMSO (Mock) served as negative control. Panel B. Huh-7 cells were transfected with pEG(MITA)SEAP and pcDNA-NS2B-GSG-NS3-
Myc followed by incubation of each compounds, and the luciferase activity was analysed after 3 days treatment. Error bars denote the means SD of three independent
ꢄ
ꢄꢄ
experiments. p < .05; p < .01.
Figure 4. DENV RdRp inhibitor 3 protected ICR-suckling mice from DENV infection. 6-day-old ICR-suckling mice were intracerebrally injected with heat-inactive DENV
(iDENV, n ¼ 5) or active DENV (DENV, n ¼ 4). Mice-receiving DENV were treated with 10 mg/kg of compound 3 (n ¼ 5) at 1, 3, 5 dpi. Panel A, clinical scores; Panel B,
body weight and Panel C, survival rate were recorded every day. Disease severity was scored as follow: 0: healthy, 1: slightly sick (reduced mobility), 2: inappetance,
3: weight loss and difficult to move, 4: paralysis, 5: death. Each group included 6 mice. Error bars denote the means SD.
Evaluation of 3 and 4 in DENV-infected ICR-suckling mouse model
inactivated DENV-2 (iDENV) served as a mock control. The results
To access the antiviral activity of compound 3 and 4 in vivo, of clinical scores (Figures 4(A) and 5(A)) and body weight
6-day-old ICR-suckling mice were inoculated with 2.5 ꢂ 105 pfu of (Figures 4(B) and 5(B)) showed that compound 3 or compound 4
DENV-2 by intracerebral injection and then were administered treatment reduced DENV-induced pathology, including ruffled fur,
with compound 3 (10 mg/kg) or 4 (1 mg/kg) by intracerebral injec- anorexia, severe paralysis, lethargy, on DENV-infected mice
tion at 1-, 3- and 5-day postinfection (dpi); inoculation of heat- within 4 to 6 dpi. Furthermore, compound 3 or 4 treatment

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1099
Figure 5. DENV protease inhibitor 4 protected ICR-suckling mice from DENV infection. Six-day-old ICR-suckling mice were intracerebrally injected with heat-inactive
DENV (iDENV, n ¼ 6) or active DENV (DENV, n ¼ 6). Mice-receiving DENV were treated with 1 mg/kg of compound 4 (n ¼ 6) at 1, 3, 5 dpi. Panel A, clinical scores, Panel
B, body weight, and Panel C, survival rate were recorded every day. Disease severity was scored as follow: 0: healthy, 1: slightly sick (reduced mobility), 2: inappetance,
3: weight loss and difficult to move, 4: paralysis, 5: death. Each group included six mice. Error bars denote the means SD.
Synergistic effect of NS5 RdRp inhibitor 3 and NS3 protease inhibi-
tor 4 against DENV replication
To examine whether combinational treatment of NS5 RdRp inhibi-
tor 3 and NS3 protease inhibitor 4 could synergistically inhibit
DENV replication, DENV-2-infected Huh-7 cells were cotreated with
3 and 4 at the indicated concentration for 3 days. As shown in
Figure 6, a synergistic inhibition of DENV replication was observed
with the 3 and 4 combination (lines 6–9), as compared to single-
compound treatment (lines 2–5).
Conclusions
We sought to design and characterise potential anti-DENV inhibi-
tors by targeting the viral enzymatic, NS5 RNA-dependent RNA
polymerase and the NS3 protease activities. We identified five
Figure 6. Combination assay of RdRp (3) and NS3 protease (4) inhibitors. Huh-7
cells were infected with DENV-2 at a multiplicity of infection (M.O.I) of 0.2 and
followed by treatment of 3 and 4 with indicated concentration for 3 days. The
DENV RNA level was analysed by RT-qPCR with specific primer targeting viral
NS5 gene, and relative viral RNA levels were normalised against cellular GADPH
mRNA levels. Error bars denote the means SD of three independent experi-
potential compounds demonstrating anti-DENV replication activity
without cytotoxicity. Two compounds targeting DENV NS3 prote-
ase and NS5 RdRp also exhibited anti-DENV activity in ICR-suckling
mouse model of DENV infection. Combination treatment exhibited
enhanced inhibition of DENV replication. Taken together, these
results demonstrate that both classes of small molecules inhibited
DENV replication and represent prototypical DAA molecules for
further development through compound modification.
ꢄ
ꢄꢄ
ments. p < .05; p < .01.
significantly protected the mice against life-threatening DENV
infection, as compared to the untreated mice (Figures 4(C) and
5(C)). Collectively, these results demonstrated that both classes of
Acknowledgements
small
molecules,
targeting
polymerase
and
protease
activities, inhibited DENV replication and represent prototypical
DAA molecules for further development through compound
modification.
The authors would like to thank Dr. Huey-Nan Wu (Institute of
Molecular Biology, Academia Sinica, Taipei, Taiwan) for giving
DENV-2 (type-2 strain 16681) and Dr. Charles Rice (Rockefeller

1100
S. PELLICCIA ET AL.
University and Apath, LCC, USA) for kindly providing human hepa- 14. Khromykh AA, Kenney MT, Westaway EG. Trans-complemen-
toma Huh-7 cells.
tation of flavivirus RNA polymerase gene NS5 by using
Kunjin virus replicon-expressing BHK cells.
J
Virol
1988;72:7270–9.
Disclosure statement
15. Rawlinson SM, Pryor MJ, Wright PJ, Jans DA. Dengue virus
RNA polymerase NS5: a potential therapeutic target? Curr
Drug Targets 2006;7:1623–38.
16. Tatem AJ, Hay SI, Rogers DJ. Global traffic and disease vector
dispersal. Proc Natl Acad Sci USA 2006;103:6242–7.
17. Halstead SB. Licensed Dengue vaccine: public health
conundrum and scientific challenge. Am J Trop Med Hyg
2016;95:741–5.
The authors report no declarations of interest. The authors alone
are responsible for the content and writing of this article.
Funding
This work was supported by grant from the Ministry of Science
and Technology of Taiwan [MOST104–2320-B-037–025-MY3], and
Kaohsiung Medical University under Aim for the Top Universities,
Taiwan [KMU-TP105H02].
18. Guy E, Lang J, Saville M, Jackson N. Vaccination against
Dengue: challenges and current developments. Ann Rev
Med 2016;67:387–404.
19. Noble CG, Chen YG, Dong H, et al. Strategies for develop-
ment of dengue virus inhibitors. Antivir Res 2010;8:5450–62.
20. Lim SP, Christian G, Noble CG, Shi PY. The dengue virus NS5
References
protein as
2015;119:57–67.
a target for drug discovery. Antiviral Res
1. Rothman AL. Immunity to dengue virus: a tale of original
antigenic sin and tropical cytokine storms. Nat Rev Immunol
2011;11:532–43.
2. Halstead SH. Dengue virus-mosquito interactions. Annu Rev
Entomol 2008;53:273–91.
21. Tomlinsona SM, Malmstroma RD, Russoa A, et al. Structure-
based discovery of dengue virus protease inhibitors.
Antiviral Res 2009;82:110–14.
22. Silvestri R, Cascio MG, La Regina G, et al. Synthesis, canna-
binoid receptor affinity and molecular modeling studies of
3. Wilder-Smith A, Ooi EE, Vasudevan SG, Gubler DJ. Update
on dengue: epidemiology, virus evolution, antiviral drugs,
and vaccine development. Curr Infect Dis Rep 2010;12:
157–64.
substituted
1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxa-
mides. J Med Chem 2008;51:1560–76.
23. La Pietra V, La Regina G, Coluccia A, et al. Design, synthesis,
and biological evaluatio of 1-phenylpyrazolo[3,4-e]pyr-
rolo[3,4-g]indolizine-4,6(1H,5H)-diones as new glycogen syn-
thase kinase-3b inhibitors. J Med Chem 2013;56:10066–78.
24. Manvar D, Pelliccia S, La Regina G, et al. New 1-phenyl-5-
(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides inhibit hepatitis
C virus replication via suppression of cyclooxygenase-2. Eur J
Med Chem 2015;90:497–506.
4. Roche C, Cassar O, Laille M, Murgue B. Dengue-3 virus gen-
omic differences that correlate with in vitro phenotype on a
human cell line but not with disease severity. Microbes
Infect 2007;9:63–9.
5. Shurtleff AC, Beasley DW, Chen JJ, et al. Genetic variation in
the 3' non-coding region of dengue viruses. Virology
2001;281:75–87.
6. Silva RLA, de Silva AM, Harris E, MacDonald GH. Genetic ana-
lysis of dengue 3 virus subtype III 50 and 30 non-coding
regions. Virus Res 2008;135:320–5.
7. Vasilakis N, Weaver SC. The history and evolution of human
dengue emergence. Adv Virus Res 2008;72:1–76.
25. Khan MF, Alam MM, Verma G, et al. The therapeutic voyage
of pyrazole and its analogs: a review. Eur J Med Chem
2016;120:170–201.
26. Corelli F, Massa S, Stefancich G, et al. Agenti antiinfiamma-
tori non steroidei. Nota V. - Sntesi di acidi 1-aril-5-(1-pirril)-
8. Zhou Y, Mammen MP, Klungthong C, et al. Comparative ana-
lysis reveals no consistent association between the second-
ary structure of the 3’-untranslated region of dengue viruses
and disease syndrome. J Gen Virol 2006;87:2595–603.
9. Dejnirattisai W, Jumnainsong A, Onsirisakul N, et al. Cross-
reacting antibodies enhance dengue virus infection in
humans. Science 2010;328:745–8.
10. Beltramello M, Williams KL, Simmons CP, et al. The human
immune response to dengue virus is dominated by highly
cross-reactive antibodies endowed with neutralizing and
enhancing activity. Cell Host Microbe 2010;8:271–83.
11. de Alwis R, Beltramello M, Messer WB, et al. In-depth ana-
lysis of the antibody response of individuals exposed to pri-
mary dengue virus infection. PLoS Negl Trop Dis
2011;6:e1188.
ꢀ
a potenziale attivita antiinfiammatoria.
pirazolil-4-acetici
Farmaco 1988;43:251–65.
27. Erbel P, Schiering N, D'Arcy A, et al. Structural basis for the
activation of flaviviral NS3 proteases from dengue and West
Nile virus. Nat Struct Mol Biol 2006;13:372–3.
28. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ.
Experimental and computational approaches to estimate
solubility and permeability in drug discovery and develop-
ment settings. Adv Drug Deliv Rev 2001;46:3–26.
29. Korb O, Stutzle T, Exner TE, PLANTS: Application of ant col-
ony optimization to structure-based drug design. In: Dorigo
M, Gambardella LM, Birattari M, Martinoli A, Poli R, Stutzle T,
eds. Ant colony optimization and swarm intelligence.
Proceedings of the 5th International Workshop, ANTS,
Lecture Notes in Computer Science, Series 4150, Berlin:
Springer; 2006:47–258.
12. Smith SA, de Alwis AR, Kose N, et al. Isolation of dengue
virus-specific memory B cells with live virus antigen from
human subjects following natural infection reveals the pres- 30. Timiri AK, Sinha BN, Jayaprakash V. Progress and prospects
ence of diverse novel functional groups of antibody clones.
on DENV protease inhibitors. Eur
J
Med Chem
J Virol 2014;88:12233–41.
2016;117:125–43.
13. Yotmanee P, Rungrotmongkol T, Wichapong K, et al. Binding 31. PyMOL, v1.8.6.0, release March 9, 2017. Available from:
specificity of polypeptide substrates in NS2B/NS3pro serine
https://www.pymol.org.
protease of dengue virus type 2: a molecular dynamics 32. Lin YT, Wu YH, Tseng CK, et al. Green tea phenolic epicate-
study. J Mol Graph Model 2015;60:24–33. chins inhibit hepatitis C virus replication via cycloxygenase-2

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1101
and attenuate virus-induced inflammation. PLoS One 40. Tannous BA. Gaussia luciferase reporter assay for monitoring
2013;8:e54466.
biological processes in culture and in vivo. Nat Protoc
33. Lee JC, Tseng CK, Wu YH, et al. Characterization of the activ-
2009;4:582–91.
ity of 2'-C-methylcytidine against dengue virus replication. 41. Tomlinson SM, Watowich SJ. Anthracene-based inhibitors of
Antiviral Res 2015;116:1–9.
dengue
virus
NS2B-NS3
protease.
Antiviral
Res
34. Wu SF, Lin CK, Chuang YS, et al. Anti-hepatitis C virus activ-
2011;89:127–35.
ity of 3-hydroxy caruilignan C from Swietenia macrophylla 42. 7-Amino-4-methylcoumarin (AMC) fluorophore-linked pep-
stems. J Viral Hepat 2012;19:364–70.
tide substrate Boc-GRR-AMC. Bachem, CA, USA; 2016.
Available from: http://www.bachem.com/ [last accessed 29
Nov 2016].
35. Mayani M, Filipe CDM, Ghosh R. Cascade ultrafiltration sys-
tems integrated processes for purification and concentration
of lysozyme. J Membrane Sci 2010;347:150–8.
36. Hartwig S, Raschke S, Knebel B, et al. Secretome profiling of
primary human skeletal muscle cells. Biochim Biophys Acta
2014;1844:1011–17.
37. Chao WW, Hong YH, Chen ML, Lin BF. Inhibitory effects of
Angelica sinensis ethyl acetate extract and major compounds
on NF-jB trans-activation activity and LPS-induced inflam-
mation. J Ethnopharmacol 2010;129:244–9.
43. Yildiz M, Ghosh S, Bell AJ, et al. Allosteric inhibition of the
NS2B-NS3 protease from dengue virus. ACS Chem Biol
2013;8:2744–52.
44. Mukhametov A, Newhouse EI, Ab Aziz N, et al.
Allosteric pocket of the dengue virus (serotype 2)
NS2B/NS3 protease: in-silico ligand screening and
molecular dynamics studies of inhibitors. J Mol Graph
Model 2014;52:103–13.
38. Hu G, Lou Z, Gupta M. The long non-coding RNA GAS5 45. Chandramouli S, Joseph JS, Daudenarde S, et al.
cooperates with the eukaryotic translation initiation factor
4E to regulate c-Myc translation. PLoS One 2014;9:e107016.
39. Tseng CK, Lin CK, Wu YH, et al. Human heme oxygenase 1 is
Serotype-specific structural differences in the protease-
cofactor complexes of the dengue virus family. J Virol
2010;84:3059–67.
a potential host cell factor against dengue virus replication. 46. Protein data bank. Available from: http://www.rcsb.org/pdb/
Sci Rep 2016;6:32176. home/home.do [last accessed 6 Dec 2016].