Synthesis, antiviral evaluation and molecular docking studies of N4-aryl substituted/unsubstituted thiosemicarbazones derived from 1-indanones as potent anti-bovine viral diarrhea virus agents

Article abstract of DOI:10.1016/j.bmc.2017.05.056

A series of N⁴-arylsubstituted thiosemicarbazones derived from 1-indanones and a set of compounds lacking such substitution in the N⁴ position of the thiosemicarbazone moiety were synthesized and evaluated for their anti-bovine viral

Full text of DOI:10.1016/j.bmc.2017.05.056

Accepted Manuscript
Synthesis, antiviral evaluation and molecular docking studies of N 4-aryl sub-
stituted/unsubstituted thiosemicarbazones derived from 1-indanones as potent
anti-bovine viral diarrhea virus agents
María C. Soraires Santacruz, Matías Fabiani, Eliana F. Castro, Lucía V.
Cavallaro, Liliana M. Finkielsztein
PII:
S0968-0896(17)30432-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.05.056
BMC 13775
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 March 2017
22 May 2017
26 May 2017
Please cite this article as: Soraires Santacruz, M.C., Fabiani, M., Castro, E.F., Cavallaro, L.V., Finkielsztein, L.M.,
Synthesis, antiviral evaluation and molecular docking studies of N 4-aryl substituted/unsubstituted
thiosemicarbazones derived from 1-indanones as potent anti-bovine viral diarrhea virus agents, Bioorganic &
Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.05.056
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Synthesis, antiviral evaluation and molecular docking studies of N4-aryl
substituted/unsubstituted thiosemicarbazones derived from 1-indanones as
potent anti-bovine viral diarrhea virus agents
c,
c
d,
María C. Soraires Santacruza,b,‡, Matías Fabiani , Eliana F. Castro , Lucía V. Cavallaro *,
‡
Liliana M. Finkielszteina,b,*.
a
Cátedra de Química Medicinal, Departamento de Farmacología, Facultad de
Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113, Ciudad
Autónoma de Buenos Aires, Argentina
b
Instituto de Química y Metabolismo del Fármaco (IQUIMEFA), Consejo Nacional de
Investigaciones Científicas y Técnicas, Facultad de Farmacia y Bioquímica,
Universidad de Buenos Aires, Junín 956, 1113, Ciudad Autónoma de Buenos Aires,
Argentina
c
Cátedra de Virología, Departamento de Microbiología, Inmunología y Biotecnología,
Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Farmacia y
Bioquímica, Universidad de Buenos Aires, Junín 956, 1113, Ciudad Autónoma de
Buenos Aires, Argentina
d
Cátedra de Virología, Departamento de Microbiología, Inmunología y Biotecnología,
Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113,
Ciudad Autónoma de Buenos Aires, Argentina.
* Corresponding authors. Tel: +541152874852, E-mail address: lfinkiel@ffyb.uba.ar
(L. Finkielsztein) and Tel: +541152874474, E-mail address: lcavalla@ffyb.uba.ar (L.
Cavallaro)
‡
These authors contributed equally to this paper

Abstract
A series of N4-arylsubstituted thiosemicarbazones derived from 1-indanones and a set of
compounds lacking such substitution in the N4 position of the thiosemicarbazone moiety
were synthesized and evaluated for their anti-bovine viral diarrhea virus (BVDV) activity.
Among these, derivatives 2 and 15 displayed high activity (EC50= 2.7 0.4 and 0.7 0.1
µM, respectively) as inhibitors of BVDV replication. Novel key structural features related to
the anti-BVDV activity were identified by structure-activity relationship (SAR) analysis. In a
previous study, the thiosemicarbazone of 5,6-dimethoxy-1-indanone (5,6-TSC) was
characterized as a non-nucleoside inhibitor (NNI) of the BVDV RNA-dependent RNA
polymerase. In the present work, cross-resistance assays were performed with the most
active compounds. Such studies were carried out on 5,6-TSC resistant BVDV (BVDV-TSCr
T1) carrying mutations in the viral polymerase. This BVDV mutant was also resistant to
compound 15. Molecular docking studies and MM/PBSA calculations were performed to
assess the most active derivatives at the 5,6-TSC viral polymerase binding site. The
differences in the interaction pattern and the binding affinity of derivative 15 either to the
wild type or BVDV-TSCr T1 polymerase were key factors to define the mode of action of
this compound.
Keywords
Thiosemicarbazones, Bovine viral diarrhea virus (BVDV), RNA-dependent RNA
polymerase, Molecular docking, Antiviral activity, Antiviral resistance.

1. Introduction
Pestiviruses are the causative agents of serious infectious diseases in animals destined to
human consumption. Such diseases are caused by the bovine viral diarrhea virus (BVDV)
in bovines, the classical swine fever virus (CSFV) in swine, and the border disease virus
(BDV) in sheep and goats. BVDV, which is one of the best characterized members of the
Pestivirus genus, is an enveloped single-stranded positive polarity RNA virus. BVDV has a
worldwide distribution and the percentages of persistently infected animals reaches 1-2%
and a 60-80% present serum anti-BVDV antibodies.1-3 In Argentina, the prevalence of anti-
BVDV antibodies in adult cattle is roughly 70%.4
The economic losses caused by BVDV infections are associated with reduced milk
production, lower conception rates, respiratory disorders and death during the acute
infection. Fetal infection during early gestation stages may cause miscarriage, congenital
defects, growth retardation, and may lead to the birth of persistently infected calves, which
eventually develop a lethal mucosal disease.5 Moreover, BVDV infections have a negative
impact on the livestock reproductive performance.6
The prevention and control of these infections combine systemic vaccination and detection
and removal of persistently infected animals. Vaccination programs are used extensively;7
however, conventional vaccines (formulations with either attenuated or inactivated virus)
may not be optimal for controlling BVDV infections.8 Consequently, there is a need for
effective and quick control tools; hence the use of antiviral agents may be useful for this
purpose.
As the genome organization, translation, replication strategy and protein functions of
pestiviruses closely resemble those of hepatitis C virus (HCV), the sole member of the
Hepacivirus genus, BVDV, has also been adopted as a surrogate model for HCV studies.9
In a previous work, we synthesized a series of sixteen thiosemicarbazones (TSCs) derived
from 1-indanones with different substitution patterns in the aromatic ring. These TSCs

were evaluated for their antiviral activity against BVDV by the viral cytopathic effect
inhibition method.10 The most potent compound was the TSC derived from 5,6-dimethoxy-
1-indanone (5,6-TSC) (Fig. 1) (EC50 = 1.8 0.6 µM) with a selectivity index (SI) against
BVDV of 80. It has been demonstrated that this compound acts by blocking the viral RNA
synthesis both in cell culture and in isolated BVDV replication complexes.11 Five
independent populations of 5,6-TSC-resistant BVDV (BVDV-TSCr; T1–5) (5,6-TSC EC50
>80.0 µM) were selected. These populations carry an NS5B N264D mutation in the NS5B
region of the viral genome encoding the BVDV RNA-dependent RNA polymerase (RdRp),
whereas BVDV-TSCr T1 also bears an NS5B A392E mutation.11
Docking studies performed with the crystal structure of BVDV RdRp (PDB entry 1S48) and
5,6-TSC have shown significant differences between the wild type (wt) and the mutated
form of the viral RdRp, both in terms of the amino acids involved in the interaction as well
as the orientation of the ligand. These studies allowed concluding that 5,6-TSC is a non-
nucleoside polymerase inhibitor (NNI) of BVDV.
Fig.1. Thiosemicarbazone derived from 5,6-dimethoxy-1-indanone (5,6-TSC)
In previous studies, a series of N4-arylsubstituted TSCs derived from 1-indanones (N4-
TSCs) were synthesized with the aim of improving the lipophilicity degree of TSCs.12 In the
present work, we investigated the anti-BVDV activity of a set of twelve N4-TSCs derived
from 1-indanones, six of which are not substituted and six, bear a 5,6-dimethoxy
substitution in the indanic ring. Six of the twelve compounds are new derivatives.
Moreover, four TSCs that did not bear any substitution in the N4 position of the
thiosemicarbazone moiety were also evaluated. The structural requirements for TSCs and

N4-TSCs optimal activity are discussed. Furthermore, cross-resistance assays of the most
active compounds against BVDV-TSCr T1 were performed. A molecular docking study was
carried out to establish the differential interactions between wild type and mutated viral
RdRp. Finally, molecular dynamics simulations were carried out to complement the
experimental data of cross-resistance assays and to provide insights into the probable
mode of action of the most active compounds.
2. Materials and methods
2.1. Chemistry
Melting points (uncorrected) were determined on a Thomas Hoover apparatus. Thin layer
chromatography (TLC) was used to monitor reactions. IR spectra were recorded as KBr
1
pellets using a Perkin Elmer Spectrum One FT-IR spectrophotometer. H and 13C NMR
spectra were recorded on a Bruker 500 MHz spectrometer. High resolution mass spectra
were acquired on Bruker micrOTOF-Q II spectrometer. 1-indanone, 5,6-dimethoxy-1-
indanone, 4-fluorophenyl isothiocyanate, 4-nitrophenyl isothiocyanate, and 4-
methoxyphenyl isothiocyanate were purchased from Sigma-Aldrich and used as received.
2.1.1. General procedure for the synthesis of N4-TSCs
Method A: In the first step, a mixture of 1-indanone or 5,6-dimethoxy-1-indanone (3.8
mmol) and hydrazine hydrate 85% (0.9 mL) in isopropanol (5 mL) was heated under reflux
for 1 h. After cooling, the solvent was evaporated and the residue was dissolved in CH2Cl2
(20 mL), dried over anhydrous Na2SO4 and evaporated again to render the crude
derivative. Hydrazones were recrystallized from methanol. The physical parameters of the
compounds obtained were in agreement with literature data.13 In the second step, a
solution of the corresponding isothiocyanate (3.0 mmol) in benzene (5 mL) was added
dropwise to a solution of the hydrazone (2.5 mmol) in benzene (10 mL). The resulting

mixture was stirred at room temperature until the hydrazone was consumed. The
hydrazone consumption was monitored by TLC. The solid obtained was filtered and
washed with benzene (5 mL) and with ethanol (2 mL).
Method B: A mixture of 1-indanone or 5,6-dimethoxy-1-indanone (0.38 mmol), 85%
hydrazine hydrate (21 µL), the corresponding isothiocyanate (0.43 mmol), acetic acid (20
µL) and isopropanol (0.5 mL) contained in a glass tube equipped with a screw cap and
magnetic agitation was placed in a microwave synthesis reactor (Microwave 300 Anton
Paar) at 90°C (30 W, 2.5 bar). Once the reaction was completed, as monitored by TLC,
the obtained mixture was suspended in water (5 mL), filtered and washed with ethanol (2
mL). N4-TSCs were recrystallized from ethanol.
2.1.1.1. Indan-1-one N-(4-fluorophenyl)thiosemicarbazone (8). Mp: 170-172°C. IR v/cm-1
(KBr): 3279 (N-H), 3199 (N-H), 1606 (C=N), 1092 (C=S). 1H NMR (CDCl3) δ ppm: 2.85 (m,
2H, CH2), 3.22 (m, 2H, CH2), 7.10 (m, 2H, H-Ar), 7.33 (t, J= 7.44 Hz, 1H, H-Ar), 7.38 (d, J=
7.59 Hz, 1H, H-Ar), 7.42 (t, J= 7.37 Hz, 1H, H-Ar), 7.62 (dd, J= 8.84, 4.81 Hz, 2H, H-Ar),
13
7.77(d, 7.72 Hz, 1H, H-Ar), 8.56 (s, 1H, NH), 9.26 (s, 1H, NH). C NMR (CDCl3) δ ppm:
26.8, 28.7, 115.7, 115.8, 121.9, 125.9, 126.7, 126.7, 127.6, 131.6, 134.1, 136.8, 149.0,
157.4, 161.7, 176.6. HRMS (ESI) m/z (M+H)+ calcd for C16H15FN3S 300.09652, found
300.09646.
2.1.1.2. Indan-1-one N-(4-nitrophenyl)thiosemicarbazone (9). Mp: 171-172°C. IR v/cm-1
(KBr): 3362 (N-H), 3218 (N-H), 1597 (C=N), 1085 (C=S). 1H NMR (CDCl3) δ ppm: 2.87 (m,
2H, CH2), 3.24 (m, 2H, CH2), 7.36 (t, J= 7.52 Hz, 1H, H-Ar), 7.40 (d, J= 7.62 Hz, 1H, H-Ar),
7.46 (t, J= 7.01 Hz, 1H, H-Ar), 7.80 (d, J= 7.71 Hz, 1H, H-Ar), 8.08 (d, J= 9.05Hz, 2H, H-
Ar), 8.28(d, J= 9.08 Hz, 2H, H-Ar), 8.61 (s, 1H, NH), 9.71 (s, 1H, NH). 13C NMR (CDCl3) δ
ppm: 27.0, 28.7, 122.0, 122.4, 124.8, 126.0, 126.5, 127.7, 132.0, 136.5, 144.1, 144.5,

149.3, 158.2, 175.1. HRMS (ESI) m/z (M+H)+ calcd for C16H15N4O2S 327.09102, found
327.09136.
2.1.1.3. Indan-1-one N-(4-methoxyphenyl)thiosemicarbazone (10). Mp: 172-173°C. IR
1
v/cm-1 (KBr): 3308 (N-H), 3210 (N-H), 1594 (C=N), 1091 (C=S). H NMR (CDCl3) δ ppm:
2.83 (m, 2H, CH2), 3.21 (m, 2H, CH2), 3.83 (s, 3H, OCH3), 6.94 (d, J= 8.21 Hz, 2H, H-Ar),
7.32 (t, J= 7.21 Hz, 1H, H-Ar), 7.38 (d, J= 7.58 Hz, 1H, H-Ar), 7.41 (t, J= 7.66 Hz, 1H, H-
Ar), 7.53 (d, J= 8.13 Hz, 2H, H-Ar), 7.77 (d, 7.42 Hz, 1H, H-Ar), 8.47 (s, 1H, NH), 9.20 (s,
1H, NH). 13C NMR (CDCl3) δ ppm: 26.7, 28.7, 55.6, 114.2, 121.9, 125.9, 126.6, 126.7,
127.5, 131.1, 131.4, 137.0, 148.9, 156.9, 157.0, 158.1, 176.8. HRMS (ESI) m/z (M+H)+
calcd for C17H18N3OS 312.11651, found 312.11651.
2.1.1.4. 5,6-Dimethoxyindan-1-one N-(4-fluorophenyl)thiosemicarbazone (14). Mp: 185-
1
186°C. IR v/cm-1 (KBr): 3300 (N-H), 3196 (N-H), 1605 (C=N), 1078 (C=S). H NMR
(CDCl3) δ ppm: 2.86 (m, 2H, CH2), 3.15 (m, 2H, CH2), 3.95 (s, 3H, OCH3), 3.96 (s, 3H,
OCH3), 6.85 (s, 1H, H-Ar), 7.12 (dd, J= 8.61, 8.52 Hz, 2H, H-Ar), 7.18 (s, 1H, H-Ar), 7.61
(dd, J= 8.76, 4.83 Hz, 2H, H-Ar), 8.52 (s, 1H, NH), 9.19 (s, 1H, NH). 13C NMR (DMSO-d6)
δ ppm: 28.41, 28.58, 56.10, 56.28, 104.9, 108.3, 115.2, 128.8, 129.9, 136.2, 143.0, 149.3,
152.6, 159.0, 160.2, 177.0. HRMS (ESI) m/z (M+H)+ calcd for C18H19FN3O2S 360.11765,
found 360.11645.
2.1.1.5. 5,6-Dimethoxyindan-1-one N-(4-nitrophenyl)thiosemicarbazone (15). Mp: 214-216
°C. IR v/cm-1 (KBr): 3273 (N-H), 3191 (N-H), 1598 (C=N), 1075 (C=S). 1H NMR (CDCl3) δ
ppm: 2.86 (m, 2H, CH2), 3.14 (m, 2H, CH2), 3.94 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 6.84
(s, 1H, H-Ar), 7.16 (s, 1H, H-Ar), 8.04 (d, J= 8.93 Hz, 2H, H-Ar), 8.26 (d, J= 8.93 Hz, 2H,
13
H-Ar), 8.56 (s, 1H, NH), 9.58 (s, 1H, NH). C NMR (DMSO-d6) δ ppm: 28.6, 28.7, 56.1,

56.3, 105.1, 108.3, 112.9, 124.7, 126.9, 129.5, 143.7, 146.1, 149.3, 152.9, 160.6, 175.7.
HRMS (ESI) m/z (M+H)+ calcd for C18H19N4O4S 387.11215, found 387.11081.
2.1.1.6. 5,6-Dimethoxyindan-1-one N-(4-methoxyphenyl)thiosemicarbazone (16). Mp: 198-
1
199 °C. IR v/cm-1 (KBr): 3312 (N-H), 3216 (N-H), 1603 (C=N), 1075 (C=S). H NMR
(CDCl3) δ ppm: 2.82 (m, 2H, CH2), 3.12 (m, 2H, CH2), 3.82 (s, 3H, OCH3), 3.92 (s, 3H,
OCH3), 3.93 (s, 3H, OCH3), 6.83 (s, 1H, H-Ar), 6.93 (d, J= 9.00 Hz, 2H, H-Ar), 7.15 (s, 1H,
H-Ar), 7.49 (d, J= 8.70 Hz, 2H, H-Ar), 8.47 (s, 1H, NH), 9.11 (s, 1H, NH). 13C NMR
(DMSO-d6) δ ppm: 28.3, 28.6, 55.8, 56.1, 56.3, 104.9, 108.3, 113.8, 128.3, 130.0, 132.7,
142.9, 149.3, 152.5, 157.5, 158.6, 177.1. HRMS (ESI) m/z (M+H)+ calcd for C19H22N3O3S
372.13764, found 372.13836.
2.2. Biology
2.2.1. Cells and viruses
Madin Darby bovine kidney cells (MDBK NBL-1; ATCC CCL-22) were grown in Eagle’s
Minimal Essential Medium (E-MEM), supplemented with 10% γ-irradiated fetal bovine
serum (FBS, Internegocios), 2 mM L-glutamine, 100ꢀµM nonessential amino acids (Gibco),
100ꢀµg/mL streptomycin, and 100ꢀIU/mL penicillin (Growing medium, GM). Bovine viral
diarrhea virus (BVDV) type 1 NADL strain, cytopathic biotype (BVDV-1, ATCC VR 534)
was provided by Dr. Laura Weber, INTA Castelar, Buenos Aires, Argentina. The 5,6-TSC-
resistant BVDV (BVDV-TSCr T1) carrying both N264D and A392E mutations in the NS5B
region had previously been selected in our laboratory.11
2.2.2. Compounds
All compounds were diluted in dimethyl sulfoxide at 5 or 1 mg/ml immediately before use.
Further serial dilutions were made in Infection Medium (IM) (E-MEM, supplemented with

2.5% donor horse serum-DHS, Gibco, 2 mM L-glutamine, 100ꢀµM nonessential amino
acids, 100ꢀ µg/mL streptomycin and 100ꢀIU/mL penicillin).
2.2.3. Cytotoxicity assay
MDBK cells were resuspended in GM and seeded at a density of 1x 104 cells per well in a
96-well cell culture plate. After 24 h of incubation at 37°C, 5% CO2, GM was removed and
serial dilutions of the test compounds were added. Mock-treated cells were included as
control (MT). Cells were allowed to proliferate for 3 days at 37°C and then cell viability was
estimated by the MTS/PES method (Cell Titer 96 Aqueous One Solution Cell Proliferation
Assay, Promega), following the manufacturer´s instructions. After incubating for 2.5 h at
37°C, the absorbance values were determined at 490 nm, which is directly proportional to
the number of viable cells. The cell viability percentage was calculated as follows: Cell
viability (%) = AbsTSC x 100/ AbsMT. The cytotoxic concentration 50% (CC50) was defined as
the compound concentration that inhibited the proliferation of exponentially growing cells
by 50% and calculated from dose-response curves using GraphPad Prism version 5.00 for
Windows. Three independent experiments were carried out in quadruplicate.
2.2.4. Plaque reduction assay for BVDV
MDBK cells were resuspended in GM and seeded at a density of 1.3 x105 cells per well in
a 24-well culture plate. After incubating for 24 h in a 5% CO2 incubator at 37°C, GM was
removed, cells were washed with Phosphate buffer saline (PBS) and then infected with
100 plaque-forming units (PFU) of BVDV. After 1 h of incubation at 37 ºC, the inoculum
was removed and cell monolayers were washed twice with PBS. After washing, 0.2 ml of
serial dilutions (1:2) of each compound in IM and 0.8 ml of overlay medium (IM
supplemented with 1% methylcellulose) were added to each well. Mock-infected cells with
and without compound and infected cells without compound (infected untreated control -

UTC), were included as controls. After incubating for 72 h at 37°C with 5% CO2, cells were
fixed with 10% formalin, stained with 0.75% crystal violet and the viral plaques counted.
The reduction of plaques (%) at each compound concentration (TSCx) was calculated as
[1-(no. plaques TSCx/no. plaques UTC)] x 100. The effective concentration 50% (EC50) was
defined as the compound concentration that caused a reduction in the number of viral
plaques by 50% with respect to the UTC and was determined from dose-response curves
using GraphPad Prism version 5.00 for Windows. Three independent experiments were
carried out in triplicate. Finally, the selectivity index (SI) was calculated as CC50/EC50.
2.2.5. Cytopathic effect reduction assay
MDBK cells were resuspended in GM and seeded at a density of 1 x 104 cells per well in a
96-well culture plate. After incubating for 24 h in a 5% CO2 incubator at 37°C, cells were
infected with BVDV at a multiplicity of infection (MOI) of 0.01 PFU/cell. Serial dilutions of
each TSC were tested in quadruplicate. Mock-infected cells (MI) and infected cells without
compound (UTC) were included as controls. After incubating for 72 h at 37°C, the
cytopathic effect was determined by measuring cell viability by the MTS/PES method (Cell
Titer 96 Aqueous One Solution Cell Proliferation Assay, Promega) as described above. The
reduction of viral cytopathic effect (CPE) was calculated as follows: CPE reduction (%) =
(AbsTSCx –AbsUTC) X100 / (AbsMI – AbsUTC). The EC50 was defined as the concentration of
compound that reduced the CPE by 50% and was calculated from dose-response curves
using GraphPad Prism version 5.00 for Windows. Two independent experiments were
performed in quadruplicate.
2.2.6. Time-of-drug-addition study: effect on viral replication
MDBK cells were resuspended in GM and seeded at a density of 1x104 cells per well in a
96-well culture plate. After incubating for 24 h in a 5% CO2 incubator at 37°C, cells were

precooled for 30 min on ice and then infected with BVDV (MOI = 1). After 90 min of
adsorption on ice, the inoculum was removed, and monolayers were washed twice with
PBS and incubated in IM. At different time points post-infection (p.i.), a 25 µM solution of
compound 2 (10 x EC50) was added. This procedure was repeated up to 12 h p.i. After
incubating for 48 h at 37°C, the CPE was determined by measuring cell viability by the
MTS/PES method (Cell Titer 96 Aqueous One Solution Cell Proliferation Assay, Promega)
as described above. Mock-infected and infected cells without compound were added as
controls. Three independent experiments were carried out in quadruplicate.
2.3. Molecular docking study
Docking studies were carried out using Autodock 4.2 program14 for 5,6-TSC and the more
potent compounds 2 and 15. To this end, the X-ray structure of the RdRp of BVDV was
obtained from Protein Data Bank (http://www.rcsb.org) (PDB entry 1S48).
Selenomethionine residues were modified back to methionine residues, polar hydrogens
were added and the water molecules were removed. The enzyme molecule was optimized
with NAMD 2.9 software15 using the CHARMM force field in order to minimize
crystallographic-induced bond clashes. The 3D structures of the compounds were
constructed using HyperChem 8.0.7 software and were pre-optimized with the MM+
procedure. The resulting geometries were refined by means of the AM1 semi-empirical
method. A full geometry optimization of minimum energy conformations was subsequently
carried out at the HF/6-31G(d) ab initio level using Gaussian 09. The ligand files were
prepared in pdbqt format using Autodock Tools 1.5.6. The putative binding site for the
selected compounds on the BVDV RdRp was determined using the metapocket 2.0
server.16 By this procedure, three putative binding sites on the surface of the enzyme were
found; however, only one was located in the region where mutations of RdRp had been
observed. Hence, this site was chosen to carry out the molecular docking procedure. An

affinity grid with a dimension of 60 x 60 x 60 points in the xyz plane and a 0.375 Å
spacing was generated with the AutoGrid and centered on the chosen site. Ligands were
fully flexible during the procedure whereas the protein remained rigid. The docking was
carried out with 1000 runs using the Lamarckian genetic algorithm (LGA). The final
selection of binding mode was made according to the estimated free binding energy and
size of clusters. The binding interactions were visualized by the Chimera 1.8 software.17
2.4. MD simulations and binding free energy calculation
Best docked poses were used as the starting structures for molecular dynamics (MD).
Simulations were performed with NAMD 2.9 software using CHARMM36 and CHARMM
general force field to represent the protein-ligand systems.18,19 Each complex was solvated
in a cubic TIP3P water20 with the minimal distance of 10 Å from the protein surface to the
edge of the box and chlorine atoms were added in order to neutralize the system. Energy
minimizations were performed with the protein being restrained, followed by second
energy minimization without restrain. The temperature was gradually increased to 300 K
during 300 ps using a NVT ensemble and then equilibrated over a period of 3 ns at the
reference pressure of 1 atm and a temperature of 300 K. The MD productions were carried
out for 6 ns under NPT conditions and an integration time step of 2 fs. The particle mesh
Ewald (PME) was used to treat long-range electrostatics interactions;21 the SHAKE
algorithm22 was applied to all hydrogen atoms and the van der Waals cutoff was set to 10
Å. The simulations trajectories were analyzed using VMD.23
The binding free energies (∆Gbind) were estimated via the widely used Molecular
Mechanic/Poisson Boltzmann Surface Area (MM/PBSA) method.24-27 According to this
approach, the free energy was calculated for each ligand/protein system as described by
the following equations:

where ∆EMM describes the molecular mechanical energies, ∆GPB and ∆GNP define the polar
and non-polar solvation energy, respectively. The entropic contribution was obtained by
performing normal mode analysis in order to be able to calculate the total ∆G.
3. Results and Discussion
3.1. Synthesis
TSCs 1-4 (Table 1)12 were synthesized with high yield from the corresponding 1-indanones
treated with thiosemicarbazide, according to the general procedure described previously.10
N4-TSCs 5-16 were obtained through two different synthetic methodologies.12 The first
approach (Method A, Scheme 1) involved a procedure comprising two steps: (1) synthesis
of hydrazone from the corresponding 1-indanone and hydrazine hydrate, (2) preparation of
the desired N4-TSCs by reaction of hydrazone with the suitable isothiocyanate. The
second one (Method B, Scheme 1) was a one-pot multicomponent coupling reaction
method performed under microwave irradiation (MW). The second route afforded N4-TSCs
in short reaction times (hours for Method A and minutes for Method B). Comparative
results obtained in both synthetic processes for new derivatives (8-10 and 14-16) are
shown in Table 2. The yields were higher when N4-TSCs were prepared by Method B,
except for compound 10. The structure of new compounds was confirmed by 1H- and 13C-
NMR, IR and HRMS data. The general structure of the TSCs and N4-TSCs studied is
shown in Table 1.

Table 1. Synthesized TSCs and N4-TSCs
Compound
R1
NO2
H
R2
H
R3
H
R4
H
R5
-
1
2
H
NO2
H
H
-
3
H
F
H
-
4
CH3
H
Br
H
CH3
H
-
5
H
H
H
6
H
H
H
H
CH3
Cl
7
H
H
H
H
8
H
H
H
H
F
9
H
H
H
H
NO2
OCH3
H
10
11
12
13
14
15
16
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
CH3
Cl
H
H
H
H
F
H
H
NO2
OCH3
H
H

Scheme 1. Synthetic routes for the obtention of N4-TSCs. Reaction conditions: (i) 85% hydrazine
hydrate/isopropanol, reflux; (ii) isothiocyanate/benzene, room temperature; (iii) acetic acid,
isopropanol, MW.
Table 2. Comparative results obtained in the synthesis of N4-TSCs by Methods A and B
Method A
Time Yield
Method B
Time Yield
(min) (%)
N4-
TSCs
R1
R2
R3
R4
R5
(h)
27
(%)
19
8
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
130
45
60
30
65
45
87
70
69
53
100
52
9
NO2
20
46
86
41
56
29
10
14
15
16
OCH3 72
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
F
96
72
NO2
OCH3 73
3.2. Antiviral activity
The antiviral activity exerted by compounds 1–16 against BVDV was evaluated by the
plaque-forming units (PFU) reduction assay and the results are shown in Table 3.
Reference compounds 5,6-TSC and Ribavirin were also included.
The TSC derived from 6-nitro-1-indanone (2) was the most active compound among the

non-substituted TSCs evaluated (1-4), with an anti-BVDV activity (EC50= 2.7 0.4 µM) that
was similar to that of 5,6-TSC (EC50= 3.8 0.4 µM).
Among the twelve N4-TSCs evaluated (5-16), those derived from the 1-indanone (5-10)
exerted a PFU reduction from 15 to 40% at the highest concentrations tested (EC50>10 µM
or EC50>20 µM). Derivatives obtained from 5,6-dimethoxy-1-indanone (11-16) were the
most active ones, with EC50 values ranging from 0.7 to 12.9 µM. Compound 15 was the
most potent of the N4-TSCs series, with an EC50 value that was almost 6-fold lower than
that of 5,6-TSC (EC50= 0.7 0.1 µM and 3.8 0.4 µM, respectively). None of the N4-TSCs
showed significant cytotoxicity at the concentrations evaluated.
For several compounds, CC50 values could not be determined due to the reduced solubility
in infection medium.

Table 3. In vitro anti-BVDV activity and cytotoxicity of TSCs and N4-TSCs derivatives
a
a
EC50
CC50
Compound
S.I.b
(µM)
(µM)
1
6.1 0.6
84.6 5.4
14.2
>9.3
5.4
>13.5
-
2
2.7 0.4
9.0 0.1
7.4 0.4
>10
>25
41.9 5.6
>100
>10
3
4
5
6
>20
>20
-
7
>20
>20
-
8
>20
>20
-
9
10
>10
>10
-
>10
>10
-
11
8.4 0.4
8.8 0.1
6.6 0.2
4.0 0.8
0.7 0.1
12.9 0.9
3.8 0.4
7.7 1.5
>10
>1.2
>2.3
>1.5
>5.0
>14.3
>1.6
>21.1
7.1
12
>20
13
>10
14
>20
15
>10
16
>20
5,6-TSC
Ribavirin
>80
54.4 3.8
EC50= Concentration of compound that reduces the number of PFU by 50% with respect to the
untreated viral control. CC50= Concentration of compound that inhibits cell viability by 50% with
a
respect to the untreated cell control. Data are expressed as mean values SD from at least 3
independent experiments. b In vitro selectivity index (CC50/EC50).
3.3. Structure-activity relationship
In our previous work neither 4 nor 6 indanic ring monosubstituted TSCs were assessed.10
Interestingly, the introduction of a nitro group in these positions led to the generation of the
most active compounds (1 and 2, respectively). The TSC bearing a fluorine atom at
position 5 was moderately active against BVDV, but it was less active than the bromine
and chlorine analogs reported previously.10 Finally, among TSCs, compound 4 showed an

activity that was similar to that of other trisubstituted compounds with different pattern of
methyl and halogen substituents.10
In the group of N4-TSCs, it was clear that the presence of the methoxy groups in the
nucleus was essential for the anti-BVDV activity. None of the N4-TSCs derived from 1-
indanone were active (5-10), whereas all N4-TSCs derived from 5,6-dimethoxy-1-indanone
exhibited anti-BVDV activity (11-16). Among N4-TSCs 11-16, the nature of the substituent
at position R5 played an important role in the anti-BVDV activity. Thus, the introduction of
electron-donating substituents in the phenyl substituent such as the methyl (12) (EC50= 8.8
0.1 µM) or the methoxyl group (16) (EC50= 12.9 0.9 µM) generated analogs with similar
or reduced antiviral activity compared to compound 11 (R5= H) (EC50= 8.4
0.4 µM).
Conversely, the presence of electron-withdrawing substituents at R5 resulted in N4-TSCs
with high anti-BVDV activity. Thus, the incorporation of a halogen atom led to improved
antiviral activity. In particular, compound 14 (R5= F) was more active (EC50= 4.0 0.8 µM)
than compound 13 (EC50= 6.6 0.2 µM), which has a chlorine atom at R5. This difference
in the antiviral activity could be explained by the greater electronegativity of the fluorine
substituent. Finally, compound 15, which presents a nitro group, a strong electron-
withdrawing substituent, at position R5, was the most active in the series of N4-TSCs
derived from 5,6-dimethoxy-1-indanone (EC50= 0.7 0.1 µM).
3.4. BVDV-TSCr T1 cross-resistance studies
Compounds 2 and 15 were evaluated against BVDV-TSCr T1, which carries two mutations
(N264D and A392E) in the RdRp coding region (NS5B).11 The CPE reduction assays
revealed that BVDV-TSCr T1 was also resistant to compound 15 at the maximal
concentration evaluated (10 µM) (Fig. 2C, Table 4) with an twenty-two fold increase in the
EC50 value when compared to the wild type virus (EC50 > 10 µM and EC50 = 0.45 0.08
µM, respectively).

However, compound 2 showed inhibition of BVDV-TSCr T1 (Fig. 2B, Table 4), and no
significant differences were observed between EC50 values determined against the wild
type virus and resistant virus.
Fig. 2. In vitro antiviral activity of 5,6-TSC (A), 2 (B) and 15 (C) against wild type and BVDV-TSCr
T1. Representative dose-response curves from one of two independent experiments. Full circles:
wild type BVDV; empty circles: BVDV-TSCr T1.
Table 4. Susceptibility of wt-BVDV and BVDV-TSCr T1 to active thiosemicarbazones.
EC50 (µM) a
Viral
NS5B
Strain
mutations
Compound 2
1.04 0.39
1.07 0.69
Compound 15
0.45 0.08
>10 (>22)b
5,6-TSC
1.95 0.54
>80 (>41)b
Wild type BVDV
BVDV- TSCr T1
-
N264D, A392E
EC50: compound concentration that reduces viral CPE by 50% with respect to the infected untreated
a
b
control (UTC). Data are mean values
SD from at least two independent experiments. The
number between parentheses represents the calculated fold decrease in susceptibility of the
resistant variant BVDV (EC50 BVDV-TSCr T1 / EC50 wt-BVDV).

3.5. Time-of-drug-addition studies.
In order to determine the time of the viral cycle at which compound 2 exerts its effects,
time-of-drug-addition experiments were carried out. At different time points post- infection
(p.i.), compound 2 at 25 µM was added. Viral replication was then evaluated as the CPE
after 48 h of incubation (Fig. 3). Compound 2 showed the strongest inhibitory effect when it
was added to the culture up to 4 h post-infection. Conversely, the addition of the
compound at later time points resulted in a gradual loss of the antiviral activity. These data
suggest that the drug may be exerting the effects after virus attachment and penetration
but before virus assembly and release. Moreover, the time point at which compound 2
exerts its activity coincided with the onset of viral RNA synthesis (i.e., at about 6 h p.i.).28
Fig. 3. Time-of-drug-addition study. MDBK cells were infected with BVDV (MOI=1), and compound
2 at 25 µM was added at different time points post-infection. After 48 h of incubation at 37 ºC, CPE
was calculated from cell viability measures (MTS/PES method).
3.6. Molecular docking study
We have previously reported the interactions observed in the docking study performed
between RdRp (PDB ID: 1S48) and 5,6-TSC close to N264 and A392, for both wild type
and mutated (N264D and A392E) BVDV RdRp .11 Herein, we explored the ligand-RdRp
interactions for the most active compounds of the new set of derivatives (compounds 2

and 15) in order to better determine the preferred binding modes and the molecular
requirements exploited by resistant mutants to escape drug inhibition.
The interaction between compound 15 and the wt-RdRp revealed (Fig. 4A): (i) two
hydrogen bonds between the N2 and the N4 atoms of the thiosemicarbazone group and
amino acids E226 and F224, respectively; (ii) a hydrogen bond between the oxygen of the
6-methoxy group and the hydroxyl group of Y289; (iii) a π-π stacking interaction between
the aromatic ring attached to N4 of the thiosemicarbazone moiety and the side chain of
F224; (iv) a close contact between the indanic ring and A222. On the other hand, the
situation in the best score pose of mutant RdRp-compound 15 complex (Fig. 4B) showed:
(i) a hydrogen bond between the N1 atom of the thiosemicarbazone group and the amino
acid A222; (ii) a hydrogen bond between the N4 of the thiosemicarbazone moiety and
G220; (iii) a close contact between the indanic ring and A222; (iv) steric hindrance
between the 5-methoxy group of the ligand and E392. For a further comparison between
wild type and mutant complexes, we presented a second orientation of the ligand to the
mutant RdRp which is similar to that adopted in the wild type RdRp (Fig. 4C). It was
observed that despite the similar orientation of the ligand in the binding site, only one
hydrogen bond is formed between N2 of the thiosemicarbazone group and E226.
Furthermore, it is noteworthy that no hydrogen bonds were formed between the 5,6-
dimethoxy group of the indanic ring and the amino acids of the mutated enzyme.
Therefore, it seems that the presence of this group plays a key role in the anti-BVDV
activity. This observation is in line with the results obtained in the cross-resistance study.
Moreover, N4-TSCs lacking the 5,6-dimethoxy group in the indanic nucleus have no
activity against BVDV.

A
B
C
Fig. 4. Docking and binding pattern of compound 15 into BVDV RdRp (PDB ID: 1S48). N4-TSC 15
is represented as a stick model according to atom types: carbons, cyan; nitrogens, blue; oxygens,
red; sulphur, yellow; hydrogens, white. Cyan dashed lines indicate hydrogen bonds. The side
chains making favorable interactions and those involved in resistant mutations are shown as stick
models. (A) Details of the highest-score docked pose of 15 into the binding pocket of the wild type
enzyme. (B) Details of the highest-score docked pose of 15 into the binding pocket of BVDV
mutated RdRp. (C) Details of the docked pose of 15 with an orientation similar to that obtained for
the wt-BVDV RdRp into the binding pocket of the mutated enzyme.
The interaction observed in the best score pose of wt-RdRp with compound 2 revealed
(Fig. 5A): (i) a hydrogen bond established between the N4 atom of the thiosemicarbazone
group and amino acid P262; (ii) two hydrogen bonds between the oxygen atoms of the
nitro group and amino acids A222 and G223; (iii) close contacts between the ring and the
amino acids F224 and T259. The interaction between compound 2 and the mutated
polymerase (Fig. 5B) showed: (i) a hydrogen bond between the N2 atom of the
thiosemicarbazone group and E392; (ii) two hydrogen bonds between the N4 of the
thiosemicarbazone moiety and E392 and P262; (iii) one hydrogen bond between the
oxygen of the nitro group and A222. Although one more hydrogen bond was observed in

the resistant mutant RdRp compared to the wild type protein, the binding mode of the TSC
2 in the wild type form showed favorable close contacts that were absent in the mutated
one. Accordingly, interactions appear to be largely maintained upon mutations. The latter
finding is in agreement with the results obtained in the cross-resistance study.
A
B
Fig. 5. Docking and binding pattern of compound 2 into BVDV RdRp (PDB ID: 1S48). TSC 2 is
represented as a stick model according to atom types: carbons, cyan; nitrogens, blue; oxygens, red;
sulphur, yellow; hydrogens, white. Cyan dashed lines indicate hydrogen bonds. The side chains
making favorable interactions, and those involved in resistant mutations are shown as stick models.
(A) Details of the highest-score docked pose of 2 into the binding pocket of the wild type enzyme.
(B) Details of the highest-score docked pose of 2 into the binding pocket of mutated BVDV RdRp.
Finally, the preferred binding modes of 5,6-TSC and derivative 15 into wt-RdRp showed
the same pattern of interactions, whereas a different pattern of interactions for compound
2 and the lead compound was observed (Fig. 6). These findings, taken together with the
results obtained in the cross-resistance assay, would suggest that this binding site is not
the molecular target for TSC 2.

Fig. 6. Comparative docking and binding patterns of compounds 5,6-TSC, 2 and 15 into BVDV
RdRp (PDB ID: 1S48). 5,6-TSC, 2 and 15 are represented as a stick model with carbons in pink,
green and orange respectively. The other atom types are represented as follows: nitrogens, blue;
oxygens, red; sulphur, yellow; hydrogens, white. Cyan dashed lines indicate hydrogen bonds.
3.7. Binding free energy analysis
The findings in the cross-resistant assay (Table 4) and the different binding poses of
compounds 2 and 15 in the pocket of the viral polymerase (Fig. 4A, 5A and 6) raise the
question of whether derivative 2 targets this binding site. Therefore, to further analyze the
ligand-protein affinities we performed MM/PBSA calculations for the two derivatives in the
wild type and N264D/A392E mutant enzyme. The calculated binding free energies are
listed in Table 5.

Table 5. Binding free energies and its components for compounds 2 and 15 on wt-BVDV
and BVDV-TSCr T1.
Compound 2
Compound 2
Compound 15
Compound 15
Wild type BVDV BVDV- TSCr T1 Wild type BVDV BVDV- TSCr T1
∆EVDW
∆EELE
∆GNP
∆GPB
-T∆S
∆Gbind
-29.66 0.10
-10.47 0.11
-3.74 0.01
26.89 0.15
12.67 0.75
-4.31
-28.71 0.11
-11.55 0.13
-3.93 0.01
27.04 0.14
12.71 0.81
-4.44
-37.08 0.12
-14.12 0.15
-4.07 0.01
32.82 0.17
14.08 0.81
-8.37
-35.69 0.13
-12.52 0.17
-4.27 0.01
31.88 0.18
14.03 0.83
-6.57
All energies are expressed in Kcal/mol. Data are mean values SD. ∆EVDW, van der Waals energy;
∆EELE, electrostatic energy; ∆GNP, nonpolar solvation energy; ∆GPB, polar solvation energy; -T∆S,
entropy contribution; ∆Gbind, binding free energy.
As indicated in Table 5, derivative 15 complexed with wt-BVDV had the most favorable
binding energy (∆Gbind =-8.37) whereas the complex with the mutated enzyme caused a
significative energetic change (∆Gbind =-6.57). On the other hand, for ligand 2 both
complexes displayed lower binding energies and, interestingly, with similar energies
between them (∆Gbind =-4.31 and -4.44).
Comparisons of the free energies components were carried out to elucidate the interaction
mechanism. In all complexes, the van der Waals energy was the most favorable
interaction for binding, pointing out the major contribution of hydrophobic groups.
Favorable interactions were also recorded in electrostatic energy and nonpolar solvation
energy. However, they were counteracted by detrimental polar solvation energy and
entropy. In addition, it should be noted that the inclusion of the entropic contribution did not
change the order of the binding energies. It is evident that the van der Waals and
electrostatic interactions were higher for derivative 15 complexed with the wild type

enzyme than that of other complexes. Thus, ∆EVDW and then ∆EELE were the main driving
forces involved in the binding of this inhibitor.
In summary, the values of ∆Gbind for compound 15 were in good agreement with the cross-
resistance assay and provide more information regarding enzyme-ligand interactions in the
selected pocket. In the case of compound 2, results suggest a different binding site or
target which may provide an explanation for the similar EC50 values in the cross-resistance
assay.
4. Conclusions
In the present study, a series of TSCs and N4-arylsubstituted TSCs derived from 1-
indanones was synthesized and evaluated as potential anti-BVDV agents. Six novel
compounds were easily prepared by one-pot multicomponent reaction under microwave
irradiation with good yields and short reaction times. The in vitro assays showed that two
new derivatives (2 and 15) display enhanced activity compared to the lead compound 5,6-
TSC. For N4-TSCs, the structure-activity relationship study demonstrated that the 5,6-
dimethoxy substitution of the indanic ring and N4-aryl substitution of the thiosemicarbazone
moiety arise as important structural features for antiviral activity as they mediate key
ligand-protein interactions in the binding pocket on the viral polymerase. Moreover, the
presence of electron-withdrawing substituents in the phenyl ring attached to N4 atom of the
thiosemicarbazone function increased the activity against BVDV. Cross-resistance assays
combined with in silico studies showed that N4-TSC 15 is highly likely to act as a non-
nucleoside inhibitor of the viral polymerase. Further studies will be carried out to elucidate
the mechanism of action of compound 2. To conclude, derivatives 2 and 15 emerge as
promising new lead compounds. The results obtained in the present work are of great
relevance and provide valuable bases for further structural design in search for novel
agents against BVDV.

Acknowledgements
We thank CONICET for scholarship to María C. Soraires Santacruz and Agencia Nacional
de Promoción Científica y Tecnológica for scholarship to Matias Fabiani. This study was
supported by the Universidad de Buenos Aires [UBACYT 2014–2017
200201130100005BA and 20020130100102BA], and by the Agencia Nacional de
Promoción Científica y Tecnológica [PICT 2012-2867].
References
1. Houe H. Epidemiological features and economical importance of bovine virus
diarrhoea virus (BVDV) infections. Vet. Microbiol. 1999; 64: 89–107.
2. Lindberg A, Brownlie J, Gunn GJ, Houe H, Moennig V, Saatkamp HW, Sandvik T,
Valle PS. The control of bovine viral diarrhoea virus in Europe: today and in the future.
Rev. Sci. Tech. 2006; 25: 961–979.
3. Presi P, Struchen R, Knight-Jones T, Scholl S, Heim D. Bovine viral diarrhea (BVD)
eradication in Switzerland--experiences of the first two years. Prev. Vet. Med. 2011; 99:
112–121.
4. Pacheco JM, Lager I. Indirect method ELISA for the detection of antibodies against
bovine diarrhea virus in bovine serum. Rev. Argent. Microbiol. 2003; 35: 19–23.
5. Al-Khaliyfa MA, Abuelzein EME, Gameel AA. Identification of cattle persistently
infected with BVDV by ear-notch testing in Saudi Arabia. Vet. Rec. 2010; 167: 660–661.
6. González Altamiranda EA, Kaiser GG, Weber N, Leunda MR, Pecora A, Malacari DA,
Morán O, Campero CM, Odeón AC. Clinical and reproductive consequences of using
BVDV-contaminated semen in artificial insemination in a beef herd in Argentina. Anim.
Reprod. Sci. 2012; 133: 146–152.

7. Bolin SR. Control of bovine viral diarrhea infection by use of vaccination. Vet. Clin.
North Am. Food Anim. Pract. 1995; 11: 615–625.
8. Van Oirschot JT, Bruschke CJ, van Rijn PA. Vaccination of cattle against bovine viral
diarrhoea. Vet. Microbiol. 1999; 64: 169–183.
9. Buckwold VE, Beer BE, Donis RO. Bovine viral diarrhea virus as a surrogate model of
hepatitis C virus for the evaluation of antiviral agents. Antiviral Res. 2003; 60: 1–15.
10. Finkielsztein LM, Castro EF, Fabian LE, Moltrasio GY, Campos RH, Cavallaro LV,
Moglioni AG. New 1-indanone thiosemicarbazone derivatives active against BVDV. Eur. J.
Med. Chem. 2008; 43: 1767–1773.
11. Castro EF, Fabian LE, Caputto ME, Gagey D, Finkielsztein LM, Moltrasio GY,
Moglioni AG, Campos RH, Cavallaro LV. Inhibition of bovine viral diarrhea virus RNA
synthesis by thiosemicarbazone derived from 5,6-Dimethoxy-1-indanone. J. Virol. 2011;
85: 5436–5445.
12. Caputto ME, Fabian LE, Benítez D, Merlino A, Ríos N, Cerecetto H, Moltrasio GY,
Moglioni AG, González M, Finkielsztein LM. Thiosemicarbazones derived from 1-
indanones as new anti-Trypanosoma cruzi agents. Bioorg. Med. Chem. 2011; 19: 6818–
6826.
13. Lansbury PT, Mancuso NR. Further studies of bond insertion and nonstereospecific
Beckmann rearrangements in 7-alkyl-l-indanone oximes. J. Amer. Chem. Soc. 1966; 88:
1205–1212.
14. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ.
AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J.
Comput. Chem. 2009; 30: 2785–2791.
15. Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, Villa E, Chipot C, Skeel RD,
Kalé L, Schulten K. Scalable molecular dynamics with NAMD. J. Comput. Chem. 2005; 26:
1781–1802.

16. Zhang Z, Li Y, Lin B, Schroeder M, Huang B. Identification of cavities on protein
surface using multiple computational approaches for drug binding site prediction.
Bioinformatics. 2011; 27: 2083–2088.
17. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin
TE. UCSF Chimera - A visualization system for exploratory research and analysis. J.
Comput. Chem. 2004; 25: 1605–1612.
18. Best RB, Zhu X, Shim J, Lopes PEM, Mittal J, Feig M, Mackerell AD. Optimization of
the additive CHARMM all-atom protein force field targeting improved sampling of the
backbone ϕ, ψ and side-chain χ(1) and χ(2) dihedral angles. J. Chem. Theory Comput.
2012; 8: 3257–3273.
19. Vanommeslaeghe K, Hatcher E, Acharya C, Kundu S, Zhong S, Shim J, Darian E,
Guvench O, Lopes P, Vorobyov I, MacKerell Jr. AD. CHARMM general force field: A force
field for drug-like molecules compatible with the CHARMM all-atom additive biological
force field. J. Comput. Chem. 2010; 31: 671–690.
20. Jorgensen WL, Chandrasekhar J, Madura JD. Comparison of simple potential
functions for simulating liquid water. J. Chem. Phys. 1983; 79: 926–935
21. Darden T, York D, Pedersen L. Particle mesh Ewald: an N⋅log(N) method for
Ewald sums in large systems. J. Chem. Phys. 1993; 98: 10089–10092.
22. Miyamoto S, Kollman PA. Settle: An analytical version of the SHAKE and RATTLE
algorithm for rigid water models J. Comput. Chem. 1992; 13: 952–962.
23. Humphrey W, Dalke A, Schulten K. VMD - Visual Molecular Dynamics. J. Molec.
Graphics. 1996; 14: 33–38.
24. Kollman PA, 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 DA, Cheatham TE 3rd. Calculating
structures and free energies of complex molecules: combining molecular mechanics and
continuum models. Acc. Chem. Res. 2000; 33: 889–897.