Identification of potent bovine viral diarrhea virus inhibitors by a structure-based virtual screening approach

Article abstract of DOI:10.1016/j.bmcl.2018.11.041

Bovine viral diarrhea virus (BVDV) is a pestivirus whose infection in cattle is globally distributed. The use of antivirals could complement vaccination as a tool of control and reduce economic losses. The RNA-dependent RNA polymerase (RdRp) of the virus

Full text of DOI:10.1016/j.bmcl.2018.11.041

Accepted Manuscript
Identification of potent bovine viral diarrhea virus inhibitorsby a structure-based
virtual screening approach
Eliana F. Castro, Juan J. Casal, María J. España de Marco, Leandro Battini,
Matías Fabiani, Gabriela A. Fernández, Ana M. Bruno, Lucía V. Cavallaro,
Mariela Bollini
PII:
S0960-894X(18)30918-1
https://doi.org/10.1016/j.bmcl.2018.11.041
BMCL 26152
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
20 August 2018
8 November 2018
21 November 2018
Please cite this article as: Castro, E.F., Casal, J.J., de Marco, J.E., Battini, L., Fabiani, M., Fernández, G.A., Bruno,
A.M., Cavallaro, L.V., Bollini, M., Identification of potent bovine viral diarrhea virus inhibitors by a structure-based
virtual screening approach, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.
2018.11.041
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Identification of potent bovine viral diarrhea
virus inhibitors by a structure-based virtual
screening approach
Leave this area blank for abstract info.
Eliana F. Castroa,&, Juan J. Casalb,&, María J. España de Marcoc, Leandro Battinib, Matías Fabiania, Gabriela A.
Fernándezb, Ana M. Brunod, Lucía V. Cavallaroc,*#, Mariela Bollinib,*#
.

Bioorganic & Medicinal Chemistry Letters
Identification of potent bovine viral diarrhea virus inhibitors by a structure-
based virtual screening approach
Eliana F. Castroa,&, Juan J. Casalb,&, María J. España de Marcoc, Leandro Battinib, Matías Fabiania, Gabriela A.
Fernándezb, Ana M. Brunod, Lucía V. Cavallaroc,*#, Mariela Bollinib,*#
.
aCátedra 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.
bLaboratorio de Química Medicinal, Centro de Investigaciones en Bionanociencias (CIBION)-CONICET, Ciudad de Buenos Aires, Argentina.
cCátedra 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.
dUniversidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Química Orgánica, Junín 956, C1113AAD, Ciudad Autónoma de Buenos
Aires, Argentina.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Bovine viral diarrhea virus (BVDV) is a pestivirus whose infection in cattle is globally
distributed. The use of antivirals could complement vaccination as a tool of control and reduce
economic losses. The RNA-dependent RNA polymerase (RdRp) of the virus is essential for its
genome replication and constitutes an attractive target for the identification of antivirals. With
the aim of obtaining selective BVDV inhibitors, the crystal structure of BVDV RdRp was used
to perform a virtual screening. Approximately 15,000 small molecules from commercial and in-
house databases were evaluated and several structurally different compounds were tested in vitro
for antiviral activity. Interestingly, of twelve evaluated compounds, five were active and
displayed EC50 values in the sub and low-micromolar range. Time of drug addition experiment
and measured intracellular BVDV RNA showed that compound 7 act during RNA synthesis.
Molecular Dynamics and MM/PBSA calculation were done to characterize the interaction of the
most active compounds with RdRp, which will allow future ligand optimization. These studies
highlight the use of in silico screening to identify a new class of BVDV inhibitors.
Keywords:
Keyword_1 BVDV inhibitors
Keyword_2 virtual screening,
Keyword_3 molecular dynamic simulation,
Keyword_4 RdRp protein
Keyword_5 antiviral
2009 Elsevier Ltd. All rights reserved.
Bovine viral diarrhea virus 1 (BVDV), named Pestivirus A
from 2017 International Committee on Taxonomy of Viruses
report belongs to the Pestivirus genus of the family Flaviviridae.
1BVDV infection is globally distributed being endemic in many
countries.2,3 The latest data reported for Argentina showed a
The viral genome consists of a single-stranded (+) RNA which
constitutes an unique open reading frame (ORF) that is flanked
by 5’ and 3’ nontranslated regions (NTRs). This ORF encodes a
3,900 amino acids long polyprotein that is co- and post-
translationally processed to the mature viral proteins by cellular
and viral proteases. From its N-terminal, the ORF encodes the
viral nonstructural protein (NS) Npro, the structural proteins
(capsid protein C, the glycoprotein Erns, and the envelope
glycoproteins E1 and E2) and the rest of the NS proteins: p7,
NS2-3 (NS2, NS3), NS4A, NS4B, NS5A and NS5B. The last
protein, is the viral RNA-dependent RNA polymerase (RdRp). 10
4
prevalence of anti-BVDV antibodies in 70% adult cattle.
BVDV
infections
can
manifest
as
generalized
immunosuppression (leading to co-infections), fertility
difficulties both in male and female cattle, and other variable
5–8
signs such as fever, diarrhea and respiratory dysfunction.
This
leads to considerable financial losses within the livestock
industry because of lower reproductive accomplishment, lower
weight gain, reduced milk production, increased mortality, early
slaughter as well as increased expenditures for veterinary
services. 9
As shown by its crystal structure, the RdRp of BVDV has the
shape of a right hand that is divided into different domains:
fingers, palm and thumb. The inner surfaces of these three
domains form a central template-binding channel; and the N-
terminal portion of and a long insert in the fingers domain
(residues 71-138 and 260-288, respectively) form the fingertip
region. 11,12
The control of BVDV infections combines vaccination, which
is not mandatory in some countries, with the detection and
removal of persistently infected animals. The vaccination
strategy has a disadvantage in terms of the time it takes to
activate the animal's immune response and the increase in the
antibody titer needed to protect against an infection. That is why
the need to have effective and rapid control tools is considered,
and the use of antivirals could be useful for this purpose.
The fingertips, associated with the thumb domain, forms the
entrance to the template-binding channel. It was also reported
that the fingertip region is involved in template/product
translocation, dimerization of the RdRp and other protein-protein
interactions that enable the assembly of an active replication
11,12
complex.
Moreover, motifs I and II, which are involved in

RNA template and NTP binding in the RNA polymerases, were
located within this region. 12
(SI) were determined as the ratio between CC50 and EC50.
Compound 3 displayed high cytotoxicity (CC50=2.11 µM) and
was evaluated to its MNCC. The evaluation of all computational
hits (Table 1) yielded five active compounds (Figure 1).
Compounds 1 and 9 showed moderate micromolar activity with
EC50 values of 9.68 and 6.40 µM, respectively. Compound 10
displayed a promising antiviral activity, with EC50 values of 2.30
μM. Finally, compounds 7 and 11 were the most active ones of
this series, showing EC50 values of 0.98 μM and 0.17 μM,
respectively. Compound 11 displayed a wide window therapeutic
index (SI=196.47) making this drug a suitable candidate for in
vivo studies.
Different selective antiviral agents against BVDV select
resistant virus that carry mutations within the fingertip region.
Previously, we described the antiviral mechanism of action of
Thiosemicabazones derived from 1-indanones (TSC), which
selected two specific mutations within NS5B: N264 and A392.13
Both mutations are located near F224, a residue that was mutated
in BVDV-resistant mutants selected with other non-nucleoside
14
15
inhibitors (NNIs) for BVDV, such as BPIP, VP32947, and
16
LZ37. The cavity where these compounds bind represents one
allosteric site of inhibition for BVDV RdRp, whose
13
hydrophobicity complements the lipophilicity of TSC and of
the other NNIs.
Computer-aided drug design (CADD) becomes increasingly
important in drug research and development. CADD contributed,
among other applications, to efficient data analysis, to the
filtering of collections of compounds to select molecules for
experimental evaluation, and to the generation of hypotheses to
understand the mechanism of action of drugs and the design of
novel chemical structures. 17–20 In this work, we have employed a
multistep High throughput docking (HTD) screening strategy to
find novel viral polymerase inhibitors using synthetic druglike
compounds (from our in-house library) and commercially
available chemical libraries, and the crystal structure of the
12
BVDV RdRp (1S48). Design was targeted against the RdRp
allosteric site of inhibition described for other NNIs (as described
above). The original ~15,000 compounds collection was filtered
based on known toxicophores and physicochemical properties.
Figure 1. Active compounds against BVDV selected by virtual
screening against viral RdRp.
We performed 60 ns MD simulation for each compound to
evaluate the role of water molecules which were discarded during
the molecular docking simulation, and to characterize the most
likely interactions with the BVDV RdRp. For the MD simulation
we started with the docked poses as initial conformation. Protein-
ligand complexes were stable in each simulation (Figure S1).
Additionally, to understand the forces involved in substrate
binding, total binding free energy and individual energy
components for each system were obtained by using the
Molecular Mechanics Poisson–Boltzmann Surface Area (MM-
PBSA) method based on the MD trajectories. The predicted
binding free energies calculated for the most active compounds
are more favorable than for inactive compounds. Active
compounds 7 and 11 display ΔGbind of -21.98 and −19.31
kcal/mol, respectively, while the ΔGbind for inactive compounds 2
and 4 are -9.85 and -9.88 kcal/mol, respectively (For the rest of
compounds see Table S1 in SI).
17,20,21
After removing metal atoms, duplicates, multiple
fragments and more than one stereocenter we obtained a database
of 13,000 in-stock compounds which were subjected to two
independent HTD. The top 1,000 structures where redocked with
22
Autodock Vina increasing the exhaustiveness to 25. The 200
ranked compounds were inspected by eye to assess scaffold
diversity, and synthetic feasibility for further modifications.
Finally, twelve compounds were synthetized (Scheme S1) or
purchased for further in vitro study (Figure S1).
First, we assayed selected compounds for cytotoxicity.
Cytotoxic concentration 50 (CC50) is defined as the concentration
of compound that reduces by 50% cell culture viability and
maximum non-cytotoxic concentration (MNCC) was the higher
concentration of compound evaluated that did not result cytotoxic
in MDBK cells. Then, their antiviral activity (EC50: concentration
of compound that reduces viral cytopathic effect by 50%) against
BVDV in cultured MDBK cells was studied. Selectivity Indexes
Table 1. In vitro cytotoxicity and anti-BVDV activity of selected compounds
CC50 ± SDa
(µM)
MNCCc
(µM)
12.50
6.00
EC50 ± SDd
(µM)
Compound
SIf
Library
1
2
3
55.90 ± 8.20
9.68 ± 0.49
5.77
-
-
UDb
Inactivee
In-house Library
2.11 ± 0.01
0.78
Inactive
4
5
6
7
8
>100
>100
>100.00
50.00
25.00
50.00
3.13
100.00
25.00
25.00
6.25
Inactive
Inactive
Inactive
0.98 ± 0.01
Inactive
6.40 ± 0.70
2.30 ± 0.70
0.17 ± 0.03
UD
-
-
-
11.24 ± 2.86
>100.00
82.30 ± 13.20
43.06 ± 2.80
33.40 ± 0.50
11.53
-
12.86
18.72
196.47
UD
Maybridge HitFinder
9
10
11
12
34.30 ± 0.10
6.25
aCC50: compound concentration that reduces cell viability by 50%; UD: undetermined; cMNCC: Maximum non-cytotoxic concentration; dEC50: compound
b
concentration that reduces viral CPE by 50%; eInactive: less than 50% of inhibition of CPE at MNCC; fSI: selectivity index (SI=CC50/EC50).

The predicted interaction of compound 7 with the hydrophobic
cite of RdRp is shown in Figure 2 A. This ligand presented
hydrogen bond between the NH of the 4-aminoquinoline and the
carbonyl O atom of the G406 with an average distance of 2.4 Å
until 1.7 ns of simulation. While this hydrogen bond is broken,
the ligand moves slightly from the initial conformation to another
conformation that remains stable during the rest of the
simulation, and a new hydrogen bond is formed between the N
atom of the 4-aminoquinoline and a NH group of R295. The
interatomic distance and angle that characterize the hydrogen
bond are closer to 2 Å and 160º (Figure S3A). Additionally, the
system was mostly stabilized by hydrophobic contacts, with
extensive interactions being established with V216, I230, T299,
P408 and L296. This is in agreement with the results obtained by
MM-PBSA calculations, in which the VDW term is the main
energy contribution (Table 2), highlighting the complementarity
between the ligand and the binding pocket. Furthermore, the
observations of the MD trajectory coincide with the per residue
energy decomposition analysis (Figure 4), as the residue with the
larger enthalpy contribution is R295, followed by V216 and I230.
A stable cation-π interaction was observed between the aromatic
ring of quinazoline and K307 throughout the simulation: the a
N+-ring centroid distance is below 6 Å, and an acceptable θ
angle of ~40° is maintained at all times during the simulation.
(Figure S4). 23
I261). The interactions with N217, I230 and V216 were kept
during the MD simulation, but the contacts with A221 and I261
disappeared due to the movement of the furan fragment of the
ligands away from these amino acids. Moreover, a weak
hydrogen bond is formed during the simulation, involving the
oxygen of the phenyl ether of 11 and the NH2 of N217, with
interatomic distances and angles closer to 2.7 Å and 120°,
respectively (Figure 2, S2B). Additionally, close contacts are
seen in the last 30 ns of the simulation between the furan and the
residues Y674 and K672 (Figure 3). This interaction correlates
with the establishment of a stable conformation of the ligand
(Figure S2B) and it is enabled because of the movement of the C-
terminal region of RdRp during the simulation, which brings
Y674 closer to the binding pocket. These observations are
consistent with the per residue energy decomposition analysis of
the MM-PBSA calculations (Figure 4), in which the amino acids
with the larger energy contribution are N217 and Y674.
Overall, molecular dynamics simulations revealed the
interaction of the active compounds with the allosteric site. It is
important to highlight that these new compounds would interact
with amino acids different from those reported for the majority of
13–16
BVDV NNIs, which includes F224, N264, I390 and A392.
In this work, the computational model shows the importance of
the interactions with Y674, R295 and N217. These three
polymerase residues were reported to interact with
24
As shown in Table 2, the VDW energy contribution is also
predominant for compound 11. In this sense, the docked complex
does not present hydrogen-bond type interactions, instead, the
predicted pose is stabilized by multiple hydrophobic interactions
with the surrounding amino acids (N217, I230, V216, A221 and
arylazoenamine derivatives,
and both R295 and Y674 were
also reported to be important for pyridoquinoxalines binding 25 to
BVDV RdRp.
Table 2. Predicted free energies for binding of 7 and 11 to RdRp by MM-PBSA binding free energies method.
Compound
ΔEvdw
ΔEELE
ΔGPB
ΔGNPB
ΔGbind
-38.18±0.42
-36.21±0.64
-7.84 ±0.39
-1.53 ±0.36
28.32 ±0.52
22.52 ±0.72
-4.28 ±0.03
-4.10 ±0.05
-21.98 ±0.62
-19.31 ±0.61
7
11
All energies are expressed in kcal/mol. Data are mean values ± SD. ΔEvdw, van der Waals energy; ΔEELE, electrostatic energy; ΔGPB,
polar solvation energy; ΔGNPB, nonpolar solvation energy; ΔGbind, binding free energy, -TΔS, entropy contribution was not considered.
B
A
Figure 2. Predicted interaction of active compounds 7 (A) and 11 (B) within the RdRp (PDB ID 1S48) protein binding site, extracted from the
molecular dynamics simulations. Color code: ligand carbons, orange; protein carbons, cyan; oxygens, red; nitrogens, blue; polar hydrogens,
white; chlorine, green. Figure prepared with UCSF Chimera 1.13. 26

A
B
Figure 3. Predicted interaction of compound 11 within the RdRp (PDB ID 1S48) protein binding site, extracted from the molecular dynamics
simulations. (A) First 30 ns simulation, (B) last 30 ns simulation. Color code: ligand carbons, orange; protein carbons, cyan; oxygens, red;
nitrogens, blue; polar hydrogens, white; chlorine, green. Figure prepared with UCSF Chimera 1.13. 26
The BVDV RdRp structure reveals the shape of a right hand
not treated with the compound (UTC). This may indicate that
compound 7 would act between 6 and 8 h p.a. Previous reports
showed that when BVDV infects MDBK cells, an increase in
ivRNA occurs between 6 and 10 h p.a, suggesting that at that
point of time occurs viral RNA synthesis.28 Taken together, and
in accordance with other non-nucleoside inhibitors of BVDV
polymerase13,14,16,28, compound 7 may act during BVDV RNA
synthesis.
composed of the fingers, palm and thumb domains. According to
12
RdRp structure,
both R295 and N217 locate in the fingers
domain. During viral RNA synthesis, the phosphate backbone of
the template interacts with residues mainly from this domain.
Interestingly, R295 is part of the motif II of the polymerase,
which is conserved between different RNA polymerases of
12,27
positive RNA viruses.
In addition, it was reported that
substitution at this residue completely abolished RNA synthesis.
25,27
On the other hand, Y674 is located within the C-terminal loop
in the thumb domain. The β-thumb region interacts with the
fingers and palm domains through this long C-terminal loop
(residues 670–679). Together with a loop between α-20 and α-21
in the thumb domain, the β-thumb reduces the volume of the
template channel, and the C-terminal loop, in combination with
the fingertip region, facilitates the translocation of the template
and product RNA. 12
Therefore, the amino acids with which compound 7 and 11
would interact would participate in essential processes during
viral RNA synthesis.
Figure 5. Effect of compound 7 addition on viral RNA
synthesis. MDBK cells were infected with BVDV (MOI of 1),
and 3.13 µM of compound 7 was added every 2 h during the first
10 hours post-adsorption (h p.a.) The incubation period continued
until 12 h p.a., when intracellular RNA was extracted and ivRNA
measured by real-time PCR. A mock-infected control (MIC) and
an infected untreated control (UTC) were included. Values are
expressed as 1/∆CT, where ∆CT=CT BVDV 5’NTR - CT β-actin
endogenous control. *p value < 0.05 vs UTC.
In conclusion, we identified potential molecules that dock into
the allosteric binding pocket of BVDV RdRp via structure-based
virtual screening approach. Twelve compounds were synthesized
or purchased and evaluated in a cell-based assay for cytotoxicity
and antiviral activities. Interestingly, five of the compounds were
active and displayed EC50 values in the sub and low-micromolar
range. Molecular dynamics studies and free energy calculation
were conducted to determine the key binding interactions
between the most active compound and the residues into the
RdRp binding site. Preliminary in vitro tests of the mechanism of
antiviral action of compound 7 reinforced its potential as
inhibitor of BVDV RdRp. Overall, these findings provide the
basis to design a novel and improved BVDV antiviral.
Figure 4. Per-residue calculated binding enthalpy decomposition
ΔH, for compounds 7 and 11 in complex with BVDV RdRp
To get insight into the mechanism of action, we selected one
of the most active compounds (7) and evaluated its effect on viral
RNA synthesis. For that purpose, we performed a time of drug
addition experiment and measured intracellular BVDV RNA
(ivRNA) at 12h post adsorption (p.a.) (Figure 5). Results showed
that when compound 7 was added during the first 8 h p.a. ivRNA
production is reduced. There was a gradual loss of inhibition
between 6 and 8 h p.a. and the addition at later time points (10 h
p.a.) led to ivRNA levels similar to that observed in infected cells

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ASSOCIATED CONTENT
Supporting Information. The Supporting Information is available
free of charge at DOI: xxx Figures S1−S5, biological assay and
organic synthesis.
20.
21.
AUTHOR INFORMATION
Corresponding Author
*Mariela Bollini, Phone: (+5411) 4899-5500 int 5622. E-mail
mariela.bollini@cibion.conicet.gov.ar, *Lucia V. Cavallaro, Phone:
(+5411) 5287-4474. E-mail lcavalla@ffy.uba.ar
22.
23.
24.
25.
Funding Sources
This work was supported by the Agencia Nacional de Promoción
Científica y Tecnológica, Argentina (PICT 2013-0078, PICT 2014-
1884, CONICET (PIP 2014 11220130100721 to MB), (PICT 2012-
2867 and UBACYT 2014-2017 20920160100170BA to LVC).
26.
27.
28.
Lai, V. C.; Kao, C. C.; Ferrari, E.; Park, J.; Uss, A. S.; Wright-
Minogue, J.; Hong, Z.; Lau, J. Y. J. Virol. 1999, 73, 10129–36.
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R.; Froeyen, M.; Ansari, I. H.; Dutartre, H.; Rozenski, J.; Gil, L. H.
V. G.; Letellier, C.; Lanford, R.; Canard, B.; Koenen, F.; Kerkhofs,
P.; Donis, R. O.; Herdewijn, P.; Watson, J.; De Clercq, E.;
Puerstinger, G.; Neyts, J. J. Virol. 2006, 80, 149–160.
Notes
The authors declare no competing financial interest.
Acknowledgments
Authors thank the TUPAC Cluster from CSC-CONICET, which is
part of SNCAD-MinCyT, for granting use of their computational
resources.
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*Highlights




Docking based virtual screening was used to
identify new inhibitors of BVDV RdRp
Five compounds displayed excellent potency
against BVDV
Molecular dynamics were carried out to identify
key RdRp-ligand interactions
In vitro experiments showed that compound 7
may act during BVDV RNA synthesis