Substituted 2,6-bis(benzimidazol-2-yl)pyridines: A novel chemical class of pestivirus inhibitors that targets a hot spot for inhibition of pestivirus replication in the RNA-dependent RNA polymerase

Article abstract of DOI:10.1016/j.antiviral.2014.03.010

2,6-Bis(benzimidazol-2-yl)pyridine (BBP/CSFA-0) was identified in a CPE-based screening as a selective inhibitor of the in vitro bovine viral diarrhea virus (BVDV) replication. The EC₅₀-values for the inhibition of BVDV-induced cytopathic (CPE) effect, viral RNA synthesis and the production of infectious virus were 0.3 ± 0.1 μM, 0.05 ± 0.01 μM and 0.3 ± 0.04 μM, respectively. Furthermore, BBP/CSFA-0 inhibits the in vitro replication of the classical swine fever virus (CSFV) with an EC ₅₀ of 0.33 ± 0.25 μM. BBP/CSFA-0 proved in vitro inactive against the hepatitis C virus, that belongs like BVDV and CSFV to the family of Flaviviridae. Modification of the substituents on the two 1H-benzimidazole groups of BBP resulted in analogues equipotent in anti-BVDV activity (EC ₅₀ = 0.7 ± 0.1 μM), devoid of cytotoxicity (S.I. = 142). BBP resistant BVDV was selected for and was found to carry the I261M mutation in the viral RNA-dependent RNA polymerase (RdRp). Likewise, BBP-resistant CSFV was selected for; this variant carries either an I261N or a P262A mutation in NS5B. Molecular modeling revealed that I261 and P262 are located in a small cavity near the fingertip domain of the pestivirus polymerase. BBP-resistant BVDV and CSFV proved to be cross-resistant to earlier reported pestivirus inhibitors (BPIP, AG110 and LZ37) that are known to target the same region of the RdRp. BBP did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). BBP interacts likely with the fingertip of the pestivirus RdRp at the same position as BPIP, AG110 and LZ37. This indicates that this region is a "hot spot" for inhibition of pestivirus replication.

Full text of DOI:10.1016/j.antiviral.2014.03.010

Accepted Manuscript
Substituted 2,6-bis(benzimidazol-2-yl)pyridines: a novel chemical class of pes-
tivirus inhibitors that targets a hot spot for inhibition of pestivirus replication in
the RNA-dependent RNA polymerase
Simone Musiu, Gerhard Pürstinger, Sylvia Stallinger, Robert Vrancken, Andy
Haegeman, Frank Koenen, Pieter Leyssen, Mathy Froeyen, Johan Neyts, Jan
Paeshuyse
PII:
S0166-3542(14)00084-9
DOI:
http://dx.doi.org/10.1016/j.antiviral.2014.03.010
AVR 3413
Reference:
To appear in:
Antiviral Research
Received Date:
Revised Date:
Accepted Date:
28 February 2013
17 March 2014
19 March 2014
Please cite this article as: Musiu, S., Pürstinger, G., Stallinger, S., Vrancken, R., Haegeman, A., Koenen, F., Leyssen,
P., Froeyen, M., Neyts, J., Paeshuyse, J., Substituted 2,6-bis(benzimidazol-2-yl)pyridines: a novel chemical class
of pestivirus inhibitors that targets a hot spot for inhibition of pestivirus replication in the RNA-dependent RNA
polymerase, Antiviral Research (2014), doi: http://dx.doi.org/10.1016/j.antiviral.2014.03.010
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Substituted 2,6-bis(benzimidazol-2-yl)pyridines: a novel chemical class of pestivirus
inhibitors that targets a hot spot for inhibition of pestivirus replication in the RNA-
dependent RNA polymerase
1
2
2
3°
Simone Musiu, Gerhard Pürstinger, Sylvia Stallinger, Robert Vrancken, Andy
3
3
1
1
1
Haegeman, Frank Koenen, Pieter Leyssen, Mathy Froeyen, Johan Neyts and Jan
1
Paeshuyse
1
Rega Institute for Medical Research, Katholieke Universiteit Leuven,
Minderbroedersstraat 10, B-3000 Leuven, Belgium
2
Institut für Pharmazie, Abteilung Pharmazeutische Chemie, Universität Innsbruck,
Innrain 80/82, A-6020 Innsbruck, Austria
3
CODA-CERVA, Groeselenberg 99, B-1180 Bruxelles, Belgium
°Current address Okapi Sciences NV, Ambachtenlaan 1, Heverlee, Belgium
*
Corresponding author: Mailing address: Rega Institute for Medical Research,
Minderbroedersstraat 10, B-3000 Leuven, Belgium. Phone: 32-16-337341. Fax: 32-16-
37340. E-mail: Johan.Neyts@rega.kuleuven.be
3
Keywords: Bovine viral diarrhea virus, RNA-dependent RNA polymerase, substituted
,6-bis(benzimidazol-2-yl)pyridine inhibitors
2
1

Abstract
,6-Bis(benzimidazol-2-yl)pyridine (BBP/CSFA-0) was identified in a CPE-based
2
screening as a selective inhibitor of the in vitro bovine viral diarrhea virus (BVDV)
replication. The EC50-values for the inhibition of BVDV-induced cytopathic (CPE)
effect, viral RNA synthesis and the production of infectious virus were 0.3 ± 0.1 µM,
0
.05 ± 0.01 µM and 0.3 ± 0.04 µM, respectively. Furthermore, BBP/CSFA-0 inhibits the
in vitro replication of the classical swine fever virus (CSFV) with an EC50 of 0.33 ± 0.25
µM. BBP /CSFA-0 proved in vitro inactive against the hepatitis C virus, that belongs like
BVDV and CSFV to the family of Flaviviridae. Modification of the substituents on the
two 1H-benzimidazole groups of BBP resulted in analogues equipotent in anti-BVDV
activity (EC50 = 0.7 ± 0.1 µM), devoid of cytotoxicity (SI = 142). BBP resistant BVDV
was selected for and was found to carry the I261M mutation in the viral RNA-dependent
RNA polymerase (RdRp). Likewise, BBP-resistant CSFV was selected for; this variant
carries either an I261N or a P262A mutation in NS5B. Molecular modeling revealed that
I261 and P262 are located in a small cavity near the fingertip domain of the pestivirus
polymerase. BBP-resistant BVDV and CSFV proved to be cross-resistant to earlier
reported pestivirus inhibitors of pestivirus replication (BPIP, AG110 and LZ37) that are
known to target the same region of the RdRp. BBP did not inhibit the in vitro activity of
recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes
(
RCs). BBP interacts likely with fingertip of the pestivirus RdRp at the same position as
BPIP, AG110 and LZ37. This indicates that this region is a “hot spot” for inhibition of
pestivirus replication.
2

Introduction
The Flaviviridae family consists of three genera: the genus Flavivirus (including human
pathogens such as dengue virus, West Nile virus and yellow fever virus), the genus
Hepacivirus (hepatitis C virus, HCV), and the genus Pestivirus (including veterinary
pathogens such as the bovine viral diarrhea virus, BVDV, and the classical swine fever
virus, CSFV).
Pestivirus infections of domesticated livestock (cattle, pigs, and sheep) cause significant
economic losses worldwide, (Houe, 2003; Hasler et al., 2012; Stegeman et al., 2000).
Classical swine fever is a highly infectious viral disease that affects domestic and wild
pigs. CSFV is included in the list of diseases notifiable to the OIE (www.oie.int). CSFV
is considered to cause one of the most devastating diseases for the pig industry
throughout the world both from an economical and sanitary point of view (Le Potier,
2
012).
The disease is endemic in Asia and is prevalent in many countries of central and South
America. In contrast to North America, where CSFV was eradicated several decades ago,
the European Union (EU) still has an ongoing progressive eradication program that
started in the early 1990s (Dong and Chen, 2007; European Union, 1980; Pereda et al.,
2
005). The most efficient vaccines currently available against CSFV are live attenuated
vaccines (Le Potier , 2012). However, recently many efforts went into the development of
new and safer marker vaccines against CSFV, along with improved diagnostic tools
(
Vannie et al., 2007; Beer et al., 2007).
BVDV is ubiquitous and causes a range of clinical manifestations (including abortion,
teratogenesis, respiratory problems, chronic wasting disease, immune system
dysfunction, and predisposition to secondary viral and bacterial infections). Certain
BVDV strains can cause acute fatal disease with mortality rates of 17–32% (Colloff et al.,
2
012; Hessman et al., 2012; Friedgut et al., 2011). BVDV is also able to establish a
persistent infection in fetuses (Peterhans et al., 2010). When born, these persistently
infected animals remain viremic throughout their lifespan and serve as continuous virus
reservoirs. Persistently infected animals may also succumb to fatal mucosal disease if
they are superinfected with a closely related BVDV strain. Vaccines are used in some
countries in an attempt to control pestivirus disease with varying degrees of success
3

(
Kalaycioglu, 2007). Other containment strategies comprise quarantine and animal
culling (Stahl and Alenius, 2012). Currently, there are no antiviral drugs to control
pestivirus infections.
Pestiviruses show greater similarity in genome structure and translation strategy to
hepaciviruses than to flaviviruses (Lindenbach and Rice, 2001). Despite of the relatively
low levels of sequence identity between BVDV and HCV, the BVDV model was widely
used in the early days of HCV drug discovery as a surrogate for HCV (Beaulieu and
Tsantrizos, 2004; Buckwold et al., 2003). Several classes of inhibitors (Finkielsztein LM
et al., 2010) with anti-BVDV activity are known; targeting either a cellular target, i.e. α-
glycosidase [which is involved during the maturation of virions (Branza-Nichita et al.,
2
001)] as well as viral encoded enzymes such as the NS3 protease and helicase/NTPase
(
Bukhtiyarova et al., 2001) or the NS5B RNA-dependent RNA polymerase (RdRp).
Polymerase inhibitors include nucleoside (Finkielsztein LM et al., 2010) and non-
nucleoside inhibitors such as N-propyl-N-[2-(2H-1,2,4-triazino[5,6-b]indol-3-
ylthio)ethyl]-1-propanamine (VP32947) (Baginski et al., 2000), a thiazole urea derivative
(
(
King et al., 2002), a cyclic urea derivative (Sun et al., 2003), imidazo-pyridines (BPIP)
Paeshuyse et al., 2006), ethyl 2-methylimidazo[1,2-a]pyrrolo[2,3-c]pyridin-8-
carboxylate (AG110) (Paeshuyse et al., 2007) and pyrazolotriazolopyrimidinamine
LZ37) (Paeshuyse et al., 2009). BVDV strains that are resistant to most of these non-
(
nucleosidic RdRp inhibitors all carry mutations in the fingertip domain of the viral RdRp.
However, most of these inhibitors do not inhibit the in vitro activity of the recombinant
viral polymerase but are able, in a dose dependent manner, to inhibit the activity of the
BVDV replicase complex (RC) (Paeshuyse et al., 2006; Paeshuyse et al., 2007). The
fingertip domain of the polymerase is crucial for the function of the polymerase and the
viral RC. This domain is thus apparently a “hot spot” binding site for selective inhibitors
of pestivirus replication (Paeshuyse et al., 2009).
Here we report on a series of benzimidazolylpyridine analogues as a novel chemical class
of inhibitors of the replication of pestiviruses. In particular, the antiviral characteristics
and mode of action of 2,6-bis(benzimidazol-2-yl)pyridine (BBP/CSFA-0) and analogues
will be discussed in detail.
4

MATERIALS AND METHODS
Compounds
Most of the analogues were prepared by reaction of (substituted) pyridine-2,6-
dicarboxylic acid with 2 equivalents of (a substituted) 1,2-phenylene diamine in PPA
(
polyphosphoric acid) at 160°C for 6 hours as depicted in Figure 1. The synthesis of
LZ37 [7-[3-(1,3-benzodioxol-5-yl)propyl]-2-(2-furyl)-7H-pyrazolo[4,3-
e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine] (Baraldi et al., 1996), BPIP (5-[(4-
bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (Puerstinger et al., 2006) and
ethyl 2-methylimidazo[1,2-a]pyrrolo[2,3-c]pyridin-8-carboxylate (AG110) (Paeshuyse et
al., 2007) were reported earlier. 3'-deoxyguanosine-5’-triphosphate (3’-dGTP) and 2’-C-
methylguanosine-5’-triphosphate (2'-C-me-GTP) were purchased from Trilink (San
Diego, CA).
Cells and viruses
Madin-Darby bovine kidney (MDBK) cells were grown in minimal essential medium
(
MEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Integro,
Zaandam, The Netherlands). BVDV NADL strain was obtained from the Veterinary and
Agrochemical Research Center (CODA-CERVA, Ukkel, Belgium). BPIP-resistant
res
BVDV (BPIP ) was produced from a pNADLp15a plasmid containing the F224S
mutation in the NS5B gene as described previously (Paeshuyse et al., 2006). Porcine
kidney cell line (PK15), in conjunction with the CSF reference-strain Alfort187 (subgroup
1
.1) were cultured as described before (Vrancken et al., 2008).
Antiviral assay
The experiments were performed as previously described (Paeshuyse et al., 2009;
Paeshuyse et al., 2006; Paeshuyse et al., 2007). In brief, MDBK cells were seeded at a
3
density of 5 x 10 per well in 96-well cell culture plates. Following 24 h incubation, at
3
7°C and 5% CO , medium was removed and 3-fold serial dilutions of the test
2
compounds were added in a total volume of 100 µL, after which the cells were infected
with BVDV NADL virus (100 CCID50). After 3 days, medium was removed and the
cytopathic effect (CPE) was quantified using the MTS/PMS method (Promega, Leiden,
The Netherlands). The 50% effective concentration (EC50) was defined as the
concentration of compound that offered 50% protection of the cells against virus-induced
5

CPE and was calculated using linear interpolation. To assess the antiviral activity against
CSFV, candidate inhibitors were evaluated using a virus-cell-based assay as described
before (Vrancken et al., 2008). In brief, the cell culture medium, obtained after incubation
with BBP, was used for the quantification of excreted viral RNA and infectious progeny
virus. Both the EC50 and CC50 values were calculated using linear interpolation and viral
titers of the medium were expressed as the 50% tissue culture infectious dose
(
TCID50/ml) using the method of Reed and Muench (Reed and Muench, 1938).
Cytotoxicity assay
Assays were carried out as described previously (Paeshuyse et al., 2009; Paeshuyse et
al., 2006; Paeshuyse et al., 2007). In brief, MDBK cells were seeded at a density of 5 x
3
1
0 per well in 96-well cell culture plates in MEM containing 5% of FBS; 24 h later,
serial dilution of the test compounds were added. Cells were allowed to proliferate for 5
days at 37°C, after which the overall metabolic activity of the cells was quantified by
means of the MTS/PMS method (Promega, Leiden, The Netherlands). The 50% cytotoxic
concentration (CC50) was defined as the concentration of compound that inhibited the
proliferation of exponentially growing cells by 50% and was calculated using linear
interpolation.
res
Isolation of BBP BVDV
res
BBP-resistant (BBP ) virus was selected as previously described (Paeshuyse et al.,
res
2
009; Paeshuyse et al., 2006; Paeshuyse et al., 2007). In brief, BBP BVDV was
generated by culturing wild-type BVDV in MDBK cells in the presence of increasing
concentrations of the compound in a 48-well plate. After 3 days of cultivation, cultures
were freeze-thawed. Lysates of infected and treated cultures that exhibited cytopathic
effect under drug pressure were used to infect new cell monolayers. These were further
incubated in the presence of increasing concentrations of the compound. The procedure
was repeated for 28 consecutive passages until drug-resistant virus was obtained.
res
Selection of BBP CSFV was done as described before (Vrancken et al., 2008). To
res
select for BBP CSFV semi-confluent PK15 cells infected with Alfort187 were passaged
at increasing concentration of BBP. The selected CSFV Alfort187 was regarded as
resistant when a 15-fold decrease in sensitivity (increase in EC50) to the antiviral effect of
BBP was observed.
6

RNA isolation
Viral RNA was isolated from cell culture supernatant using a QIAamp viral RNA
minikit (Qiagen, Venlo, The Netherlands). Total cellular RNA was isolated from cells
using an RNeasy minikit (Qiagen, Venlo, The Netherlands) according to the
manufacturer’s instructions. To extract CSFV RNA, the total cellular RNA content was
isolated from infected cell cultures using the RNeasy Mini kit (QIAgen,Venlo,
Netherlands) according to the manufacturer’s instructions.
RT-qPCR
RT-quantitative PCR (RT-qPCR) was performed as previously described (Paeshuyse et
al., 2009; Paeshuyse et al., 2006; Paeshuyse et al., 2007). In brief, a 25 µL RT-qPCR
reaction was composed of 12.5 µL 2 x reaction buffer (Eurogentec, Seraing, Belgium),
6
.3 µL H O, 300 nmol/L forward and reverse primer, 300 nmol/L TaqMan probe and 5
2
µL total cellular or viral RNA extract. The RT step was performed at 48°C for 30 min,
denatured for 15 min at 95°C and subsequent PCR amplification of 40 cycles of
denaturation at 94°C for 20 s and annealing and extension at 60°C for 1 min in an ABI
7
500 FAST Real-Time PCR System. The EC50 was defined as the concentration of
compound that reduced the amount of viral RNA by 50% as compared to an infected
untreated control and was calculated using linear interpolation. CSFV RNA levels were
quantified by a CSFV-specific, probe-based real-time RT-PCR assay using the TaqVet
Kit (LSI, Lissieu, France) as described before (Vrancken et al., 2009b; Vrancken et al.,
2
009a). The assay detects a 90 bp sequence located in the 5’-UTR, with a limit of
detection of 2.2 ± 1.2 equivalent genome copies (EGC) and uses β-actin as an internal
control. In each assay, positive and negative controls were included.
Sequencing
PCR fragments that cover the entire nonstructural protein coding region of the BVDV
genome were generated and analyzed using the cycle sequencing method (ABI Prism
BigDye Terminator Cycle Sequencing Ready Reaction Kit). Both DNA strands were
sequenced. Sequence data were obtained using an ABI 373 Automated Sequence
Analyser (Applied Biosystems), and sequences were analyzed using the Vector NTI
software package (Invitrogen, Merelbeke, Belgium). To sequence the genomic region of
the CSFV polymerase, a fragment of 2 419 b, comprising the NS5B gene was amplified
7

by combining the forward primer [5’-GGA GGT TGG TGC AAA AGT GT-3’]
designed using Primer 3; (Rozen and Skaletsky, 2000)), in combination with the
(
reversed primer HB32 (Bjorklund et al., 1998) using the SuperScriptTM One-Step RT-
PCR System for Long Templates (Invitrogen, Merelbeke, Belgium). The sequences of the
PCR fragments were determined by an ABIPRISM Sequence Analyser 310 (Applied
Biosystems, Foster City, CA, USA) using the Big Dye Termination v3.1 Cycle
Sequencing kit (Applied Biosystems, Foster City, CA, USA) and analysed using the
Chromas 2.3 application (http://www.technelysium.com.au/chromas.html).
RNA-dependent RNA polymerase assay
BVDV (NADL) RNA-dependent RNA polymerase (RdRp) was expressed and purified
as described before (Zhong et al., 1998). These experiments were performed as
previously described (Paeshuyse et al., 2009; Paeshuyse et al., 2006; Paeshuyse et al.,
2
007). In brief, the purified BVDV polymerase (100 nM) was mixed with 100 µM GTP
3
(
containing 8.3 µM of [ H]GTP, Perkin Elmer) and increasing concentration of the
inhibitor (0.2 µM, 0.7 µM, 2 µM, 6 µM, 19 µM, 56 µM, 165 µM or 500 µM) in 50 mM
Hepes pH 8.0, 10 mM KCl, 10 mM DTT, 1 mM MgCl , 2mM MnCl and 0.5% igepal
Sigma). Enzyme mix and inhibitors were pre-incubated in order to favor enzyme-
2
2
(
inhibitor interactions before RNA binding, was allowed to proceed, in case of
competition for RNA binding site. Reactions were started by the addition of 100 nM of
poly(C) (about 500 nt in size) template. Reactions were incubated at 30°C and stopped by
addition of 50 mM EDTA. Samples were transferred onto DE-81 filters, washed with 0.3
M ammonium formate solution and dried. Radioactivity bound to the filter was
determined by liquid scintillation counting.
Replication complex assay
The replication complex (RC) assay is similar to the one described before (Paeshuyse et
al., 2009; Paeshuyse et al., 2007). In brief, BVDV-infected MDBK cells were suspended
in ice-cold hypotonic buffer A (10 mM Tris, 10 mM NaCl pH 7.8) and were further
disrupted by 50 strokes with a Dounce homogenizer. The disrupted cells were pelleted by
centrifugation at 1,000 x g for 5 min at 4°C. The supernatant fraction, containing
cytoplasmic material and plasma membranes, was concentrated by high-speed
8

centrifugation at 200,000 x g for 20 min at 4°C. The pellet was resuspended in 120 µl of
hypotonic buffer B (10 mM Tris, 10 mM NaCl pH 7.8 and 15% glycerol) and used for an
RNA polymerase assay. Replicase reactions were carried out as described before with
3
3
some modification (Paeshuyse et al., 2009; Paeshuyse et al., 2007). The [α- P]UTP
3
3
(
3,000 mCi/mmol) was replaced by [α- P]CTP (3,000 mCi/mmol). The following
incubation, RNA extraction, RNA analysis and quantification were performed as
previously described (Paeshuyse et al., 2009; Paeshuyse et al., 2007).
Molecular modeling
The published X-ray structure of the BVDV RdRp [PDB entry 1S48 (Choi et al.,
2
004)] was used for the docking experiments. Selenium atoms in the selenomethionine
residues were modified back to sulphur atoms to get methionine residues. Target structure
and ligand BBP was prepared for docking by Autodocktools (Gasteiger charges were
added, atom types assigned for use with autodock 4.2). The docking site for BBP was
defined as a cube centered at Ile261A.CD1, dimensions 60x60x60, spacing 0.375 Å
(
Morris et al., 1998). Flexible dockings of BBP with 2 active torsion angles were
performed using Autodock 4.2. Several runs (100) using a Lamarckian genetic algorithm
were performed and the best docking (lowest energy score) was selected.
9

Results
Antiviral activity of BBP (CSFA-0)
BBP (CSFA-0) was identified as an inhibitor of BVDV (NADL) replication in a
multiple infection cycle assay in MDBK cells. BBP (CSFA-0) was found to inhibit virus-
induced CPE formation in a dose-dependent manner (Fig.2A) with an EC50 of 0.3 ± 0.1
µM. To further characterize the anti-BVDV activity of BBP, the effect of the compound
on viral RNA synthesis (Fig. 2B, C) and infectious virus yield (Fig. 2D) was determined.
Overall, comparable inhibitory effects on viral RNA synthesis and infectious viral yield
production were obtained (Fig.2A, B, C and D). The EC50 for inhibition of intracellular
viral RNA replication and release of viral RNA in the culture supernatant was 0.05 ± 0.01
µM and 0.05 ± 0.02 µM respectively. The inhibitory effect of BBP (CSFA-0) produced
an EC50 of 0.3 ± 0.04 µM for the inhibition of infectious virus yield in culture
supernatant. BBP (CSFA-0) did also inhibit the in vitro replication of the CSFV strain
Alfort187 in a dose-dependent manner. An EC50 of respectively 0.33 ± 0.25; 0.37 ± 0.19
and 0.38 ± 0.12 µM was calculated for a standard anti-CSFV assay or when viral RNA or
infectious virus yield was quantified, respectively. A CC50 of > 100 µM (maximum tested
concentration) was determined. BBP (CSFA-0) did not inhibit the hepatitis C virus, the
yellow fever virus (YFV), the dengue virus (DENV) nor the replication of a panel of
unrelated DNA viruses (herpes simplex virus 1 & 2, vaccinia virus and cytomegalovirus)
or RNA viruses (respiratory syncytial virus, vesicular stomatitis virus, Coxsackie virus
B4, Sindbis virus, reovirus-1 and parainfluenza-3 virus) (data not shown).
Structure-activity relationship
In an attempt to optimize the antiviral activity of BBP (CSFA-0), additional analogues
were synthesized. First, the importance of the benzimidazolyl substituents was assessed
by either omitting one of the benzimidazolyl side chains (1) or by modification of the
benzimidazolyl ring system (2-7). From Table 1, it is apparent that both the nature of the
benzimidazolyl side chain and the number, i.e. two, of side chains on position 2 and 6 of
the pyridine ring are essential for the anti-BVDV activity of this class of compounds.
10

Next, the importance of the central pyridine ring for the anti-BVDV activity was
assessed. As shown in Table 2, the substitution of the pyridine by benzene (8) results in
reduced anti-BVDV activity. The introduction of a hydroxyl group on position 4 of the
pyridine (9) or the substitution of the pyridine ring by a thiophene (10) also yields
inactive derivatives. For the pyridine N-oxide (11) a reduced cytotoxicity as compared to
8
and 9 and a 5-fold increase in antiviral activity were observed.
To further understand the structural requirements for the antiviral activity within this
class of compounds, a third set of analogues was synthesized. Here, different substituents
1
2
(
R and R ) were introduced onto the two 1H-benzimidazole groups (compounds 12-25,
Table 3). Some substituents that were introduced on position 4 and 5 (compound 12) and
in position 4(7) (compounds 13 and 14, Table 3) resulted in a decrease in anti-BVDV
activity as compared to the parent compound [BBP (CSFA-0)], with compound 12 being
cytotoxic. Further modifications of the two 1H-benzimidazole groups were introduced at
position 5(6) of the benzimidazole group (compounds 15-24, Table 3); in particular a
fluorine in position 5(6) of the benzimidazole groups (16) resulted in a 10-fold increase in
anti-BVDV activity as compared to BBP (CSFA-0) but is still associated with cytotoxic
effects. Analogue 22 had an equipotent anti-BVDV as BBP (CSFA-0) and was not
cytotoxic to the cells. The addition of 5(6)-methoxy substituents on the two 1H-
benzimidazole groups (22) was well tolerated by the cells, much better than other
substituents (-CH
3
, -Cl, -CF , -I, 5,6-benzo).
3
Isolation and characterization of drug-resistant viruses
To identify the target of BBP, we isolated and characterized BBP-resistant virus
res
res
(
BBP ). BBP virus was selected by propagating BVDV (strain NADL) for 28 passages
in increasing concentration of the drug (from 1.8 to 30 µM). Drug resistant BVDV
variants selected have comparable replication kinetics and no gross differences in
replication fitness (supplemental file). The resulting drug-resistant virus proved 55-fold
less susceptible to the inhibitory effect of BBP as compared to the parent wild-type strain
and about 6.7-fold, >11.3-fold and 186-fold less susceptible to the known BVDV
inhibitors AG110, LZ37 or BPIP, respectively (Table 4). Conversely, BBP proved to be
about 6-fold less active on the replication of recombinant BVDV carrying only the single
11

res
mutation F224S in the polymerase. By comparing the BBP virus genome sequence to
the parental wild-type strain (GenBank accession no. AJ781045), we identified a
transition of A to G at position 10975 in the viral RNA. This point mutation results in an
amino acid change of isoleucine (I) to methionine (M) at amino acid residue 261 in the
res
RdRp. For CSFV, three independently selected cultures of BBP CSFV were obtained
following 11 consecutive passages of the wild type virus (strain Alfort187) in the presence
of gradually increasing concentrations of BBP. All three drug-resistant virus cultures
proved almost completely refractory to inhibition of viral replication by BBP (EC50 > 100
µM) and were thus regarded as BBP-resistant (Table 4). Susceptibility of BBP-resistant
viruses and other resistant variants studied to 2’-C-methylcytidine was comparable to or
more than WT (Table 4).
res
Moreover, BBP proved to be cross-resistant with the earlier selected BPIP -CSFV
(
Vrancken et al., 2008) (EC50 > 100 µM, Table 4). Since the molecular target of BPIP in
CSFV replication has been shown to be the RdRp, the sequence of the CSFV NS5B-gene
res
of the selected BBP CSFV was determined and aligned to the sequence of the viral
population before the start of the resistance selection and with wild-type CSFV strain
Alfort187 (NCBI accession number X87939). Two of the three independently selected,
drug-resistant viruses carried a T to A transversion at position 10 695 resulting in the
substitution of an isoleucine (I) to asparagine (N) at position 261 of the NS5B protein.
The other drug-resistant virus carried a C to G transversion at position 10 697 resulting in
the substitution of a proline (P) to an alanine (A) at position 262 of the NS5B protein.
Effect of BBP (CSFA-0) on the BVDV RdRp and Replication complexes.
Since the BBP-resistant virus carries mutations in RdRp, the inhibitory effect of BBP on
the BVDV polymerase activity was evaluated using a recombinant BVDV RdRp making
use of a poly(C) template. For the reference inhibitor 3΄-dGTP, the 50% inhibitory
concentration for the BVDV polymerase activity was <1 µM. BBP however had no effect
on the activity of the viral polymerase (Fig. 3). Because BBP did not inhibit the activity
of the purified BVDV RdRp, the effect of the compound on viral replication complexes
(
RCs), isolated from MDBK cells that had been infected with the wild-type virus or with
res
the selected BBP BVDV strain, was assessed. BBP inhibited the activity of the BVDV
12

WT replication complexes in a dose-dependent manner with a maximum inhibition of
~80% at 5 µM (Fig. 4A and B). By contrast, the activity of BBP against RCs isolated
res
from MDBK cells that had been infected with the BBP BVDV, was 4-fold less potent
compared to the inhibitory effect of BBP on the RCs derived from cells infected with
BVDV WT (Fig. 4A and B).
Computational docking of BBP in the BVDV RdRp crystal structure
A closer examination of the BVDV RdRp crystal structure revealed that the mutation
I261M is located in a small cavity near the fingertip domain of the BVDV polymerase at
a distance of only 8 Ǻ from F224 and 12 Ǻ from E291, (Fig 5). The larger side chain of
methionine M261 may be involved in resistance against VP32947, BPIP, LZ37 and
AG110, respectively (Baginski et al., 2000; Paeshuyse et al., 2009; Paeshuyse et al.,
2
006; Paeshuyse et al., 2007). Docking studies in this cavity revealed the following
possible interactions between the polymerase and BBP: (i) hydrophobic contacts of BBP
with T160, F224, P262, K263, N264, I287 and A392; and (ii) a hydrogen bond from a N
in the 5-membered ring to the main chain O atom in P262 (Fig. 5). The larger side chain
of methionine may clash with the BBP, thereby interfering with its binding.
13

DISCUSSION
In a large scale screening effort aimed to identify novel pestivirus inhibitors, we
identified 2,6-bis(benzimidazol-2-yl)pyridine [BBP (CSFA-0)], as a selective in vitro
inhibitor of the replication of pestiviruses. BBP proved active against BVDV-1 as well as
against CSFV, but proved inactive against the related HCV and flaviviruses (YFV and
DENV) or a panel of unrelated RNA and DNA viruses.
In an attempt to improve the antiviral activity/selectivity, 25 analogues were
synthesized. A structure-activity relationship was derived from the data obtained with
these compounds in virus-cell-based assays. We did not succeed in optimizing the
potency of this class of molecules. In particular analogue 22 was shown to have similar
activity/selectivity as the lead compound.
res
To unravel the mechanism of antiviral activity, the genotype of in vitro selected BBP
BVDV was determined. An isoleucine (I) to methionine (M) mutation was detected at
amino acid residue 261 (I261M) of the NS5B, the gene that encodes the viral RNA-
dependent RNA polymerase. Similar observations were made for CSFV for which two
individual point mutations, i.e. I261N and P262A, were identified in the viral polymerase.
The crystal structure of the RdRp reveals a right-handed structure made up of fingers, a
palm and a thumb domain. A special feature of the pestivirus RdRp is that the fingers
domain contains an extension called fingertips domain, which interacts with the thumb
domain, with which it forms an entrance to the template-binding channel. The fingertip
domain is believed to be involved in the template/product translocation, dimerization of
RdRp within the RC, or protein-protein interactions, enabling the assembly of an active
RC (Choi et al., 2004; Uttenthal et al., 2005; Morris et al., 1998; Choi et al., 2006).
Moreover, the fingertip region contains the polymerase motifs I and II, which are
involved in RNA template and nucleoside triphosphate (NTP) binding. Motif I has been
reported to be located near the initiation NTP binding site and to bind the incoming NTP
(
Choi et al., 2004). Interestingly, the I261M mutation is located in the fingertip region
within this motif. This mutation, selected under antiviral pressure with BBP, might thus
cause conformational changes in the fingertip region of RdRp, disturbing one of the
aforementioned functions.
14

res
Despite the fact that only (a) mutation(s) in the RdRp was (were) found in both BBP
BVDV and CSFV, provides strong evidence that the viral polymerase is the target, BBP
had no effect on the activity of the purified recombinant BVDV RdRp. The viral
polymerase assay was validated by demonstrating that 3'-dGTP inhibited the BVDV
RdRp activity. Likewise, also BPIP (Paeshuyse et al., 2006), AG110 (Paeshuyse et al.,
2
007) and LZ37 (Paeshuyse et al., 2009) also had no inhibitory effect on the highly
purified RdRp. However, in the context of the host cell, the Pestivirus NS5B interacts
with other viral and cellular proteins and together with the RNA genome, assembles in a
membrane-associated replication complex that is involved in viral replication (Uttenthal
et al., 2005). The replication activity of the latter was shown to be inhibited by BPIP
(
Paeshuyse et al., 2006), AG110 (Paeshuyse et al., 2007) and LZ37 (Paeshuyse et al.,
2
009). We therefore evaluated the effect of BBP on RCs isolated from MDBK cells that
res
had been infected with either the wild-type virus or the BBP BVDV. BBP inhibited the
function of the wild-type RC 4-fold more than that of the mutant strain. This may be
explained by (i) a specific conformation of the viral RdRp in the RC that is needed for a
productive interaction with the RNA template, or (ii) a protein-protein interaction
between NS5B and the others viral non-structural proteins and host protein blocking
compound binding sites on the viral polymerase finger domain (Paeshuyse et al., 2007).
The observation that BBP inhibits the function of RC but not that of the purified RdRp
may be explained by the fact that, following binding to the NS5B, the compound might
disturb the proper formation of a functional replication complex. We hypothesize that
BBP may inhibit the protein-protein interactions and stability between the different non-
structural proteins that compose the replication complex, interfering with its correct
functioning.
The previously reported BVDV inhibitors BPIP, AG110, and LZ37 proved to be cross-
res
res
resistant with BBP-resistant BVDV. BPIP and LZ37 BVDV were shown earlier to
carry the F224S and F224Y mutation (Paeshuyse et al., 2009; Paeshuyse et al., 2006),
res
respectively in their polymerase; while AG110 BVDV carry the E291G mutation
(
Paeshuyse et al., 2007). The cross-resistance of these different classes of inhibitors might
be explained by the proximity (7 Å) of E291, F224 and I261 in the RdRp structure.
Furthermore, it was shown that a singular mutation, namely F224S, was sufficient to
15

cause a ~6-fold reduction in activity of BBP (see Table 4). The I261M mutation might
influence the structure of RdRp in the close vicinity of residues 291 and 224, causing
conformational changes in this cavity of the polymerase, thus explaining the cross-
resistant to these different inhibitors.
The findings made for BVDV were paralleled by observations made with CSFV.
res
Although BPIP belongs to a structurally different class of compounds, BPIP -virus
proved also insensitive to BBP (EC50 > 100 µM). Despite the obvious cross-resistance of
BPIP and BBP, the mutations in the CSFV RdRp (i.e. I261N and P262A) are different
from the previously reported mutation (i.e. T259S) that confers resistance to the
imidazopyridine BPIP. However, residues I261 and P262 are located in close proximity
to both T259 and F224 (Paeshuyse et al., 2006; Vrancken et al., 2008). Considering the
clear cross-resistance and very close vicinity of the residues I261 and P262 to T259 and
F224, it is reasonable to assume that BBP may interact with NS5B of CSFV at a site very
close to (if not identically to) the binding-site of BPIP. Combining these observations
suggests that the cross-resistance among several structurally unrelated classes of
pestivirus inhibitors are the result of mutations that lead to conformational changes in this
“cavity” of the viral polymerase.
In conclusion, we identified a highly potent and selective inhibitor of pestiviruses
replication. Structural analogues of this molecule also exhibit anti-pestivirus activity.
BBP possibly interacts with the fingertip of the BVDV-RdRp region that is a “hot spot”
for inhibition of pestivirus replication.
16

ACKNOWLEDGEMENTS
We thank Carolien De Keyzer, Stijn Delmotte and Tom Bellon for excellent technical
assistance, Suzanne Kaptein for performing the YFV and DENV assays and Leentje
Persoons for performing the screen against unrelated DNA and RNA viruses. This work
was supported by a postdoctoral fellowship from the Research Foundation Flanders-FWO
to Jan Paeshuyse, the IWT-SBO project #100042, KU Leuven grant (GOA/10/014) and
by grant G.0728.09N of the Research Foundation Flanders-FWO.
17

1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
FIG.1. Schematic representation of the synthesis of analogues 0-25.
FIG.2. (A) Effect of BBP on BVDV (NADL)–induced CPE formation in MDBK cells
(bars) and on the proliferation of exponentially growing MDBK cells (diamonds). (B)
Inhibitory effect of BBP on release of intracellular viral RNA. (C) Inhibitory effect of
BBP on release of extracellular viral RNA. (D) Inhibitory effect of BBP on infectious
virus yield. Data are mean values ± standard deviations from three independent
experiments. UTC, untreated control. Antiviral activity and cytotoxicity were obtained in
separate experiments.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
FIG.3. Effect of BBP (open triangles) and 3'-dGTP (open diamonds) on the activity of
the purified BVDV RdRp. Data are from a typical experiment and are expressed as the
percentage of untreated control.
FIG.4. Effect of BBP on the activity of RCs isolated at 14 h post-infection from MDBK
res
cells that had been infected with wild-type BVDV (NADL) or with the selected BBP
BVDV strain. Densiometric analysis of the autoradiographs; black bars represent activity
of wild-type BVDV (NADL) RCs, and open bars represent activity of RCs from cell
res
infected with BBP virus. Data are mean values ± SD of 2 independent experiments.
UTC, untreated control; WT, wild-type.
FIG.5. Modeling of BBP near the position I261M in the RNA-dependent RNA
polymerase. The different domains of the BVDV polymerase are colored as follows: the
N-terminal domain is in yellow (residues 92 to 138), the finger domain is in blue
(residues 139 to 313 and residues 351 to 410), the palm domain is in green (residues 314
to 350 and residues 411 to 500), and the thumb domain in red (thumb, residues 501 to
679). Carbons of residue I261I are colored orange and the I261M mutation has magenta
carbons. Residues F224 and E291 are also made visible in stick representation. The
ligand carbon atoms are colored green. Hydrophobic interactions are visualized by a cyan
color. BBP has a hydrogen bond from an N in the 5 membered ring to the main chain O
atom in P262.
18

1
2
3
4
5
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1
1
1
1
1
2
5
6
7
8
9
0
22

FIG.1.
1
R
1
R
2
R
H N
2
N
N
PPA
N
2
2
O
O
+
R
R
N
N
N
H N
2
H
H
OH
OH

FIG.2.
A
B
1
00
8
6
4
2
0
0
0
0
0
0
.01
0.04
0.1
0.4
1
Concentration of BBP (µM)
C
D
1
00
100
80
60
40
20
0
8
6
4
2
0
0
0
0
0
0
.01
0.04
0.1
0.4
1
0.1
0.4
1
3
10
Concentration of BBP (µM)
Concentration of BBP (µM)

FIG.3.

FIG.4.
1
00
8
6
4
2
0
0
0
0
0
0
0.6
1.3
2.5
5
Concentration of BBP (µM)

FIG.5.

Table 1. Structure, in vitro anti-BVDV activity and cytotoxicity of compounds 0-7.
a
a
b
Compound
Structure
EC50 (µM)
CC50 (µM)
S.I.
0
0.5 ± 0.2
>100
200
1
2
>33
>33
>100
>100
n.a.
n.a.
3
4
5
>33
>33
>33
>100
73 ± 5.6
>100
n.a.
n.a.
n.a.
6
7
>33
>33
>100
>100
n.a.
n.a.
The EC50 (50 % effective concentration) is the concentration of compound that inhibits virus-induced
cytopathic effect by 50%. The CC50 (50% cytostatic concentration) is the concentration that inhibits the
proliferation of exponentially growing cells by 50%.
a
Data are mean values ± SD for at least 4 independent experiments.
b
In vitro selectivity index (CC /EC ); n.a., not applicable.
5
0
50

Table 2. Structure, in vitro anti-BVDV activity and cytotoxicity of compounds 8-11.
a
a
b
Compound
Structure
EC50 (µM)
CC50 (µM)
S.I.
8
4.0 ± 1.4
4.6 ± 0.9
1.1
1
9
2.6 ± 0.5
2.6 ± 0.5
1
1
0
1
>33
>100
3.0
0.1 ± 0.6
15 ± 3.2
150
The EC50 (50 % effective concentration) is the concentration of compound that inhibits virus-induced
cytopathic effect by 50%. The CC50 (50% cytostatic concentration) is the concentration that inhibits the
proliferation of exponentially growing cells by 50%.
a
Data are mean values ± SD for at least 4 independent experiments.
b
In vitro selectivity index (CC /EC ); n.a., not applicable.
5
0
50