Structure-based virtual screening, synthesis and SAR of novel inhibitors of hepatitis C virus NS5B polymerase

Article abstract of DOI:10.1016/j.bmc.2010.05.030

Hepatitis C virus (HCV) NS5B polymerase is a key target for the development of therapeutic agents aimed at the treatment of HCV infections. Here we report on the identification of novel allosteric inhibitors of HCV NS5B through a combination of structure-

Full text of DOI:10.1016/j.bmc.2010.05.030

Bioorganic & Medicinal Chemistry 18 (2010) 4630–4638
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure-based virtual screening, synthesis and SAR of novel inhibitors
of hepatitis C virus NS5B polymerase
Tanaji T. Talele a, , , Payal Arora b, Shridhar S. Kulkarni a, Maulik R. Patel a, Satyakam Singh a,
*
Maksim Chudayeu b, Neerja Kaushik-Basu b, ,
*
a Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St. John’s University, 8000 Utopia Parkway, Jamaica, NY 11439, USA
b Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 March 2010
Revised 8 May 2010
Accepted 11 May 2010
Available online 15 May 2010
Hepatitis C virus (HCV) NS5B polymerase is a key target for the development of therapeutic agents aimed
at the treatment of HCV infections. Here we report on the identification of novel allosteric inhibitors of
HCV NS5B through a combination of structure-based virtual screening, synthesis and structure–activity
relationship (SAR) optimization approach. Virtual screening of 260,000 compounds from the ChemBridge
database against the tetracyclic indole inhibitor binding pocket of NS5B (allosteric pocket-1, AP-1),
sequentially down-sized the library by 4 orders of magnitude to yield 23 candidates. In vitro evaluation
of the NS5B inhibitory activity of the in-silico selected compounds resulted in 17% hit rate, identifying
two novel chemotypes. Of these, compound 3, bearing the rhodanine scaffold, proved amenable for pro-
ductive SAR exploration and synthetic modification. As a result, 25 derivatives that exhibited IC50 values
Keywords:
NS5B polymerase
Virtual screening
Rhodanine
Imidazocoumarin
SAR
ranging from 7.7 to 68.0 lM were developed. Docking analysis of lead compound 28 within the tetracy-
clic indole- and benzylidene-binding allosteric pockets (AP-1 and AP-3, respectively) of NS5B revealed
topological similarities between these two pockets. Compound 28, a novel rhodanine analog with
NS5B inhibitory potency in the low micromolar level range may be a promising lead for future develop-
ment of more potent NS5B inhibitors.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
teins.11–13 Nonstructural protein 5B (NS5B), a 66 kDa RNA-depen-
dent RNA polymerase (RdRp) is an important therapeutic target,
Hepatitis C virus (HCV), the etiological agent of parenteral non-
A, non-B hepatitis, often causes the development of malignant
chronic disease, including liver cirrhosis and hepatocellular carci-
noma, which frequently ends in liver failure.1–4 An estimated 200
million cases of HCV infections exist worldwide, of which over
4.1 million infections occur in the United States.5 At present, nei-
ther a vaccine nor a therapy with effective broad spectrum mode
of action against all genotypes of HCV is available.6–8 Current
since it plays a pivotal role in replicating the HCV RNA genome
and the host lacks its functional equivalent.14–16 The availability of
several high-resolution three-dimensional crystal structures of
NS5B either alone or in complex with ribonucleotide substrates or
non-nucleoside inhibitors, has resulted in the identification of five
distinct NS5B inhibitor binding pockets to-date.8,17–19 Three of these
correspond to NS5B allosteric inhibitor binding pockets: allosteric
pocket (AP)-1, -2, and -3.17–20 AP-1 is located on the surface of the
thumb domain adjacent to the allosteric GTP-binding site.21,22
Inhibitors identified against this pocket include benzimidazole,23
indole,24 quinoxaline,25 thieno[3,2-b]pyrrole,26 and coumestan27
derivatives. AP-2 is located in the thumb domain, next to
AP-1.28–30 Chemotypes of AP-2 binding analogs include the thiazo-
lone,31 N,N-disubstituted phenylalanine,32 thiophene-2-carboxylic
acid,33 pyranoindole,34 dihydropyranone,35 and thiazolidin-
4-one36 derivatives. AP-3 is located adjacent to the active site and
includes inhibitors such as benzothiadiazine,37 benzylidene,38
proline sulfonamide,39 anthranilic acid,40 acrylic acid,41 and pyrrol-
idine42 derivatives. Crystallographic analyses of these NS5B-inhibi-
tor complexes in combination with high-throughput-screening
(HTS), have uncovered several diverse chemical scaffolds of HCV
NS5B non-nucleoside inhibitors (NNIs).17–20 In recent years, virtual
HCV therapy comprising of pegylated interferon
a (PEG-IFN-a) in
combination with ribavirin has found limited patient compliance
due to severe adverse effects.9,10 Therefore, there is a need to de-
velop novel anti-HCV agents with high therapeutic index, reduced
side-effects, and a convenient route of administration.
HCV is an enveloped, positive-stranded RNA virus with ꢀ9.6 kb
genome that encodes three structural (Core, E1, and E2) and seven
nonstructural (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) pro-
* Corresponding authors. Tel.: +1 718 990 5405; fax: +1 718 990 1877 (T.T.T.);
tel.: +1 973 972 8653; fax: +1 973 972 5594 (N.K.-B.).
E-mail addresses: talelet@stjohns.edu (T.T. Talele), kaushik@umdnj.edu (N.
Kaushik-Basu).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.030

T. T. Talele et al. / Bioorg. Med. Chem. 18 (2010) 4630–4638
4631
screening has been emerging as a complementary approach to HTS
in an effort to identify potential leads in the drug discovery
process.43
Previously, we have reported on the utility of 3D QSAR method-
ologies such as the comparative molecular field analysis (CoMFA)
and comparative molecular similarity indices analysis (CoMSIA)
for investigating and optimizing benzimidazole HCV NS5B poly-
merase inhibitors which bind to AP-1 of NS5B.44 In another study,
we identified 2,3-disubstituted-4-thiazolidinone derivatives, as
modest inhibitors of NS5B polymerase, and predicted that they
bind to AP-2 of NS5B.36 In the present study, we have employed
virtual screening tool to identify novel small molecule NNIs of
NS5B and further optimized the 5-benzylidene rhodanine-3-acetic
acid hit based on the crystal structure of NS5B in complex with a
tetracyclic indole inhibitor (PDB ID: 2dxs).24
2. Results
2.1. Validity of docking and virtual screening approach
Figure 1. Virtual screening parameters employed for identifying potential NS5B
allosteric inhibitors from the ChemBridge database.
As a first step, we validated the ability of the docking algorithm
to reproduce the co-crystallized pose of the tetracyclic indole, thia-
zolone and benzylidene inhibitors within their respective allosteric
pockets on NS5B. This yielded a good agreement between the
docked and crystal structures as is evident from the root mean
square (rms) deviation values of 0.827, 0.332, and 0.111 Å for tetra-
cyclic indole, thiazolone, and benzylidene inhibitors, respectively.
This confirms the reliability of the Glide docking procedure in
reproducing the experimentally observed binding mode for NS5B
inhibitors. Therefore, the parameters set for Glide docking appear
reasonable to provide meaningful insight into the predicted binding
mode for hitherto untested NS5B inhibitors.
We next evaluated whether this methodology was amenable to
discriminate between known inhibitors and randomly chosen
drug-like molecules from the ChemBridge database. For this we
docked the ‘validation set’ into the tetracyclic indole binding site
of NS5B, and analyzed the list of the ‘hit’ molecules for the top
10% highest scoring compounds. This investigation yielded 11 of
the 20 known NNIs as the top 10% scoring molecules (Supplemen-
tary data, Fig. S1). Of these 11 compounds, the most potent benz-
imidazole analog was identified as the top ranked hit with a VS
(virtual screening) GlideScore of À7.07. It was interesting to note
that approximately eight compounds with low micromolar to
sub-micromolar NS5B inhibitory activity exhibited a VS GlideScore
of ꢀÀ5.00. Since the majority of these known NS5B NNIs seeded in
the ‘validation dataset’ as test standards yielded VS GlideScores
ranging between À5.0 and À7.0 (Supplementary data, Fig. S1),
we set VS GlideScore À5.0 as a threshold parameter to identify di-
verse hit molecules.
Figure 2. Representative key pharmacophoric features employed for visual inspec-
tion and selection of compounds against tetracyclic indole inhibitor binding
allosteric pocket of NS5B. The pharmacophoric features include hydrophobic
groups pointing to HP-1 and/or HP-2, hydrogen bond acceptor or negatively
charged groups and the central heterocyclic scaffold to hold these features together.
Throughput Virtual Screening (HTVS) workflow. This second
screening resulted in the elimination of ꢀ98% molecules based
on the application of VS GlideScore selection cut-off of À5.0. The
remaining 650 compounds were visually inspected based on
our previous in-silico44 and wet lab27,36 experience for improba-
ble docking orientations in AP-1 of NS5B. The ensuing compounds
were further analyzed for their interactions with the amino
acid residues which are important for binding of indole and
benzimidazole allosteric inhibitors,44 in context of the following
parameters: (1) possess at least one hydrophobic group pointing
towards hydrophobic pocket-1 (HP-1) formed by residues Val37,
Ala393, Ala396, Leu492, and Val494 or hydrophobic pocket 2
(HP-2) formed by residues Leu392, Ala395, Ala396, Ile424,
Leu425, His428, and Phe429, (2) form at least one hydrogen bond
with His428, Ser431, and Gly493 or ionic interaction with the
guanidine group of Arg503, and (3) display structural uniqueness
(to avoid redundancy among chemically similar hits) (Fig. 2).
This rigorous in-silico screening criteria ultimately filtered out
627 compounds, thus yielding a set of 23 compounds bearing
structural diversity and good docking orientations for biological
evaluation.
2.2. Virtual screening of compounds against AP-1 of HCV NS5B
A step-wise strategy for virtual screening was employed to
identify allosteric inhibitors of NS5B polymerase. Towards this
end, a ChemBridge database comprising of 260,000 compounds
was filtered according to the criteria described in the Methods sec-
tion, to generate a new database comprising of 52,000 3D com-
pounds (Fig. 1). Irrespective of whether the ligands were treated
as neutral or ionized, did not change the outcome of the screening
investigations. However, upon ionization, the ligands exhibited rel-
atively more negative binding energies and their ionizable groups
such as –COOH oriented towards the guanidine group of Arg503
as opposed to the backbone of Gly493 as observed in their neutral
state. The new database comprising of 52,000 3D compounds was
docked in AP-1 of NS5B polymerase, employing the Glide’s High

4632
T. T. Talele et al. / Bioorg. Med. Chem. 18 (2010) 4630–4638
Table 1
Anti-NS5B RdRp activity of compounds 1–4
HOOC
O
HOOC
O
NC
NC
N
N
N
N
OH
N
O
S
N
S
O
O
CF3
1
2
3
4
Compounda
MW
283
282
347
262
C log Pb
% inhibition at 250
l
Mc
IC50 (l
M)d
1
2
3
4
1.73
3.24
2.31
2.89
55 2.8
48.6 4.9
71 7.0
ꢀ200
ꢀ250
55.2 1.10
60.2 1.17
62.5 7.7
a
b
c
Compounds acquired from ChemBridge were reported to have a purity P90% by 1H NMR.
C log P was calculated using QikProp v3.0.
Percent inhibition was determined at 250 lM concentration of the indicated compound and represents an average of at least two independent measurements in
duplicate. NS5B RdRp activity in the absence of the inhibitor was taken as 100% after subtraction of residual background activity.
d
The IC50 values of compounds 3 and 4 were determined from dose–response curves using 8–12 concentrations of each compound in duplicate in two independent
experiments. Curves were fitted to data points using nonlinear regression analysis and IC50 values were interpolated from the resulting curves using GraphPad Prism 3.03
software.
2.3. In vitro screening of inhibitors
resulted in
IC50 = 19.9
carboxyl group in compound 8 was replaced with bioisosteric
sulfonic acid group (compound 9, IC50 = 23.5 M). Separation of
carboxyl group from the rhodanine core by a propylene linker
resulted in substantial loss of activity (compound 10, IC50
58.0 M), in contrast the activity improved by 3.6-fold when the
pentylene linker was used (compound 11, IC50 = 16.1 M). Several
additional analogs such as: 5-(2,4-dichlorobenzylidene)-3-ethyl-
2-thioxothiazolidin-4-one, 2-(4-oxo-2-thioxothiazolidin-3-yl)acetic
acid, and 2-(5-benzylidene-4-oxo-2-thioxothiazolidin-3-yl)acetic
acid lacking (a) a carboxyl group at N3-substituent, (b) benzylidene
moiety at C5-position, and (c) substituents on benzylidene moiety,
respectively, were found to be either inactive or poorly active
(Supplementary data, compounds 29–34, Table S1). In addition,
analogs with electron donating substituents on the benzylidene
moiety (Supplementary data, compounds 35 and 36, Table S1)
exhibited poor activity, whereas electron withdrawing 3-cyano
and 3-carboxy substituents on the benzylidene moiety (Supple-
mentary data, compounds 37 and 38, Table S1) exhibited higher
NS5B inhibitory activity.
a
marginal decrease in activity (compound 8,
l
M). Further decrease in activity was observed when
To investigate the inhibitory activity of the virtually screened
23 candidates against NS5B, in vitro NS5B RNA-dependent RNA
polymerase (RdRp) inhibition assay was carried out as described
in Section 5.27,36 The RdRp reaction was performed on poly rA/
U12 template-primer (TP), employing recombinant HCV NS5B
(genotype 1b) with an N-terminal His-tag and C-terminal 21-ami-
l
=
l
l
no acid deletion (NS5BC
D
21).27,36 LQB34, a coumestan derivative,
previously characterized by us as a NS5B inhibitor was included
as an internal reference standard.27 To identify candidates belong-
ing to a wider range of structural scaffolds, preliminary screening
was conducted at a concentration of 250 lM for each compound.
This analysis yielded four compounds exhibiting P50% inhibition
of NS5B RdRp activity (Table 1), thus demonstrating a 17% hit rate.
Of these, compounds 3 (rhodanine analog) and 4 (imidazocouma-
rin analog) exhibited IC50 values of 55.2 and 60.2 lM, respectively.
Further exploration of SAR around imidazocoumarin analog 4 re-
sulted in either inactive or poorly active analogs (Supplementary
data, compounds 39–43, Table S1). Thus we pursued compound
3 for further SAR investigations.
The 1-naphthylidene substituent at 5-position of the rhodanine
ring was detrimental to the NS5B inhibitory activity when the N3-
2.4. Development of SAR around rhodanine scaffold
substituent was either an acetic acid (compound 12, IC50
61.4 M) or -methyl acetic acid (compound 13, IC50 = 68.1 M),
in contrast to a bulkier -isopropyl acetic acid substituent (com-
pound 14, IC50 = 16.9 M) which was beneficial. We next obtained
compounds 15–17, which similar to compound 14 carried the
-isopropyl acetic acid substituent at 3-position of the rhodanine
=
Following the initial rhodanine hit 3, commercially available
rhodanine analogs (compounds 5–22) were obtained for explora-
tion of SAR around 3- and 5-position of the rhodanine scaffold,
leading to the development of detailed SAR data (Table 2) and
l
a
l
a
l
identification of potent analog (compound 22, IC50 = 10.6
l
M) as
a
described below.
ring, but harbored a series of substituted benzylidenes in lieu of
Moving –CF3 group on the 5-benzylidene moiety of
(IC50 = 55.2 M) from para to the meta position (compound 5,
IC50 = 50.9 M) led to a marginal enhancement of inhibitory activ-
ity. Bioisosteric replacement of the –CF3 group in compound 5 with
–Br group (compound 6, IC50 = 20.2 M) resulted in a 2.5-fold in-
3
1-naphthylidene moiety. While 4-fluorobenzylidene (compound
l
l
15, IC50 = 42.9
lM) and 2-chlorobenzylidene (compound 16,
IC50 = 36.1 M) analogs proved to be approximately two- to three-
l
fold less active compared to compound 14, comparable inhibitory
activity was observed in the presence of 2,4-dichlorobenzylidene
l
crease in inhibitory activity. Further improvement in inhibitory
substituent (compound 17, IC50 = 14.1 lM).
activity was obtained when 3-CF3 benzylidene was replaced with
2,4-dichlorobenzylidene moiety (compound 7, IC50 = 17.9 lM).
We purchased compounds 8–11, to explore the effect of 3-position
substituents on rhodanine ring in the presence of 2,4-dichloroben-
zylidene substituent at 5-position of the rhodanine ring. Separating
Based on these findings, we procured compounds 18–22
bearing 2,4-dichlorobenzylidene substituent at 5-position and a
various substituents at 3-position of the rhodanine ring. While
a
-propylacetic acid (compound 18, IC50 = 28.2
thioethyl acetic acid (compound 19, IC50 = 29.2
resulted in twofold decrease in inhibitory activity, the bulkier
l
M) and
a-methyl-
lM) substituents
a-carboxyl group from the rhodanine core by ethylene bridge

T. T. Talele et al. / Bioorg. Med. Chem. 18 (2010) 4630–4638
4633
Table 2
Structure–activity relationship of compound 3 derivatives
R
N
S
O
S
Ar
5 28
-
Compounda
R
Ar
MW
C log Pb
IC50
(lM)
c
5
6
7
8
9
–CH2COOH
–CH2COOH
–CH2COOH
–CH2CH2COOH
–CH2CH2SO3H
–CH2CH2CH2COOH
–(CH2)5COOH
–CH2COOH
–CH(COOH)CH3
–CH(COOH)CH(CH3)2
–CH(COOH)CH(CH3)2
–CH(COOH)CH(CH3)2
–CH(COOH)CH(CH3)2
–CH(COOH)CH2CH2CH3
–CH(COOH)CH2CH2SCH3
–CH(COOH)CH2CH(CH3)2
–CH(COOH)CH3
–CH(COOH)CH2Ph
–CH2COOH
3-CF3Ph
3-BrPh
347
358
348
362
398
376
404
329
343
371
339
355
390
390
422
404
362
438
324
324
297
313
348
371
2.31
3.02
3.40
3.96
3.20
4.31
5.10
3.60
3.94
4.64
4.13
4.18
4.73
4.71
5.26
5.28
3.94
5.77
2.10
2.01
2.65
2.82
3.39
4.26
50.9 1.4
20.2 1.2
17.9 1.2
19.9 1.3
23.5 1.2
58.0 1.5
16.1 1.3
61.4 1.3
68.1 1.5
16.9 1.2
42.9 1.3
36.1 1.2
14.1 1.2
28.2 1.1
29.2 1.2
18.9 1.1
13.3 1.2
10.6 1.5
16.2 1.4
21.9 1.3
42.1 1.0
18.4 1.2
8.4 1.5
2,4-ClPh
2,4-ClPh
2,4-ClPh
2,4-ClPh
2,4-ClPh
Naphthalene-1-yl
Naphthalen-1-yl
Naphthalen-1-yl
4-FPh
2-ClPh
2,4-ClPh
2,4-ClPh
2,4-ClPh
2,4-ClPh
2,4-ClPh
2,4-ClPh
2-NO2Ph
4-NO2Ph
4-FPh
3-ClPh
3,4-ClPh
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
–CH2COOH
–CH2COOH
–CH2COOH
–CH2COOH
–CH2COOH
3-PhenoxyPh
7.7 1.4
a
Compounds 5–8, and 12 acquired from ChemBridge and compounds 9–11 and 13–22 acquired from Sigma were reported to have a purity P90% by 1H NMR, compounds
23–28 were synthesized in the present study.
b
C log P was calculated using QikProp v3.0.
The IC50 values of compounds 5–28 were determined from dose–response curves using 8–12 concentrations of each compound in duplicate in two independent
c
experiments. Curves were fitted to data points using nonlinear regression analysis and IC50 values were interpolated from the resulting curves using GraphPad Prism 3.03
software.
a
-isobutyl acetic acid analog (compound 20, IC50 = 18.9
the less bulky -methyl acetic acid analog (compound 21,
IC50 = 13.3 M) exhibited activities in a comparable range to com-
pound 17. Finally, compound 22 having -benzyl acetic acid group
at 3-position (IC50 = 10.6 M) proved to be the most potent com-
lM) and
a
l
a
l
pound of the commercially available rhodanine series (Table 2).
With the SAR information gathered from the in vitro evaluation
of commercially available rhodanine analogs against NS5B, we
sought to synthesize novel rhodanine analogs in order to identify
more potent compounds. We chose rhodanine-3-acetic acid as
the scaffold for further SAR exploration aiming at variations at
C5-benzylidene substituent. This choice was based on the following
Scheme 1. Preparation of compounds 23–28.
the 2-position of the benzylidene moiety may be favorable for nitro
rationale: (1) reasonably active compounds 6 (IC50 = 20.2
lM) and
substituent. Replacement of nitro group in compound 24 with
7 (IC50 = 17.9 M) both bearing rhodanine-3-acetic acid functional-
l
fluoro (compound 25, IC50 = 42.1
activity indicating the importance of the size of the electron with-
drawing group. The 3-chlorobenzylidene analog (compound 26,
IC50 = 18.4 lM) exhibited comparable activity to compound 24.
Finally, more than twofold enhancement of inhibitory activity
was obtained when 3-chlorobenzylidene was replaced with either
3,4-dichlorobenzylidene (compound 27, IC50 = 8.4
oxybenzylidene (compound 28, IC50 = 7.7 M), which led to the
l
M) led to ꢀ2-fold decrease in
ity; (2) absence of stereogenic center; (3) easy availability of
starting material and (4) one step synthetic derivatization at
C5-position through Knoevenagel condensation leading to target
compounds. Addition of arylalkylidene moiety at C5-position of
the rhodanine-3-acetic acid was accomplished by a Knoevenagel
condensation to afford target compounds 23–28 as shown in
Scheme 1. This condensation reaction with aromatic aldehyde pro-
vided only the Z isomer, as determined by the chemical shift of the
methylene proton ranging from 7.9 to 8.1 as a singlet.45,46 The
synthesized compounds 23–28 were subjected to in vitro NS5B
evaluation (Table 2). The 2-nitrobenzylidene analog (compound
lM) or 3-phen-
l
identification of the two most potent compounds from the in house
synthetic efforts on rhodanine analogs. These data illustrate that
carboxyl group at
a-position of the N3-substituent and electron
withdrawing group bearing benzylidene moiety at C5-position
are essential pharmacophoric features of the rhodanine series of
compounds for NS5B polymerase inhibitory activity.
23, IC50 = 16.2
zylidene analog (compound 24, IC50 = 21.9
l
M) exhibited higher activity versus the 4-nitroben-
M), suggesting that
l

4634
T. T. Talele et al. / Bioorg. Med. Chem. 18 (2010) 4630–4638
Table 3
Binding energies of compounds 3, 22, 27, 28, Amgen 5o, and Valeant 6 in their ionized state within each of the allosteric pockets of NS5B
O
O
HN
N
COOH
COOH
O
COOH
O
S
F
N
N
N
S
HN
S
S
S
O
O
S
S
S
O
O
Cl
Cl
C2H5
Valeant 6
Cl
27
Cl
Amgen 5o
28
Compounds
XP-GlideScore (kcal/mol)
AP-2
IC50 (lM)
AP-1
AP-3
3
22
27
28
À6.00
À5.50
À5.29
À5.14
À3.80
À3.49
À5.92
À4.79
À5.18
À4.66
À3.30
À7.54
À5.46
À3.70
À8.00
À6.37
À4.79
À7.10
55.2
10.6
8.4
7.7
0.2
Amgen 5o
Valeant 6
3.0
Figure 3. Macromodel hydrophilic (orange color) and hydrophobic (yellow color) surface representations of AP-1 (panel A) and AP-3 (panel B) of NS5B in the presence of
compound 28.
2.5. Docking analysis
fied through screening for AP-1 binding molecules. This aroused
our curiosity to examine the binding energies for compound 3
and compound 22, the most potent compound of the commercially
available series, within each of the three allosteric pockets of NS5B.
Consistent with our screening analysis, both these compounds
exhibited a better fit in AP-1 versus the other two pockets (Table
We have identified a novel rhodanine analog (compound 3) as a
‘hit’ molecule by virtual screening against AP-1 of NS5B. To gain a
better insight into the potential binding mode of the optimized
derivatives of this initial hit, we docked compounds 27 and 28 into
each of the three reported HCV NS5B NNI binding sites represented
by AP-1,24 AP-2,31 and AP-338 (Table 3). For comparison, we also
docked two structurally related compounds, Amgen 5o (AP-3
bound inhibitor)38 and Valeant 6 (AP-2 bound inhibitor)31 within
AP-1, AP-2, and AP-3 of NS5B (Table 3). Since the ionized state of
the ligands at physiological pH is known to influence their binding
modes and energetics of binding at the target sites, we have used
the ionized form of these compounds for docking experiments.43
Consistent with their experimental binding modes, Amgen 5o
exhibited tighter binding in AP-3 versus AP-1 and AP-2 of NS5B,
while Valeant 6 bound much tighter in AP-2 compared to AP-1
or AP-3, as deduced from their relative XP-GlideScore values (Table
3). Surprisingly compound 28 appeared to bind more favorably in
AP-3 (XP-GlideScore = À6.37) versus AP-1 (XP-GlideScore = À5.14),
whereas AP-2 (XP-GlideScore = À4.66) appeared to be the least fa-
vored. A similar binding trend was observed with compound 27.
This observation was rather intriguing given that compounds 27
and 28 were derived from compound 3, our ‘hit’ molecule identi-
3). It may be noted that Amgen 5o (IC50 = 0.2
lM), although sev-
eral-fold more potent than compound 28 (IC50 = 7.7
l
M), exhibited
weaker binding energetics compared to compound 28 in AP-3
(Table 3). This discrepancy is not surprising in view of the observa-
tion that Amgen 5o forms a covalent bond with Cys366 in the NS5B
co-crystal structure.38 Consistent with this parameter, when the
Amgen 5o bound structure (C5-benzylidene moiety linked to the
rhodanine ring through a single bond) was docked into AP-3 it
yielded XP-GlideScore value of À7.94 (through space interaction,
data not shown).
Further analysis of the Macromodel hydrophilic (orange color)/
hydrophobic (yellow color) surface area representation of AP-1
(Fig. 3A) and AP-3 (Fig. 3B) of NS5B upon docking of compound
28 indicates topological similarities between these two pockets.
For example, the 3-phenoxybenzylidene moiety of compound 28
appears to be completely engulfed within the yellow hydrophobic
surface area in both AP-1 and AP-3 of NS5B. The rhodanine acetic
acid moiety is surrounded by hydrophilic surface area in both

T. T. Talele et al. / Bioorg. Med. Chem. 18 (2010) 4630–4638
4635
Figure 4. XP-Glide predicted binding mode of compound 28 within AP-1 (panel A) and AP-3 (panel B) of NS5B. Important amino acids are depicted as sticks with the atoms
colored as carbon—green, hydrogen—white, nitrogen—blue, oxygen—red, and sulfur—yellow) whereas the inhibitor is shown as ball and stick model with the same color
scheme as above except carbon atoms are represented in orange. Dotted black lines indicate either hydrogen bonding or ionic interaction.
AP-1 and AP-3 of NS5B. This finding may thus explain why com-
pound 28 binds within both pockets, albeit with much stronger
interaction in AP-3 versus AP-1, as indicated by its corresponding
XP-GlideScores within each of these pockets (Table 3).
size that a salt-bridge interaction between the e-amino group of
Lys141 and the carboxylate group of Glu143 might have destabi-
lized due to the formation of salt-bridge interaction between the
e-amino group of Lys141 and the carboxylate group of compound
As shown in Figure 4A, compound 28 appears to span the entire
AP-1 pocket (i.e., hydrophobic contacts with HP-1 and HP-2 and io-
nic interaction with Arg503). The solvent-exposed acetic acid
group at N3-position of the rhodanine ring forms ionic interaction
with the guanidine group of Arg503 (–COOÁ Á ÁH2N–Arg503, 1.71 Å).
A hydrogen bond was seen between the C4 carbonyl oxygen atom
of the rhodanine ring and the guanidine group of Arg503
(–C@OÁ Á ÁH2N–Arg503, 1.82 Å, 151.6°). The rhodanine ring is stabi-
lized by the side chains of Pro495 and Trp500. The phenyl ring of
the benzylidene group at C5-substituent forms hydrophobic con-
tacts with several residues present in HP-2 such as Leu392,
Ala395, Ala396, Ile424, Leu425, His428, Phe429, and Val494
whereas the terminal phenyl ring is stabilized by hydrophobic
interactions with residues from HP-1 such as Val37, Ala393,
Ala396, Leu492, and Val494.
28. The C4 carbonyl oxygen atom of the rhodanine ring forms water
mediated hydrogen bonding interactions with the backbone
atoms of Gln446 and Gly449 (–C@OÁ Á ÁH2O1432, 2.24 Å, 129.7°;
1432OH2Á Á ÁO@C–Gln446, 2.36 Å, 169.9°; 1432H2OÁ Á ÁHN–Gly449,
2.13 Å, 169.3°). The rhodanine ring is stabilized by the side chains
of Arg158, Gln446 and Ser556. The phenyl ring of the benzylidene
group at C5-substituent forms hydrophobic contacts with Phe193,
Cys366 and Tyr448, whereas the terminal phenyl ring is extensively
stabilized by hydrophobic interactions with residues Leu384,
Met414, Tyr415, and Tyr448. Apart from hydrophobic interactions,
the terminal phenyl ring is also stabilized through
p-cation type
interaction with the guanidine group of Arg200. These models thus
provide insight into the binding of compound 28 in AP-1 and AP-3
of NS5B.
Figure 4B shows the binding mode of compound 28 within AP-3
of NS5B. The carboxyl group forms ionic interaction with the
3. Discussion
e
-amino group of Lys141 (–COOÁ Á ÁH3N–Lys141, 1.79 Å). In addition,
In this study, we have employed the virtual screening approach
in an effort to identify and further design new inhibitors of HCV
NS5B, a validated target for antiviral therapy against HCV. A num-
ber of previous studies have reported the utility of this strategy to-
wards identifying novel lead molecules targeting HCV NS5B.47–51
the carboxyl oxygen atom forms water1307 mediated hydrogen
bonding interaction with the side chain of Glu143 and water1249
(–COOÁ Á ÁH2O1307, 1.69 Å, 159.6°; 1307OH2Á Á ÁOOC–Glu143,
1.88 Å, 174.4°; 1307H2OÁ Á ÁH2O1249, 2.12 Å, 166.6°). We hypothe-

4636
T. T. Talele et al. / Bioorg. Med. Chem. 18 (2010) 4630–4638
These screening campaigns have explored several commercially
available diversity set chemical libraries such as the 3D Pharmo
DB™ (Equispharm, Korea), National Cancer Institute (NCI), Lead-
Quest, Maybridge, French National Library and Prestwick through
pharmacophore or docking-based virtual screening approach with
equal success.47–51 Notably, these studies have highlighted the
cost-effectiveness of this strategy towards identifying potential
leads by virtue of downsizing the library by several orders of mag-
nitude. In our studies, we have explored 260,000 molecules from
the ChemBridge compound library through virtual screening
against the tetracyclic indole inhibitor binding pocket (AP-1) of
NS5B,24 and identified 23 candidates (Fig. 1), thus downsizing the
library by 4 orders of magnitude, consistent with afore-mentioned
reports.
acid side chains with appropriate stereochemistry to optimize
the pharmacokinetic profile of this series of compounds.
Analysis of the XP-Glide predicted binding pose of compound
28 in AP-3 (Fig. 4B), suggests that 4, 5, and 6 position of the phenyl
ring of the C5-benzylidene substituent can be substituted with
small hydrophobic groups. Similarly, branching at the methine
group of the C5-benzylidene substituent with small hydrophobic
groups such as methyl, ethyl, or cyclopropyl may be well tolerated
without compromising the inhibitory potency of the parent com-
pounds. Further, the terminal phenyl ring can accommodate small
groups such as methyl and halogens at ortho, meta, and para posi-
tions. The vacant region around a-carbon of N3-substituent similar
to that found in AP-1, provides further opportunity for optimizing
the amino acid side chains in terms of bulk, hydrophobicity,
charge, polarity and also the stereochemistry of the resulting chiral
center.
Among the two novel chemotypes identified upon in vitro RdRp
screening of the in-silico selected 23 compounds (Table 1), pursuit
of SAR around the imidazocoumarin scaffold (compound 4) had to
be abandoned as it yielded either inactive or poorly active analogs
(Supplementary data, compounds 39–43, Table S1). In contrast,
compound 3, bearing the rhodanine scaffold proved to be a suitable
‘hit’ for development of more potent novel analogs, through SAR
optimization and chemical synthesis (Table 2). Previous studies
have reported the IC50 values for lead molecules identified through
4. Conclusions
The ChemBridge database consisting of 260,000 compounds
was screened against the tetracyclic indole inhibitor binding allo-
steric pocket (AP-1) of NS5B to identify novel inhibitors. Thus, a to-
tal of 25 inhibitors belonging to the rhodanine scaffold with IC50
virtual screening, ranging from 20
to 1 M for compounds originating from the French National Li-
l
M for the 3D Pharmo DB™ set51
values in the range of 7.7–68.0 lM were identified through a com-
l
bined use of virtual screening, SAR analysis, synthesis and biolog-
ical evaluation. Current efforts are underway to establish the
experimental binding mode of compound 28 with NS5B. In the in-
terim the docking models of compound 28 within AP-1 and AP-3 of
NS5B may serve as a guide for future NS5B inhibitor development
endeavor.
brary/Prestwick commercial library and the NCI library put to-
gether.50 Our ‘hit’ compound 3, though it exhibited a higher IC50
value of 55
derivatization around a selected parent heterocyclic core of com-
pound 7, ultimately yielded compound 28 (IC50 = 7.7 M), the most
lM, a systematic SAR exploration and one step synthetic
l
potent NS5B inhibitor of this series. These findings clearly illustrate
the applicability of the virtual screening protocol to develop novel
analogs with a clear improvement in their biological activity.
Our two lead molecules, compounds 27 and 28, share some
structural features with previously reported NS5B polymerase
inhibitors from Amgen38 and Valeant.31 These may be compared
and contrasted here as follows. For instance, Amgen 5o possesses
an arylsulfonamide group at the N3-position as opposed to (un)-
branched carboxyalkyl groups present in our compounds (Table
3). Valeant 6 on the other hand harbors a thiazolone core in lieu
of the 4-oxo-2-thioxothiazolidine core present in our compounds
and in addition, bears 4-fluorophenylglycine at 2-position of the
thiazolone ring instead of (un)branched carboxyalkyl moiety at
N3-position as seen in our compounds (Table 3). Thus, our lead
molecules appear to be more closely related to Amgen 5o, an AP-
3 bound inhibitor,38 an aspect consistent with their predicted bind-
ing mode in AP-3 of NS5B (Table 3). However, in the absence of a
co-crystal structure for these compounds, one cannot rule out their
binding in AP-1, given that these analogs were originally identified
through screening against this pocket. We speculate that topolog-
ical similarities between AP-1 and AP-3 of NS5B upon docking of
compound 28 as indicated by the Macromodel hydrophilic/hydro-
phobic surface area representation (Fig. 3) may potentially account
for the identification of our leads through AP-1 of NS5B.
5. Materials and methods
5.1. Virtual screening
A ChemBridge database comprising of (a) the first 220,000 com-
pounds from the 430,000 compounds present in Express Pick, Feb-
ruary 2008, (b) 30,000 compounds from MW set, and (c) 10,000
compounds from Kinase set (ChemBridge Corporation) was ini-
tially prefiltered according to Lipinski’s ‘Rule of Five’,52 use of no
tautomers, removal of metals from salts and one stereoisomer
per ligand. Since previous studies have treated the ligands as either
neutral or ionized during virtual screening experiments,53 in this
study we treated ligands independently as neutral and ionized.
These filtering criteria resulted in filtering out ꢀ80% of the com-
pounds from the original 2D databases to generate 52,000 3D mol-
ecules using LigPrep (Schrodinger, LLC: New York), a ligand
preparation software which generates a minimized conformation
of each ligand.
Crystal structures of NS5B polymerase in complex with tetracy-
clic indole (PDB ID: 2DXS),24 thiazolone (PDB ID: 2I1R),31 and ben-
zylidene (PDB ID: 2AX1)38 inhibitors, representing AP-1, AP-2 and
AP-3 pockets, respectively, were used in the present study. AP-1
was used to perform the entire virtual screening operation, while
AP-2 and AP-3 were employed as controls to evaluate the compar-
ative binding potential of the newly identified analogs. The proton-
ation states of residues in the allosteric site were adjusted to the
dominant ionic forms at pH 7.4. Water molecules of crystallization
were removed from all complexes except 2AX1, where the bound
ligand was stabilized through hydrogen bonding with water mole-
cule. The resultant protein was minimized for docking using the
protein preparation facility implemented in Maestro v8.0 and Im-
pact program v4.5 (Schrodinger, LLC: New York).
Given the feasibility that these compounds may bind either AP-
1 or AP-3 of NS5B, we have examined the binding characteristic of
compound 28 within both these pockets, in order to gain insight
for future design and development of more potent novel NS5B
inhibitors. As shown in Figure 4A, the phenyl ring of the benzyli-
dene group at C5-substituent of compound 28 bound to HP-2 in
AP-1 offers room for further modifications with small hydrophobic
groups such as methyl and halogens at 4, 5, and 6 positions. Simi-
larly, the terminal phenyl ring of compound 28 bound to HP-1 can
tolerate substituents such as methyl and halogens. The
a-carbon
To validate the accuracy of our docking approach, we deter-
mined ‘the lowest energy pose’ (binding conformation) predicted
atom of the substituents at the N3-position of the rhodanine ring,
being solvent-exposed, may be branched with a variety of amino

T. T. Talele et al. / Bioorg. Med. Chem. 18 (2010) 4630–4638
4637
by the XP-GlideScore function, versus the experimental binding
mode as determined by X-ray crystallography. Towards this end,
we removed the afore-mentioned crystallographic bound inhibi-
tors from their binding sites, minimized and then re-docked them
into their respective binding site on HCV NS5B polymerase. To
establish the specificity of the Glide (Grid-based ligand docking
with energetics) methodology (Schrodinger, LLC: New York) in dis-
criminating known inhibitors from the randomly chosen drug-like
molecules taken from ChemBridge database, the ‘validation
dataset’ comprising of 1000 molecules, of which 20 were known
NS5B NNIs belonging to benzimidazole,23 indole,24 and quinoxa-
line25 series, were docked into the tetracyclic indole binding
AP-1 of NS5B.
5.2.1.3. 2-(5-(4-Fluorobenzylidene)-4-oxo-2-thioxothiazolidin-
3-yl)acetic acid (25). Yellow solid. Yield = 0.33 g, (72%), mp 256–
259 °C; Rf = 0.54 (DCM/MeOH 80:20), 1H NMR (DMSO-d6) d 4.75
(2H, s), 7.41 (2H, d, J = 8.76 Hz), 7.75 (2H, d, J = 5.52 Hz), 7.93
(1H, s), 13.50 (1H, s). Anal. Calcd. for C12H8FNO3S2: C, 48.48; H,
2.71; N, 4.71. Found: C, 48.57; H, 2.67; N, 4.73.
5.2.1.4. 2-(5-(3-Chlorobenzylidene)-4-oxo-2-thioxothiazolidin-
3-yl)acetic acid (26). Yellow solid. Yield = 0.36 g, (73%), mp 220–
223 °C; Rf = 0.62 (DCM/MeOH 80:20), 1H NMR (DMSO-d6) d 4.75
(2H, s), 7.61 (3H, s), 7.79 (1H, s), 7.91 (1H, s), 13.50 (1H, s). Anal.
Calcd for C12H8ClNO3S2: C, 45.93; H, 2.55; N, 4.47. Found: C,
45.97; H, 2.43; N, 4.50.
A flowchart depicting the various steps of virtual screening (VS),
including database filtration and subsequent high throughput
docking (default settings) studies is shown in Figure 1.
5.2.1.5. 2-(5-(3,4-Dichlorobenzylidene)-4-oxo-2-thioxothiazoli-
din-3-yl)acetic acid (27). Yellow solid. Yield = 0.47 g, (83%), mp
263–266 °C; Rf = 0.35 (DCM/MeOH 80:20), 1H NMR (DMSO-d6) d
4.74 (2H, s), 7.61 (1H, d, J = 8.80 Hz), 7.83 (1H, d, J = 8.44 Hz),
7.90 (1H, s), 8.00 (1H, s), 13.51 (1H, s). Anal. Calcd for
C12H7Cl2NO3S2: C, 41.39; H, 2.03; N, 4.02. Found: C, 41.48; H,
1.96; N, 4.01.
5.2. Chemistry
Melting points (mp) of the synthesized compounds were deter-
mined on a Thomas–Hoover capillary melting point apparatus and
are uncorrected. 2-nitrobenzaldehyde and 4-nitrobenzaldehyde
were obtained from Sigma–Aldrich (Saint Louis, MO, USA); rhoda-
nine-3-acetic acid, 3-chlorobenzaldehyde, 4-fluorobenzaldehyde,
3,4-dichlorobenzaldehye, and 3-phenoxybenzaldehyde were ob-
tained from VWR (Bridgeport, NJ, USA) and TCI America (Portland,
OR, USA) and were used as received. All compounds were checked
for their homogeneity by TLC using silica as stationary phase. The
1H NMR spectra were recorded on a Bruker 400 Avance DPX spec-
trometer outfitted with a z-axis gradient probe. The chemical shifts
are reported as parts per million (d ppm) downfield from tetra-
methylsilane (TMS) as an internal standard. Data are reported as
follows: chemical shift, multiplicity (s) singlet, (d) doublet, (t)
triplet, (m) multiplet. The C, H, and N analyses were performed
by Atlantic Microlabs, Inc., (Norcross, GA, USA) and the observed
values were within 0.4% of calculated values.
5.2.1.6. 2-(5-(3-Phenoxybenzylidene)-4-oxo-2-thioxothiazoli-
din-3-yl)acetic acid (28). Brown solid. Yield = 0.45 g, (87%), mp
178–180 °C; Rf = 0.62 (DCM/MeOH 80:20), 1H NMR (DMSO-d6) d
4.73 (2H, s), 7.10 (2H, d, J = 8.52 Hz), 7.22 (2H, m), 7.28 (1H, s),
7.45 (3H, t, J = 7.68 Hz), 7.58 (1H, t, J = 7.98 Hz), 7.90 (1H, s),
13.46 (1H, s). Anal. Calcd for C18H13NO4S2Á1/2H2O: C, 56.83; H,
3.71; N, 3.68. Found: C, 57.10; H, 3.75; N, 3.72.
5.3. Determination of the inhibition constant
The compounds were dissolved in dimethylsulfoxide (DMSO) as
a 10 mM stock solution and stored at À20 °C for no more than
1 week. Serial dilutions were made in DMSO immediately prior
to the assay. Recombinant NS5BC
the plasmid pThNS5BC 21 expressed in Escherichia coli DH5
Ni–NTA column chromatography as described previously.27,36
NS5BC
D
21 protein was purified from
D
a
, by
5.2.1. General procedure for the preparation of 5-benzylidene
rhodanine-3-acetic acid derivatives
D
21 enzyme stocks were stored in aliquots at À80 °C in buf-
fer containing 50 mM Tris–HCl [pH 8.0], 100 mM NaCl, 5 mM
MgC12 and 50% glycerol. The effect of the compounds on the RdRp
To a three neck reaction flask equipped with reflux condenser,
rhodanine-3-acetic acid (0.3 g, 1.57 mmol) in 6 mL anhydrous
toluene was added, followed by addition of ammonium acetate
(1.60–3.20 mmol) and 1.57 mmol of the corresponding aromatic
aldehydes. The reaction mixture was refluxed for 1–2 h under inert
condition. The reaction was monitored by TLC. The reaction mix-
ture was evaporated under vacuum, and the residue was dissolved
in water. Aqueous solution was then acidified with concd HCl and
precipitated product was extracted into ethyl acetate, dried over
sodium sulfate and evaporated under vacuum to obtain the puri-
fied target compounds.
activity of NS5BC
dependent elongation assays as previously described.27,36 Briefly,
enzymatic reaction mixtures (25 L) containing 20 mM Tris–HCl
(pH 7.0), 100 mM NaCl, 100 mM sodium glutamate, 0.01% BSA,
0.01% Tween 20, 5% glycerol, 20 U/mL of RNase Out, 0.5 M of poly
rA/U12, 25 M UTP, 2–5 Ci [ 21,
32P]UTP, 300–500 ng of NS5BC
D21 was evaluated by the standard primer-
l
l
l
l
a
-
D
and 0.5 mM MnCl2 in the presence or absence of the inhibitor were
incubated for 1 h at 30 °C. Reactions were terminated by the addi-
tion of ice cold 5% (v/v) trichloroacetic acid (TCA) containing
0.5 mM pyrophosphate. Precipitated RNA products were evaluated
by GF-B filter binding assay and quantified on a liquid scintillation
counter (Packard). RNA synthesis in the absence of the inhibitor
was set at 100% (control), and that in the presence of the inhibitor
was expressed relative to this control. The concentration of the
compounds inhibiting 50% of NS5B RdRp activity (IC50) were calcu-
lated from the inhibition curve as a function of inhibitor concentra-
tions employing GraphPad Prism 3.03 software (GraphPad
Software, Inc., San Diego, CA).
5.2.1.1. 2-(5-(2-Nitrobenzylidene)-4-oxo-2-thioxothiazolidin-3-
yl)acetic acid (23). Brown solid. Yield = 0.37 g, (72%), mp 201–
204 °C; Rf = 0.74 (DCM/MeOH 80:20), 1H NMR (DMSO-d6) d 4.76
(2H, s), 7.78 (2H, d, J = 7.48 Hz), 7.92 (1H, t, J = 7.58 Hz), 8.15 (1H,
s), 8.25 (1H, d, J = 8.36 Hz) 13.49 (1H, s). Anal. Calcd for
C12H8N2O5S2: C, 44.44; H, 2.47; N, 8.64. Found: C, 44.65; H, 2.49;
N, 8.44.
5.2.1.2. 2-(5-(4-Nitrobenzylidene)-4-oxo-2-thioxothiazolidin-3-
yl)acetic acid (24). Yellow solid. Yield = 0.39 g, (76%), mp 259–
262 °C, (lit. 240–243 °C)54; Rf = 0.52 (DCM/MeOH 80:20), 1H NMR
(DMSO-d6) d 4.77 (2H, s), 7.95 (2H, d, J = 8.76 Hz), 8.03 (1H, s),
8.37 (2H, d, J = 8.76 Hz), 13.54 (1H, s). Anal. Calcd for C12H8N2O5S2:
C, 44.44; H, 2.47; N, 8.64. Found: C, 44.21; H, 2.33; N, 8.44.
Acknowledgments
This work was supported by the National Institute of Health
Research Grant DK066837 to N.K.B. T.T.T. was supported by
start-up funds and resources from the College of Pharmacy of St.
John’s University. The authors thank Dr. Santosh Khedkar from

4638
T. T. Talele et al. / Bioorg. Med. Chem. 18 (2010) 4630–4638
30. Wang, M.; Ng, K. K.; Cherney, M. M.; Chan, L.; Yannopoulos, C. G.; Bedard, J.;
Morin, N.; Nguyen-Ba, N.; Alaoui-Ismaili, M. H.; Bethell, R. C.; James, M. N. J.
Biol. Chem. 2003, 278, 9489.
31. Yan, S.; Appleby, T.; Larson, G.; Wu, J. Z.; Hamatake, R.; Hong, Z.; Yao, N. Bioorg.
Med. Chem. Lett. 2006, 16, 5888.
Harvard Medical School, Dr. V. Hariprasad from Huntsman Cancer
Institute, and Dr. Narayanan Ramasubbu from University of Medi-
cine and Dentistry of New Jersey for many helpful suggestions.
32. Chan, L.; Reddy, T. J.; Proulx, M.; Das, S. K.; Pereira, O.; Wang, W.; Siddiqui, A.;
Yannopoulos, C. G.; Poisson, C.; Turcotte, N.; Drouin, A.; Alaoui-Ismaili, M. H.;
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Moinet, C.; Bethell, R.; Hamel, M.; L’Heureux, L.; David, M.; Nicolas, O.;
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.05.030.
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