Development of potent inhibitors of the coxsackievirus 3C protease.

Abstract

Coxsackievirus B3 (CVB3) 3C protease (3CP) plays essential roles in the viral replication cycle, and therefore, provides an attractive therapeutic target for treatment of human diseases caused by CVB3 infection. CVB3 3CP and human rhinovirus (HRV) 3CP have a high degree of amino acid sequence similarity. Comparative modeling of these two 3CPs revealed one prominent distinction; an Asn residue delineating the S2' pocket in HRV 3CP is replaced by a Tyr residue in CVB3 3CP. AG7088, a potent inhibitor of HRV 3CP, was modified by substitution of the ethyl group at the P2' position with various hydrophobic aromatic rings that are predicted to interact preferentially with the Tyr residue in the S2' pocket of CVB3 3CP. The resulting derivatives showed dramatically increased inhibitory activities against CVB3 3CP. In addition, one of the derivatives effectively inhibited the CVB3 proliferation in vitro.

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Development of potent inhibitors of the coxsackievirus 3C protease
Eui Seung Leea, Won Gil Leea, Soo-Hyeon Yunb, Seong Hwan Rhoa, Isak Ima,
Sung Tae Yanga, Saravanan Sellamuthua, Yong Jae Leea, Sun Jae Kwonc,
Ohkmae K. Parkc, Eun-Seok Jeonb, Woo Jin Parka,*, Yong-Chul Kima,*
aDepartment of Life Science, Gwangju Institute of Science and Technology, 1 Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea
bDepartment of Medicine, Sungkyunkwan University School of Medicine, Cardiac and Vascular Center, Samsung Medical Center,
Seoul 135-710, Republic of Korea
cSchool of Life Science and Biotechnology, Korea University, Seoul 136-701, Republic of Korea
Received 9 March 2007
Available online 20 April 2007
Abstract
Coxsackievirus B3 (CVB3) 3C protease (3CP) plays essential roles in the viral replication cycle, and therefore, provides an attractive
therapeutic target for treatment of human diseases caused by CVB3 infection. CVB3 3CP and human rhinovirus (HRV) 3CP have a high
degree of amino acid sequence similarity. Comparative modeling of these two 3CPs revealed one prominent distinction; an Asn residue
delineating the S20pocket in HRV 3CP is replaced by a Tyr residue in CVB3 3CP. AG7088, a potent inhibitor of HRV 3CP, was mod-
ified by substitution of the ethyl group at the P20position with various hydrophobic aromatic rings that are predicted to interact pref-
erentially with the Tyr residue in the S20pocket of CVB3 3CP. The resulting derivatives showed dramatically increased inhibitory
activities against CVB3 3CP. In addition, one of the derivatives effectively inhibited the CVB3 proliferation in vitro.
? 2007 Elsevier Inc. All rights reserved.
Keywords: Coaxsackievirus; 3C protease; Inhibitor
Coxsackievirus B3 (CVB3), a member of the picornavi-
rus family, is a primary causative agent of viral myocarditis
in humans [1–3]. Acute myocarditis is a principle cause of
heart failure in children and adolescents, and often pro-
gresses to chronic myocarditis and dilated cardiomyopa-
thy, which are responsible for approximately 50% of the
heart transplantationsregistered
[4,5]. No effective antiviral therapies for either prevention
or treatment of the diseases caused by CVB3 infection
are currently available. Like other picornaviruses including
polioviruses, human rhinoviruses (HRV), and hepatitis A
virus, the positive strand RNA genome of CVB3 is trans-
lated into a large polyprotein precursor which undergoes
annually worldwide
a series of proteolytic cleavages to generate functional viral
proteins. Processing of the polyprotein is essential for the
production of new, infectious virions, and is primarily
dependent on two virally encoded proteases 2A and 3C
[2,4,6]. Therefore, these enzymes are attractive therapeutic
targets for development of antiviral therapeutic agents [7].
In this study, we sought to develop potent inhibitors of
CVB3 3C protease (3CP). CVB3 3CP is highly homologous
to HRV 3CP in their amino acids sequences (approxi-
mately 64%). Utilizing information from the crystal struc-
ture of HRV 3CP [8,9] in complex with its inhibitor,
AG7088 [10], and the comparatively modeled structure of
CVB3 3CP, we rationally designed various AG7088 deriv-
atives. We report here that several derivatives are indeed
highly potent inhibitors of CVB3 3CP, and that one of
the compounds was highly effective in preventing prolifer-
ation of CVB3 in vitro.
0006-291X/$ - see front matter ? 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2007.03.208
*Corresponding authors. Fax: +82 62 970 2484.
E-mail addresses: wjpark@gist.ac.kr (W.J. Park), yongchul@gist.ac.kr
(Y.-C. Kim).
www.elsevier.com/locate/ybbrc
Biochemical and Biophysical Research Communications 358 (2007) 7–11

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Materials and methods
Comparative modeling of CVB3 3CP. A model for the structure of
CVB3 3CP was generated based on information from the structure of
HRV 3CP [8,9] in complex with the inhibitor AG7088 (PDB# 1CQQ) [10].
The two 3CPs have a high degree of amino acid sequence similarity.
Modeling was performed using MODELLER software [11]. The model
with the lowest MODELLER restraint energy was further refined by
energy minimization using GROMACS software [12].
Synthesis of inhibitors. All reagents used for inhibitor synthesis were
purchased from Sigma–Aldrich. The synthetic scheme for the CVB3 3CP
inhibitors is outlined in Fig. 2 [13]. Alcoholic compounds 1 (3.06 mmol,
2.0 equiv) was added to a solution of diethylphosphonoacetic acid
(1.53 mmol, 1.0 equiv) and PyBOP (2.30 mmol, 1.5 equiv) in dichloro-
methane (30 ml) at room temperature (RT), and the mixture was stirred
for 2 h. After concentrating the solution by evaporation of dichloro-
methane, the resulting solution was partitioned between ethyl acetate
(30 ml) and de-ionized water (2 · 20 ml). The combined organic layers
were dried over Na2SO4, filtered, concentrated under reduced pressure,
and purified by silica gel column chromatography (40% EtOAc in hexane)
to yield compounds 2a–f (>50.0%).
Sodium bis(trimethylsilyl)amide (1.0 M solution in THF, 0.67 mmol,
1.5 equiv) was added to a solution of compound 2 (0.54 mmol, 1.2 equiv)
in THF (20 ml) at ?78 ?C, and the mixture was stirred for 20 min.
Compound 3 [13] was added to the mixture, and stirring was continued for
2 h at the same temperature. The solution was then warmed to 0 ?C for
10 min, and then partitioned between 0.5 M HCl (20 ml) and a 1:1 mixture
of hexane and EtOAc (2 · 20 ml). The combined organic layers were dried
over Na2SO4, filtered, concentrated under reduced pressure, and purified
by silica gel column chromatography (33% EtOAc in hexane) to yield
compounds 4a–f (>60.0%).
A solution of L-valine (15.74 mmol, 1.0 equiv), 5-methylisoxazole-3-
carboxylic acid (15.74 mmol, 1.0 equiv), and PyBop (15.74 mmol,
1.0 equiv) in DMF (100 ml) was stirred for 5 h at 40 ?C. After concen-
trating the solution by evaporation of DMF under vacuum, the mixture
was partitioned between 1.0 M HCl (50 ml) and EtOAc (2 · 80 ml). The
combined organic layers were dried over Na2SO4, filtered, concentrated
under reduced pressure, and purified by silica gel column chromatography
(3.0% MeOH in chloroform) to yield compound 5 (76.4%).
A solution of 4-fluoro-Phe-methyl ester (1.00 mmol, 1.0 equiv), com-
opund 5 (1.00 mmol, 1.0 equiv), and PyBop (1.52 mmol, 1.5 equiv) in
dichloromethane (20 ml) was stirred at RT for 2 h. After concentrating the
solution by evaporation of dichloromethane, the mixture was partitioned
between ethyl acetate (2 · 20 ml) and de-ionized water (20 ml). The com-
bined organic layers were dried over Na2SO4, filtered, concentrated under
reduced pressure, and purified by silica gel column chromatography (33%
EtOAc in hexane) to yield compound 6 (80.3%), which was subsequently
hydrolyzed in 1 N NaOH in MeOH (20 ml) to yield compound 7 (93%).
To remove the Boc group of compounds 4a–f, 4 N HCl in 1,4-dioxane
(5 ml) was added to a solution of compounds 4a–f (0.15 mmol, 1.0 equiv)
in the same solvent (5 ml) at RT. After 3 h, the solvent was evaporated
under reduced pressure. Dichloromethane (10 ml) was added to the
resulting residue containing compound 7 (0.15 mmol, 1.0 equiv). HOBt
(0.45 mmol 3.0 equiv),DIPEA (0.90 mmol,
(0.45 mmol, 3.0 equiv) were added and the mixture was stirred overnight
at RT. After concentrating the solution by evaporation of dichlorometh-
ane, the residue was partitioned between ethyl acetate (2 · 20 ml) and de-
ionized water (20 ml). The combined organic layers were dried over
Na2SO4, filtered, concentrated under reduced pressure, and purified by
silica gel column chromatography (40% EtOAc in hexane) to yield com-
pounds 8a–f (>30.0%).
A solution of 20% TFA in dichloroethane was added to compounds
8a–f (0.02 mmol, 1.0 equiv) at RT. After 1 h, the solvent was evaporated
under reduced pressure, and compounds 9a–f (>80.0%) were purified by
preparative TLC (12% MeOH in chloroform).
Determination of the IC50values of the inhibitors. The substrate peptide
(TTLEALFQGPPVY) for CVB3 3CP was purchased from AnyGen
5.0 equiv), andEDC
(Gwangju, Korea). The CVB3 3CP coding region was amplified by PCR
and subcloned into the pTYB12 expression vector (New England Bio-
Labs). Escherichia coli strain BL21(DE3) was then transformed with the
resulting plasmid. The CVB3 3CP was purified as previously described
[14].
In the reaction buffer (40 mM Tris–HCl, pH 8.0, 1 mM dithiothreitol,
and 0.1% Nonidet P-40), 150 lM substrate peptide, various concentra-
tions of inhibitors, and 1 lM CVB3 3CP were added sequentially in a total
volume of 100 ll, and then incubated for 1 h at 30 ?C. Reactions were
quenched by addition of 200 ll of 0.1% trifluoroacetic acid. A 200 ll ali-
quot of the mixture was subjected to reverse-phase HPLC on a 10 cm
Vydac C18column using a 0–95% 30 min linear gradient of acetonitrile in
0.1% trifluoroacetic acid. The CVB3 3CP activity in the absence of
inhibitor was defined as 100%.
In vitro antiviral assay. HeLa-UVM cells were obtained from Sally
Huber (University of Vermont) and maintained in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. The
H3 variant of the Woodruff strain of coxsackievirus B3 (CVB3-H3) was
obtained from a cDNA copy [15]. Virus titers were determined using a
plaque forming unit (pfu) assay on HeLa-UVM cells.
HeLa-UVM cells were infected with CVB3-H3 at a multiplicity of
infection (MOI) of 100. CVB3 3CP inhibitors were added to the cultures at
various final concentrations (0.5–100 lM) and incubated for 6–24 h. At
the end of the incubation period, 15 ll of the proliferation-dye reagent
which was supplied with Cell Counting Kit 8 (Dojindo Lab, Tokyo,
Japan) was added. Only viable cells with intact mitochondria are stained
with this reagent. Absorbance at 450 nm was measured using an ELISA
plate reader. Non-infected HeLa-UVM cells were used as a control and
proliferation of these cultures was defined as 100%. Data are presented as
means ± SD from three independent experiments.
GFP detection and Western blot analysis. Previously, a GFP-CVB3
fusion gene was constructed in which a green fluorescent protein (GFP)
gene was placed immediately upstream of theVP0 capsid protein of CVB3
with a cleavage site for CVB3 3CP positioned between GFP and VP0.
GFP is cleaved from the viral polyprotein by 3CP during viral replication
[16,17]. HeLa-UVM cells were infected with GFP-CVB3 (MOI, 100) for
6 h, and GFP expression was analyzed either by fluorescence microscopy
or by Western blotting. The fluorescence of functional GFP in the infected
cells was observed without fixation using the FITC filter on a fluorescence
microscope. Total protein extracts from the cultures (10 lg) were frac-
tionated on a 10% SDS–PAGE gel and then blotted onto Hybond-ECL
membranes (Amersham). Protein bands with positive antibody reactions
against GFP and tubulin were visualized by chemiluminescence (Amer-
sham). GFP expression (n = 4) was quantified by densitometric analysis
using ImageLab (version 2.2.4) and normalized to the control level (non-
infected cells).
Results and discussion
Comparative modeling and synthesis of inhibitors
The structure of CVB3 3CP was modeled, and com-
pared with that of HRV 3CP (Fig. 1). The conformation
and amino acids demarcating the substrate binding pocket
were found to be conserved in these proteases with the
exception that Asn22 in HRV 3CP was replaced by
Tyr22 in CVB 3CP. These amino acids appear to be critical
for determining the P20specificity of the substrates [8,9].
AG7088 is a potent peptidomimetic inhibitor of HRV
3CP that was developed by structure-based drug design
approaches [18], and is currently being evaluated in a Phase
1 clinical trial in humans [10]. This inhibitor is composed of
a substrate-derived tri-peptide binding determinant with
affinity for the target protease and a Michael acceptor
8
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moiety which irreversibly forms a covalent adduct with the
active site cysteine residue of HRV 3CP [18]. The ethyl pro-
penoate Michael acceptor moiety corresponds to the P20
position of AG7088 (boxed in compounds 9a–f in Fig. 2).
We reasoned that replacing this Michael acceptor moiety
with one containing aromatic rings would significantly
increase the inhibitory activity of the compound against
CVB3 3CP, because the aromatic rings would provide
additional hydrophobic or p–p interactions with Tyr22.
An analog of AG7088 was synthesized (compound 9a),
which contains a glutamine residue instead of a lactam ring
in the P1 position (shaded in compounds 9a–f). This substi-
tution greatly simplified the synthesis of the compounds.
Five derivatives (9b–f) of AG7088 which contain various
aromatic rings instead of the ethyl moiety at the P20posi-
tion were synthesized (Fig. 2). Detailed procedures for
Fig. 1. Comparative modeling of CVB3 3CP. Modeling of CVB3 3CP was performed using the HRV 3CP structure as a reference. Structures of HRV 3CP
and CVB3 3CP docked with a HRV 3CP inhibitor, AG7088, are shown. Note the presence of Tyr22 at S20of CVB3 3CP (right panel). We hypothesized
that Tyr22 would interact preferentially with aromatic R groups in potential inhibitors. The electrostatic calculation of models and the figure preparation
were performed using the PyMol software [19].
EtO
P
O
EtO
OH
O
EtO
P
O
EtO
O
O
R
HN
Boc
H
HN
O
TrtO
HN
Boc
H
HN
O
Trt
O
O
R
NH
HHN
O
Trt
O
O
R
O
N
H
F
N
O
H
N
O
O
N
O
H
N
O
O
O
O
HN
N
O
H
N
O
O
OH
F
2a-f

4a-f
5
N
O
H
N
O
O
OH
O
HN
F
67
3
(a)
(b)
(c)
(d)
(e), (f)
(g)
1a-f
HO R
HO
HO
HO
HO
HO
F
HO
1a 1b 1c 1d 1e 1f
8a-f
NH
H
O
O
R
O
N
H
F
N
O
H
N
O
O
9a-f
H2N
O
Fig. 2. Synthesis of CVB3 3CP inhibitors. Reagents and conditions are as follows: (Tr = CPh3) (a) 2.0 equiv of alcohol compound, 1.5 equiv of Pybop,
DCM, 2 h, 56.3%; (b) 1.5 equiv of sodium bis(trimethylsilyl)amide, THF, ?78 ?C, 2 h, 63.0%; (c) 1.0 equiv of Boc-L-Phe-OMe, 1.5 equiv of Pybop, DCM,
2 h, 80.3%; (d) 1 N NaOH in MeOH, 30 min, 93.0%; (e) 2 N HCl in 1,4-dioxane, 3 h; (f) 3.0 equiv of HOBt, 3.0 equiv of EDC, 5.0 equiv of DIPEA, DCM,
overnight, 32.8%; (g) 20% TFA in DCE, 1 h, 80.2%.
E.S. Lee et al. / Biochemical and Biophysical Research Communications 358 (2007) 7–11
9

Page 4

the synthesis of these derivatives are described in the Mate-
rials and methods.
Determination of IC50values
IC50values of the AG7088 analog and its derivatives
were determined using purified recombinant CVB3 3CP
in vitro (Table 1). Compound 9a, an analog of AG7088,
possessed moderate inhibitory activity against CVB3
3CP, which is consistent with previous reports assessing
AG7088 [10]. All five derivatives containing aromatic rings
at the P20position showed significantly enhanced inhibi-
tory activities against CVB3 3CP, and the compound 9b
derivative carrying a naphthalene ring was the most effi-
cient inhibitor with an IC50value of 93 nM. These results
demonstrate the validity of our rational approach for
designing potent inhibitors of CVB3 3CP.
Antiviral activity assay
We examined whether inhibiting CVB3 3CP activity is
able to prevent the proliferation of CVB3. None of the syn-
thesized compounds had direct cytotoxicity when used at
concentrations up to 100 lM (data not shown). In an initial
experiment, compound 9b showed the most prominent
antiviral activity (data not shown), which was well corre-
lated with its inhibition of the protease activity in vitro.
Therefore, compound 9b was analyzed further. HeLa-
UVM cells were transfected with CVB3-H3 (MOI, 100)
in the presence of various concentrations of compound
9b, and cell viability was determined after 24 h. Compared
to CVB3-H3 infected cultures incubated in the absence of
compound 9b, the viability was significantly greater in cul-
tures incubated in the presence of 5–10 lM compound 9b,
and was completely restored to control levels in the pres-
ence of 50 lM compound 9b (Fig. 3A). We then tested
whether the recovery in viability in CVB3-H3 infected cul-
tures that were incubated with compound 9b was associ-
ated with the inhibition of CVB3 3CP activity. A CVB3
3CP-dependent GFP assay was performed as described
previously [17]. HeLa-UVM cells were transfected with a
GFP-CVB3 fusion construct in which a CVB3 3CP cleav-
age site is placed between GFP and the CVB3 polyprotein.
Cleavage by CVB3 3CP releases the GFP from the fusion
protein, which can be observed directly by fluorescence
microscopy or by Western blotting. Compound 9b signifi-
cantly reduced the number of the GFP positive cells
(Fig. 3B) and reduced the amount of GFP detected by
Western blotting (Fig. 3C) in a dose dependent manner.
These data indicate that compound 9b inhibits CVB3
3CP activity and CVB3 proliferation in vivo in a dose-
dependent manner.
In conclusion, we have successfully designed and synthe-
sized a potent CVB3 3CP inhibitor using a structure-based
rational design approach. This inhibitor will serve as a plat-
form to generate more effective and specific CVB3 3CP
Table 1
IC50values
CompoundsRFormulaIC50(nM)
9a
9b
9c
9d
9e
9f
CH2CH3
CH2C10H7
CH(C6H5)2
CH2C6H5
CH2CH2C6H5
CH2CH2(p-F-C6H5)
C28H36FN5O7
C37H40FN5O7
C39H42FN5O7
C33H38FN5O7
C34H40FN5O7
C34H39F2N5O7
48,000
93
480
650
950
1000
Compound 9a is an analog of AG7088, a potent inhibitor of HRV 3CP.
Compounds 9b–f are derivatives of AG7088 containing aromatic groups
at the Michael acceptor position instead of an ethyl group.
A
B
C
μ
μ
μμ
μμ
μ
Fig. 3. Inhibition of viral proliferation by compound 9b. (A) Antiviral
activity of compound 9b. HeLa-UVM cells were transfected with CVB3-
H3 in the presence of various amounts of compound 9b for 24 h. Cell
viabilities are presented as means ± SD from three independent experi-
ments. (B,C) Inhibition of CVB3 3CP by compound 9b. HeLa cells were
infected with GFP-CVB3 (MOI, 100), and treated with various amounts
of compound 9b. The CVB3 3CP activity was monitored by the expression
of GFP using fluorescence microscopy (B), and Western blotting (C).
Tubulin was used as a loading control.
10
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inhibitors and contribute to our understanding of the
pathology of CVB3.
Acknowledgments
During this work, W.J.P. was supported by a GRL
Grant (M6-0605-00-0001) from the Ministry of Science
and Technology, Korea, YCK was supported by a grant
of the Korea Health 21 R&D Project, from the Ministry
of Health & Welfare, Republic of Korea (Grant No.
A040042), and OKP was supported by a grant from the
Plant Signaling Network Research Center funded by the
Korea Science and Engineering Foundation.
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