Binding interaction of quercetin-3-β-galactoside and its synthetic derivatives with SARS-CoV 3CLpro: Structure-activity relationship studies reveal salient pharmacophore features

Article abstract of DOI:10.1016/j.bmc.2006.09.014

The 3C-like protease (3CL^pro) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is one of the most promising targets for discovery of drugs against SARS, because of its critical role in the viral life cycle. In this study,

Full text of DOI:10.1016/j.bmc.2006.09.014

Bioorganic & Medicinal Chemistry 14 (2006) 8295–8306
Binding interaction of quercetin-3-b-galactoside and its synthetic
derivatives with SARS-CoV 3CLpro: Structure–activity relationship
studies reveal salient pharmacophore features
Lili Chen,a, Jian Li,a, Cheng Luo,a Hong Liu,a Weijun Xu,b Gang Chen,b Oi Wah Liew,b
b,
a,
a
Weiliang Zhu,a,* Chum Mok Puah, Xu Shen and Hualiang Jiang
*
*
aDrug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
bTechnology Centre for Life Sciences, Singapore Polytechnic, 500 Dover Road, 139651 Singapore, Singapore
Received 21 June 2006; revised 2 September 2006; accepted 8 September 2006
Available online 12 October 2006
Abstract—The 3C-like protease (3CLpro) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is one of the
most promising targets for discovery of drugs against SARS, because of its critical role in the viral life cycle. In this study, a natural
compound called quercetin-3-b-galactoside was identified as an inhibitor of the protease by molecular docking, SPR/FRET-based
bioassays, and mutagenesis studies. Both molecular modeling and Q189A mutation revealed that Gln189 plays a key role in the
binding. Furthermore, experimental evidence showed that the secondary structure and enzymatic activity of SARS-CoV 3CLpro
were not affected by the Q189A mutation. With the help of molecular modeling, eight new derivatives of the natural product were
designed and synthesized. Bioassay results reveal salient features of the structure–activity relationship of the new compounds: (1)
removal of the 7-hydroxy group of the quercetin moiety decreases the bioactivity of the derivatives; (2) acetoxylation of the sugar
moiety abolishes inhibitor action; (3) introduction of a large sugar substituent on 7-hydroxy of quercetin can be tolerated; (4)
replacement of the galactose moiety with other sugars does not affect inhibitor potency. This study not only reveals a new class
of compounds as potential drug leads against the SARS virus, but also provides a solid understanding of the mechanism of inhi-
bition against the target enzyme.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
gic measures to manage the impact and severity of fu-
ture outbreaks.7
The emergence of severe acute respiratory syndrome
(SARS) in November 2002 followed by its rapid trans-
mission posed serious challenges to public health global-
ly.1–5 By July 31, 2003, a total of 8096 SARS cases and
774 SARS-related fatalities were reported worldwide.6
Although there have been no further reports of new
SARS cases since April 2004, the disease should still
be regarded as a potential serious health threat. There-
fore, there is still need to study the biology, epidemiolo-
gy and pathogenesis of the SARS-CoV and develop
effective therapeutics for its treatment as part of strate-
SARS-CoV, which bears similarities to other coronavi-
ruses, is an enveloped, positive-strand RNA virus that
contains a single-stranded RNA genome of ꢀ29,700
nucleotides.8,9 This large viral genome is predominated
by two overlapping open reading frames that are con-
nected by a ribosomal frame shift site to encode two rep-
licase polyproteins, pp1a and pp1ab, that mediate viral
replication. The 3C-like protease (3CLpro
10
)
plays a
key role in the proteolytic release of replicative proteins
from their replicase precursor polyproteins. In view of
its essentiality in viral replication, the 3CLpro represents
an attractive target for the development of therapeutics
against SARS-CoV.
Keywords: SARS-CoV 3CLpro; Virtual screening; Quercetin-3-b-galac-
toside; Molecular modeling; Structure–activity relationships.
Since the emergence of SARS, much concerted efforts had
been made in solving the crystal structure of SARS-CoV
*
Corresponding authors. Tel.: +86 21 50806600; fax: +86 21
50807088; e-mail addresses: wlzhu@mail.shcnc.ac.cn; puah@sp.
edu.sg; xshen@mail.shcnc.ac.cn
3CLpro
,
characterizing the structural determinants
11–13
of enzyme catalysis or maturation mechanism,14–18
These two authors contributed equally to this work.
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.09.014

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L. Chen et al. / Bioorg. Med. Chem. 14 (2006) 8295–8306
performing structure-based inhibitor design, virtual
screening,19–22 and biological activity assays against
SARS-CoV 3CLpro or viral entry.7,13,23–30 To date, a
number of small molecules with inhibitory activity to-
ward SARS-CoV 3CLpro or viral replication have been
identified. However, no effective small molecule agent
has thus far been validated as a drug to treat SARS.
Hence, the identification of new chemical classes of inhib-
itors and characterization of their interaction mode with
SARS-CoV 3CLpro remains a relevant pursuit in the
search for chemotherapeutic agents for the disease.
2. Results and discussion
2.1. Molecular docking result and possible inhibition
mechanism of quercetin-3-b-galactoside against SARS-
CoV 3CLpro
A bioflavanoid, quercetin-3-b-galactoside (1), was dis-
covered by virtual screening as a potent binder to the
catalytic pocket of SARS-CoV 3CLpro. The predicted
binding free energy, inhibitory constant, and the predict-
ed LogP are ꢁ9.24 kcal/mol, 1.70 · 10ꢁ7, and 0.43,
respectively. Figure 1 shows the details of the specific
interactions between quercetin-3-b-galactoside and the
enzyme active site. Clearly, the molecule forms hydro-
Quercetin, first reported in 1955,31 is a water soluble
flavanoid that serves as a building block for other mem-
bers of the flavonoid family. This natural compound has
been reported to have ameliorative effects on a host of
disorders including cancer, renal, and cardiovascular
disease,32–35 and have inhibitory activity toward
SARS-CoV 3CLpro or viral replication.36,37
phobic interactions with residues Leu141
, ,
Asn142
Met165, and Glu166 of SARS-CoV 3CLpro (Fig. 1A).
Meanwhile, the inhibitor forms six hydrogen bonds with
residues in the vicinity of the catalytic binding pocket
(Fig. 1). The N atom of main chain of Glu166 forms
two hydrogen bonds with quercetin-3-b-galactoside with
e1
˚
In this work, quercetin-3-b-galactoside (1) was identified
as a potent inhibitor of SARS-CoV 3CLpro by molecular
docking studies and enzymatic inhibition assays. This
compound showed inhibitory activity against SARS-
CoV 3CLpro with IC50 of 42.79 4.97 lM in a compet-
itive mode. Molecular modeling strongly suggested that
the residue Q189 plays a key role in the binding between
quercetin-3-b-galactoside and SARS-CoV 3CLpro. To
confirm this binding model, site-directed mutagenesis
of the 3CLpro protease was performed. Comparative
studies between SARS-CoV 3CLpro and SARS-CoV
3CLpro Q189A using CD spectroscopy, fluorescence
spectra, and enzyme activity assays were conducted to
determine the mechanistic effects of the Q189A mutation
on enzyme–inhibitor interaction. The results indicated
that while SARS-CoV 3CLpro Q189A retained the same
level of biological activity as its wild-type counterpart,
the inhibitory potency of quercetin-3-b-galactoside on
SARS-CoV 3CLpro Q189A was decreased due to a
reduction in binding affinity. To further explore the
mode of binding interaction between the natural prod-
uct and SARS-CoV 3CLpro, and discover more potent
inhibitors, we systematically designed, with the aid of
structural information from molecular modeling, and
synthesized eight quercetin-3-b-galactoside derivatives
(2a–h). We then tested their inhibitory activities against
SARS-CoV 3CLpro. The results showed that removing
the hydroxy groups on the quercetin moiety (2f–h) sub-
stantially decreased inhibitory activity; acetoxylation of
the sugar moiety (2a) was unfavorable for the inhibitory
activity of the compound; and introduction of a large
sugar substituent on 7-hydroxy of quercetin (2e)
did not adversely affect the ability of the inhibitor to
functionally disrupt the SARS-CoV 3CLpro protease
activity. All the experimental results were in good agree-
ment with the molecular binding model. Thus, this work
not only demonstrates quercetin-3-b-galactoside and its
derivatives as a new class of SARS-CoV 3CLpro inhibi-
tors, but also clarifies the mode of binding interaction
between the flavonoids and SARS-CoV 3CLpro. The elu-
cidation of a legible pharmacophore of SARS-CoV
3CLpro inhibitors lays the foundation for future rational
drug design.
distances of 2.88 and 3.28 A, respectively, while the O
and Ne2 atoms of the Gln189 side chain form four
hydrogen bonds with distances of 2.62, 2.69, 3.44, and
˚
3.68 A, respectively. This binding model points to
Gln189 as a crucial residue providing the key interaction
elements for the binding of quercetin-3-b-galactoside
with SARS-CoV 3CLpro. Logically then, substitution
of the polar Gln189 residue to an aliphatic alanine resi-
due would reduce the number of hydrogen bond interac-
tion sites and thus significantly weaken the binding
affinity. From the crystal structure of SARS-CoV
12
3CLpro
,
it is shown that Gln189 lies in the long loop
region (residues 185–200), separated from the active
sites of SARS-CoV 3CLpro. Therefore, the Q189A muta-
tion was not expected to adversely affect the catalytic
capability of the enzyme but rather lead to a significant
drop in its binding affinity to quercetin-3-b-galactoside.
Thus, this mutational model provides a good index for
assessing the correctness of our proposed docking re-
leased binding mechanism. In addition, it can be ob-
served from Figure 1B that there is ample spatial
capacity within the binding pocket of the enzyme
around the 7-hydroxy group of quercetin, suggesting
that this site can accommodate significant structural
modification with larger chemical moieties around 7-hy-
droxy of quercetin (2e).
2.2. Characterization of SARS-CoV 3CLpro or SARS-
CoV 3CLpro Q189A
The His-tag recombinant SARS-CoV 3CLpro and
SARS-CoV 3CLpro Q189A were successfully expressed
in Escherichia coli strain M15 and purified by affinity
chromatography to yield 12 and 16 mg of the purified
proteins per liter of culture, respectively. The purity
and molecular weight of the two proteases were assessed
by SDS–PAGE. CD spectra of SARS-CoV 3CLpro and
SARS-CoV 3CLpro Q189A were recorded on a JASCO-
810 spectropolarimeter collected with a thermal control-
ler. Far-UV spectra of the two proteins give a positive
peak at 196 nm that is characteristic of a b-sheet struc-
ture. Negative peaks at 209 and 222 nm obtained for
SARS-CoV 3CLpro and SARS-CoV 3CLpro Q189A,

L. Chen et al. / Bioorg. Med. Chem. 14 (2006) 8295–8306
8297
A
B
Figure 1. The main interactions between quercetin-3-b-galactoside and SARS-CoV 3CLpro. (A) The binding conformations of quercetin-3-b-
galactoside and SARS-CoV 3CLpro. The N atom of main chain of Glu166 forms two hydrogen bonds with quercetin-3-b-galactoside. The side chain
of Gln189 accepts four strong hydrogen bonds with quercetin-3-b-galactoside, respectively. (B) Three dimensional representation of the binding
between quercetin-3-b-galactoside and SARS-CoV 3CLpro. The blue dash denotes hydrogen bonds.
respectively, are typical of an a-helical structure
(Fig. 2A). Thus, the CD spectra demonstrated that the
E-Edans according to the published method.28 Based
on the double reciprocal plots of the initial velocity
(v0) and substrate concentration of SARS-CoV 3CLpro
and SARS-CoV 3CLpro Q189A (Figs. 3A and B), the
kinetic parameters (Km and kcat) were obtained. The
determined Km values of SARS-CoV 3CLpro and
SARS-CoV 3CLpro Q189A are 49.38 5.80 and 38.17
2.60 lM, respectively. The kcat values of SARS-CoV
3CLpro and SARS-CoV 3CLpro Q189A are 0.16 0.01
and 0.14 0.01 minꢁ1. Therefore, the kcat/Km values of
SARS-CoV 3CLpro and SARS-CoV 3CLpro Q189A
(3.2 0.2 and 3.7 0.3 mM minꢁ1) are very close to
each other, indicating that the activities of the two
secondary structure of SARS-CoV 3CLpro Q189A is
similar to that of SARS-CoV 3CLpro
.
The evidence
14
from CD analysis excludes the possibility of structural
misfolding resulting from the mutagenesis of Gln189.
Figure 2B shows the fluorescence spectra of the two pro-
teases to be identical with a major peak at the wave-
length of 330 nm.
The enzymatic activity of SARS-CoV 3CLpro and
SARS-CoV 3CLpro Q189A was determined by cleavage
of the fluorogenic substrate Dabcyl-KNSTLQSGLRK

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L. Chen et al. / Bioorg. Med. Chem. 14 (2006) 8295–8306
Figure 2. Characterization studies of SARS-CoV 3CLpro and SARS-
CoV 3CLpro Q189A proteases. (A) CD spectra of SARS-CoV 3CLpro
and SARS-CoV 3CLpro Q189A Far-UV CD spectra of SARS-CoV
3CLpro (–j–) and SARS-CoV 3CLpro Q189A (–s–) at 25 ꢁC. Protein
concentration used in CD experiments is 10 lM, and the two protein
samples were prepared in 20 mM Tris–HCl, pH 7.4, 120 mM NaCl. (B)
Fluorescence spectra of SARS-CoV 3CLpro and SARS-CoV 3CLpro
Q189A fluorescence spectra of SARS-CoV 3CLpro (—) and SARS-
CoV 3CLpro Q189A (. . .) at 25 ꢁC. Protein concentration used in the
experiment is 10 lM, and two protein samples were prepared in
20 mM Tris–HCl, pH 7.4, 120 mM NaCl.
Figure 3. Kinetic parameters determination of SARS-CoV 3CLpro and
SARS-CoV 3CLpro Q189A. The enzymatic activity of SARS-CoV
3CLpro and SARS-CoV 3CLpro Q189A was measured by the fluores-
cence resonance energy transfer (FRET) method. SARS-CoV 3CLpro
or SARS-CoV 3CLpro Q189A (1 lM) and the fluorogenic substrate
with different concentrations from 5 to 200 lM were incubated at
25 ꢁC. The fluorescence intensity was monitored continuously for 1 h.
The Km and kcat values were calculated by Lineweaver–Burk plot. (A)
The double reciprocal plot of SARS-CoV 3CLpro, (B) the double
reciprocal plot of SARS-CoV 3CLpro Q189A.
curves of quercetin-3-b-galactoside with SARS-CoV
3CLpro or SARS-CoV 3CLpro Q189A are shown in Fig-
ure 4. For the SARS-CoV 3CLpro (Fig. 4A), quercetin-3-
b-galactoside results in a significant and dose-dependent
increase in SPR response units (RU) and presents char-
acteristic fast-binding and slow-dissociation curves. The
concentration series of quercetin-3-b-galactoside are fit-
ted by the 1:1 Langmuir binding model in the Bia-
core3000 evaluation software for binding affinity
determination. The dissociation constant (KD) between
quercetin-3-b-galactoside and SARS-CoV 3CLpro was
determined as 38.41 2.06 lM. This result is consistent
with our molecular modeling prediction that Q189A
mutation would lead to a significant drop in binding
affinity. While the residue is essential for the binding
of the natural product, it is not a key residue for the
function of the 3CL protease. Therefore, the modeled
interaction mode between the natural product and the
protease is accurate.
proteinases are similar in vitro. Hence, experimental
data from CD spectroscopy, fluorescence spectra, and
enzyme activity assay indicated that SARS-CoV 3CLpro
mutant was structurally and functionally similar to the
wild-type protease.
2.3. Binding affinity of quercetin-3-b-galactoside to
SARS-CoV 3CLpro or SARS-CoV 3CLpro Q189A
The binding affinity of quercetin-3-b-galactoside to
SARS-CoV 3CLpro or SARS-CoV 3CLpro Q189A in vi-
tro was determined using surface plasmon resonance
(SPR) biosensor technology. For kinetic analysis on
the Biacore3000 instrument, various concentrations of
quercetin-3-b-galactoside were injected for 120 min at
a flow rate of 30 lL/min to allow for interaction with
SARS-CoV 3CLpro or SARS-CoV 3CLpro Q189A
immobilized on the sensor chip surface. The binding

L. Chen et al. / Bioorg. Med. Chem. 14 (2006) 8295–8306
8299
Figure 4. Binding affinity of quercetin-3-b-galactoside to SARS-CoV 3CLpro and SARS-CoV 3CLpro Q189A determined by SPR. Specificity and
concentration-dependent binding and real-time binding affinity measurement of quercetin-3-b-galactoside to immobilized SARS-CoV 3CLpro and
SARS-CoV 3CLpro Q189A (A and B) using Biacore 3000. For SARS-CoV 3CLpro, the concentrations of quercetin-3-b-galactoside were 80, 56, 39.2,
27.4, 19.2, 13.4, and 9.4 lM, while for SARS-CoV 3CLpro Q189A, the concentrations of quercetin-3-b-galactoside were 200, 140, 98, 68.6, 48.0, 33.6,
23.5, and 16.5 lM from top to bottom. Quercetin-3-b-galactoside was injected for 60 s, and dissociation was monitored for more than 120 s,
respectively.
2.4. Design and synthesis of quercetin-3-b-galactoside
derivatives (2a–h)
procedures and structural characterization of the com-
pounds 1 and 2a–h are depicted in Schemes 1–3, and de-
scribed in Section 4.
With the validation of our binding model between quer-
cetin-3-b-galactoside and SARS-CoV 3CLpro, and tak-
ing into account the structural and functional
similarity but contrasting binding interaction mode of
the protease and its mutant (Fig. 1), we designed and
synthesized compound 2a (Scheme 1) to determine if
unprotected hydroxy sites on the sugar moiety are nec-
essary for ligand–enzyme interaction, compounds 2f–h
(Schemes 2 and 3) for demonstrating the significance
of the four hydroxy groups of the quercetin compound
1 (Scheme 1) as key interactive elements, and compound
2e for proving that significant spatial volume within the
binding pocket of the enzyme around the 7-hydroxy
group of quercetin available to accommodate large
chemical moieties. Meanwhile, compounds 2b–d were
prepared to determine if the type of sugar moiety would
affect inhibitory activity. The details of the synthesis
2.5. Inhibitory activity of quercetin-3-b-galactoside (1)
and its derivatives 2a–h
The inhibitory activities of quercetin-3-b-galactoside (1)
toward the proteolytic activity of SARS-CoV 3CLpro or
SARS-CoV 3CLpro Q189A were measured by FRET as-
say using a peptide substrate labeled with a pair of flu-
orogenic dyes. The inhibition of SARS-CoV 3CLpro
slowly increased in the concentration range from 0 to
500 lM of quercetin-3-b-galactoside (Fig. 5A). The
curve finally reached steady state, which was dependent
on the concentration of inhibitors. The IC50 value of the
natural product in inhibiting the catalytic activity of
SARS-CoV 3CLpro was calculated to be 42.79
4.97 lM by fitting the dose–response curve using a logis-
tic derivative equation in Origin 7.0.28 However, the

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L. Chen et al. / Bioorg. Med. Chem. 14 (2006) 8295–8306
OH
O
O
O
O
Ph
Ph
Ph
Ph
HO
O
R1O
O
O
HO
O
O
a
OH
b
OH
OR1
OH
OH
O
OH
OH
6
3
4
c,d
b
OH
OH
OH
OH
O
Ph
Ph
HO
O
R2O
O
HO
O
O
c
OR1
OR2
OR1
OH
O
2a
OH
O
OH
O
5
OAc
R =
1
AcO
AcO
2e
OH
R2 =
HO
HO
O
O
d
OAc
OH
OH
OH
D-Galactose
1
OH
2b
OH
HO
R2 =
R2 =
R2 =
R2 =
HO
HO
HO
O
O
O
OH
O
OH
OR2
L-Fucose
D-Galactose
OH
2c
2d
OH
HO
HO
1, 2b-d
O
OH
HO
HO
O
OH
D-Glucose
D-Arabinose
Scheme 1. Synthesis of compounds 2a–2e. Reagents and conditions: (a) Ph2CCl2, 180 ꢁC, 5 min; (b) O-acetylglycosyl bromide, K2CO3, DMF; (c)
10% Pd/C, H2; (d) i—MeONa, MeOH, ii—H+ resin.
O
O
a
OH
O
Cl
N
+
N
OH
O
7
O
c
b
OH
O
HO
OH
8
HO
O
OH
2g
OH
HO
HO
O
O
R2 =
HO
O
HO
HO
d
e
O
OR1
OR2
OH
OH
D-Galactose
OH
O
OH
OH
O
2g
10
9
Scheme 2. Synthesis of compound 2g. Reagents and conditions: (a) Et3N, CH2Cl2; (b) HCl (g), Et2O; (c) i—Bz2O, PhCO2Na, ii—KOH, EtOH, CO2
(g); (d) acetobromo-a-D-galactose, K2CO3, DMF; (e) i—MeONa, MeOH, ii—H+ resin.
IC50 value of quercetin-3-b-galactoside against SARS-
CoV 3CLpro Q189A was estimated to be
127.89 10.06 lM (Fig. 5B), which is 2-fold larger than
that of SARS-CoV 3CLpro. Meanwhile, the double reci-
procal plot of velocity as a function of substrate at vary-
ing inhibitor concentrations shown in Figure 6 suggests
that quercetin-3-b-galactoside inhibits SARS-CoV
3CLpro in a competitive mode as well.
as described for compound 1. The results was summa-
rized in Table 1. For the primary assay, the percent inhi-
bitions of the compounds at 50 lM were measured. Of
the synthetic derivatives tested, four of these—namely
2b, 2c, 2d, and 2e, displayed remarkable inhibitory ac-
tion on the catalytic activity of SARS-CoV 3CLpro (ex-
pressed as percent inhibition at 50 lM > 50%),
indicating that these are good candidate inhibitors of
SARS-CoV 3CLpro. The IC50 values determined for
these compounds were 24.14 4.32, 31.62 2.43,
48.85 8.15, and 61.46 9.13 lM, respectively.
The inhibition assays of compounds 2a–h against
SARS-CoV 3CLpro were performed in the same manner

L. Chen et al. / Bioorg. Med. Chem. 14 (2006) 8295–8306
8301
R3
R3
R3
OH
R3
R3
CHO
a
O
O
O
b
+
R3
OR1
OH
O
R3 = H or OBz
O
11
12
R4
R4
R4
R4
2f
OH
R2 =
R2 =
R4 = OH
HO
HO
O
O
O
O
d
c
OH
OR2
OR1
D-Galactose
O
2h
OH
R4 = H
HO
O
R4 = H or OH
2f and 2h
HO
13
OH
D-Galactose
Scheme 3. Synthesis of compounds 2f and 2h. Reagents and conditions: (a) i—KOH, EtOH, dioxane, ii—NaOH, 30% H2O2, EtOH; (b) acetobromo-
a-D-galactose, K2CO3, DMF; (c) 10% Pd/C, H2; (d) i—MeONa, MeOH, ii—H+ resin.
Figure 6. Enzyme kinetic analysis of SARS-CoV 3CLpro by quercetin-
3-b-galactoside. The reaction was performed in the presence of
different concentrations of quercetin-3-b-galactoside (m, 0 lM; n,
10 lM; s, 50 lM; d, 100 lM) at varying concentrations of the
fluorogenic substrate (from 5 to 100 lM). Lineweaver–Burk plots of
kinetic data were fitted to obtain the Ki value of 35.66 4.57 lM.
Table 1. The inhibitory activities and IC50 values of compounds 1 and
2a–h against SARS-CoV 3CLpro
Compound
Inhibitory rate at 50 lM (%)
IC50 (lM)
2a
2b
2c
2d
2e
2f
2g
2h
1
12.4
57.4
49.4
57.5
53.0
30.1
18.7
NA
41.8
—
24.14 4.32
31.62 2.43
48.85 8.15
61.46 9.13
—
—
—
42.79 4.97
Figure 5. Dose–response plot for quercetin-3-b-galactoside on the
proteolytic activity inhibition of SARS-CoV 3CLpro and SARS-CoV
3CLpro Q189A. Inhibition of cleavage was measured by FRET using a
peptide substrate labeled with a pair of fluorogenic dyes. IC50 values
were calculated from the curves using nonlinear regression analysis.
IC50 value for quercetin-3-b-galactoside on SARS-CoV 3CLpro is
42.79 4.97 lM (A). IC50 value for quercetin-3-b-galactoside on
SARS-CoV 3CLpro Q189A is 127.89 10.06 lM (B).
2.6. Structure and activity relationship of quercetin-3-b-
galactoside derivatives (2a–h)
From the foregoing, the binding model of quercetin-3-b-
galactoside with SARS-CoV 3CLpro provided valuable

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L. Chen et al. / Bioorg. Med. Chem. 14 (2006) 8295–8306
insights into the structural determinants involved in
compound–target interaction, leading to rational design
and synthesis of eight quercetin-3-b-galactoside deriva-
tives containing systematic chemical variations. Biologi-
cal evaluation of inhibitory activities of these analogues
(Table 1) provides the basis to formulate the structure–
activity relationship (SAR) The key elements observed
are: (1) removing the hydroxy groups of the quercetin
moiety (2f–h) substantially decreases the inhibitory
activity of the derivatives, (2) acetoxylation of sugar
moiety (2a) abolishes inhibitory functions, (3) introduc-
tion of a large sugar substituent on 7-hydroxy of querce-
tin (2e) can be tolerated, and (4) replacement of the
galactose moiety with other sugars, such as fucose
(2b), arabinose (2c), and glucose (2d), had no evident ef-
fect on inhibitor potency. These observations were in
good agreement with the molecular binding model.
chased from Qiagen. The chelating column and pro-
tein molecular weight marker for SDS–PAGE were
from Amersham Pharmacia Biotech. Amicon Ultra-4
centrifugal filter devices were purchased from Milli-
pore. The QuickChange site-directed mutagenesis kit
was purchased from Stratagene (La Jolla, Calif). Ana-
lytical thin-layer chromatography (TLC) was per-
formed on HSGF 254 (150–200 lm thickness, Yantai
Huiyou Company, China). Yields were not optimized.
Melting points of the compounds were measured in a
capillary tube on a SGW X-4 melting point apparatus
without correction. Nuclear magnetic resonance
(NMR) spectra were obtained from
a Brucker
AMX-400 NMR (IS as TMS). Chemical shifts were
reported in parts per million (ppm, d) downfield from
tetramethylsilane. Proton coupling patterns were de-
scribed as singlet (s), doublet (d), triplet (t), quartet
(q), multiplet (m), and broad (br). Low- and high-res-
olution mass spectra (LRMS and HRMS) were ob-
tained using electrospray (ESI) produced by LCQ-
DECA spectrometer.
3. Conclusion
A natural compound, quercetin-3-b-galactoside that
has been shown to be effective for treating allergies
and preventing heart disease and cancer, was identified
as a new class of inhibitors against SARS-CoV 3CLpro
through molecular docking, SPR and FRET bioassays,
and mutagenesis studies. Both molecular modeling and
G189A mutation revealed that Gln189 plays a key role
in the binding of quercetin-3-b-galactoside to SARS-
CoV 3CLpro. Furthermore, comparative studies of
SARS-CoV 3CLpro and SARS-CoV 3CLpro Q189A
employing CD spectroscopy, fluorescence spectra, and
enzyme activity assay revealed no evidence that second-
ary structure and biological activity of the protease are
affected by the mutation. Eight new derivatives of the
quercetin-3-b-galactoside containing systematic chemi-
cal variations to binding elements were rationally de-
signed under the guidance of molecular modeling and
successfully synthesized. The bioassay data obtained
from these new compounds demonstrate that the four
hydroxy groups of the quercetin moiety are key deter-
minants of the bioactivity of this type of inhibitors,
that the spatial dimensions of the binding pocket in
the vicinity of the sugar moiety and 7-hydroxy site of
quercetin can accommodate large structural modifica-
tions, and that the hydroxyl groups of the sugar moiety
should be unprotected to allow compound–target inter-
action by hydrogen bonding. Our data provide new in-
sights into the essential interactions of quercetin-3-b-
galactoside with the binding pocket of SARS-CoV
3CLpro, thus leading to a legible pharmacophore of
SARS-CoV 3CLpro inhibitors that will aid future inhib-
itor design.
4.2. Virtual screening
The crystal structure of SARS-CoV 3CLpro (PDB en-
try: 1UK4) was used as the target for virtual screening
on our in-house drug-like filtered ACD database
(17,000 molecules) by using DOCK4.0.38 Residues
around the binding site were isolated for constructing
the grids for docking screening, and the pocket com-
posed by these residues is large enough to include res-
idues of the binding pocket. During the docking
calculations, Kollman-all-atom charges39 were assigned
to the protein, and Geisterger–Huckel charges40 were
¨
assigned to the small molecules in the database. The
conformational flexibility of the compounds from the
database was considered during the docking. In the
DOCK simulation, the ligand–receptor binding energy
was taken to be approximately the sum of the van der
Waals and electrostatic interaction energies. After the
initial orientation and scoring evaluation, grid-based
rigid body minimization was carried out for the ligand
to locate the nearest local energy minimum within the
SARS-CoV 3CLpro binding site. The top-3000 mole-
cules obtained from the DOCK search were selected
for further analyses. These molecules were re-scored
by Sybyl/Cscore and the scoring function of Auto-
Dock3.0.41 Finally, a natural product, quercetin-3-b-
galactoside, was selected for bioassay, based on the
scoring results.
4.3. Synthesis of quercetin-3-b-galactoside and its
derivatives
4.3.1. 2-(2,2-Diphenylbenzo[d][1,3]dioxol-5-yl)-3,5,7-tri-
hydroxy-4H-chromen-4-one (4). A mixture of quercetin
3 (1 g, 3 mmol) and Ph2CCl2 (1.7 mL, 8.9 mmol) was
stirred at 180 ꢁC for 10 min. The reaction mixture was
purified by flash chromatography on silica gel, eluted
with a mixture of EtOAc/petroleum ether (1:4, v/v), to
afford 4 (0.5 g, 37%) as a yellow solid. Mp 239–240 ꢁC,
1H NMR (DMSO) d: 6.22 (1H, d), 6.50 (1H, d), 7.26
(1H, d), 7.44–7.60 (10H, m), 7.80–7.83 (2H, m).
4. Experimental procedures
4.1. Materials
All solvents and reagents with reagent grade or ultra-
pure quality were purchased commercially and were
used without further purifications. The vector pQE30
and the bacterial strain E. coli strain M15 were pur-

L. Chen et al. / Bioorg. Med. Chem. 14 (2006) 8295–8306
8303
4.3.2. 2-(2,2-Diphenylbenzo[d][1,3]dioxol-5-yl)-5,7-dihy-
droxy-3-tetraacetylgalactosyl-4H-chromen-4-one (5) and
2-(2,2-diphenylbenzo[d][1,3]dioxol-5-yl)-5-hydroxy-3,7-
di(tetraacetylgalactosyl)-4H-chromen-4-one (6). A mix-
ture of 4 (0.3 g, 0.64 mmol), acetobromo-a-D-galactose
(0.4 g, 0.97 mmol), and K2CO3 (0.12 g, 0.9 mmol) in
DMF (5 mL) was stirred at rt for 10 h under argon. The
reaction mixture was poured into H2O and extracted with
EtOAc. The combined organic layer was washed, dried,
filtered, and condensed. The crude product obtained
was purified by flash chromatography on silica gel, eluted
with a mixture of EtOAc/petroleum ether (1:4, 1:2 v/v), to
afford 5 (0.16 g, 31%) and 6 (0.18 g, 25%), respectively.
MS-ESI 797 [M+H]+ and MS-ESI 1127 [M+H]+.
romo-a-D-glucose, yield 48% (last step). Mp 172–
174 ꢁC, 1H NMR (DMSO) d: 3.1–3.8 (7H, m), 5.49
(1H, d), 6.22 (1H, d), 6.43 (1H, d), 6.87 (1H, d), 7.61
(2H, m). MS-ESI 465 [M+H]+, HRMS (ESI) m/z: Calcd
C21H21O12 [M+H]+ 465.1033. Found: 465.1067.
4.3.7. 2-(30,40-Dihydroxyphenyl)-5-hydroxy-3,7-di(b-D-
galactosyl)-4H-chromen-4-one (2e). In the same manner
as described for 1, 2e was prepared from 6, yield 56%
(two steps). Mp 165–168 ꢁC, 1H NMR (DMSO): d
3.30–3.70 (12H, m), 5.06 (1H, d), 5.43 (1H, d), 6.46
(1H, s), 6.76 (1H, s), 6.86 (1H, d), 7.57 (1H, s), 7.70
(1H, d); ESI-MS m/z: 627 [M+H]+. HRMS (ESI) m/z:
Calcd C27H31O17 [M+H]+ 627.1561. Found: 627.1560.
4.3.3. 2-(30,40-Dihydroxyphenyl)-5,7-dihydroxy-3-b-D-ga-
lactosyl-4H-chromen-4-one (1). A mixture of 5 (160 mg,
0.2 mmol), 10% palladium on charcoal (20 mg), and
EtOH (20 mL) was stirred at 25 ꢁC for 24 h in an atmo-
sphere of hydrogen. The catalyst was filtered and the fil-
trate was concentrated to dryness. The crude product
obtained was purified by flash chromatography on silica
gel, eluted with a mixture of EtOAc/petroleum ether (1:1
v/v), to afford 2a (0.1 g, 79%). Mp 108–110 ꢁC, 1H
NMR(CDCl3) d: 1.89 (3H, s), 2.01(3H, s), 2.10 (3H,
s), 2.26 (3H, s), 3.89–4.02 (3H, m), 5.15 (1H, dd), 5.42
(2H, m), 5.82 (1H, d), 6.26 (1H, s), 6.37 (1H, s), 6.98
(1H, d), 7.51 (1H, d), 7.79 (1H, s), MS-ESI 633 [M+H]+.
4.3.8. Cyanomethyl benzoate (7). A mixture of benzoic
acid (4.9 g, 40 mmol), 2-chloroacetonitrile (4.5 g,
60 mmol), and Et3N (8 g, 80 mmol) in CH2Cl2 (25 mL)
was stirred at rt for 48 h. The reaction mixture was
poured into H2O, and extracted with EtOAc. The com-
bined organic layer was washed, dried, filtered, and con-
densed, to afford 7 (5.2 g, 81%).
4.3.9. 3,5,7-Trihydroxy-2-phenyl-4H-chromen-4-one (9).
Compound 8 was synthesized from compound 7 and
phloroglucinol using Chen’s method.42 A mixture of 8
(2 g, 6.9 mmol), benzoic anhydride (8.1 g, 35.8 mmol),
and sodium benzoate (1.6 g, 11.2 mmol) was stirred at
170 ꢁC for 3 h. To the reaction mixture were added
KOH (5 g) and EtOH (51 mL). After refluxing for
0.5 h, the greater part of the alcohol was evaporated,
the residue was dissolved in water and subsequently pre-
cipitated as a brown solid by saturating the liquid with
carbon dioxide, collected, washed, and dried, to afford
To a solution of the 2a (50 mg, 0.079 mmol) in methanol
(10 mL) was added NaOMe (5 mg, 0.09 mmol) and the
resultant solution was stirred at rt for 1 h. The solution
was neutralized by passage down a Dowex 50 (H+) ion-
exchange resin. The resin was filtered and the filtrate was
concentrated to afford 1 (30 mg, 81%). Mp 212–215 ꢁC,
1H NMR(DMSO) d: 3.20–3.62 (7H, m), 5.37 (1H, d),
6.19 (1H, d), 6.40 (1H, d), 6.82 (1H, d), 7.51 (1H, d),
7.66 (1H, d). MS-ESI 465 [M+H]+, HRMS (ESI) m/z:
Calcd C21H21O12 [M+H]+ 465.1053. Found: 465.1067.
1
9 (1.1 g, 59%). Mp 175–176 ꢁC, H NMR (DMSO) d:
6.25 (1H, d), 6.50 (1H, d), 7.61 (3H, m), 8.20 (2H, m).
4.3.10.
2-Phenyl-5,7-dihydroxy-3-b-D-galactosyl-4H-
chromen-4-one (2g). In the same manner as described
for 1, 2g was prepared from 9, yield 66% (last step).
1
4.3.4.
2-(30,40-Dihydroxyphenyl)-5,7-dihydroxy-3-b-L-
Mp 162–164 ꢁC, H NMR (DMSO): d 3.30–3.58 (5H,
fucosyl-4H-chromen-4-one (2b). In the same manner as
described for 1, 2b was prepared from 4 and acetob-
romo-a-L-fucose, yield 80% (last step). Mp 172–
m), 3.66 (1H, d), 5.46 (1H, d), 6.26 (1H, d), 6.48 (1H,
d), 7.54 (3H, m), 8.17 (2H, m); ESI-MS m/z: 433
[M+H]+. HRMS (ESI) m/z: Calcd C21H21O10 [M+H]+
433.1135. Found: 433.1138.
1
174 ꢁC, H NMR (DMSO) d: 1.02 (3H, d), 3.30–3.60
(5H, m), 5.36 (1H, d), 6.23 (1H, d), 6.44 (1H, d), 6.86
(1H, d), 7.56 (1H, d), 7.71 (1H, d). MS-ESI 449
[M+H]+, HRMS (ESI) m/z: Calcd C21H21O11 [M+H]+
449.1084. Found: 449.1087.
4.3.11.
2-(30,40-bis(Benzyloxy)phenyl)-3-hydroxy-4H-
chromen-4-one (11). Compound 11 was synthesized
using the Frederique’s method43 from 2-hydroxyace-
tophenone and 3,4-dibenzyloxybenzaldehyde.
4.3.5.
2-(30,40-Dihydroxyphenyl)-5,7-dihydroxy-3-b-D-
arobinosyl-4H-chromen-4-one (2c). In the same manner
as described for 1, 2c was prepared from 4 and acetob-
romo-a-D-arabinose, yield 60% (last step). Mp 42–
44 ꢁC, 1H NMR(DMSO) d: 3.20–3.80 (6H, m), 5.30
(1H, d), 6.23 (1H, d), 6.44 (1H, d), 6.87 (1H, d), 7.66
(1H, d), 7.69 (1H, d). MS-ESI 435 [M+H]+, HRMS
(ESI) m/z: Calcd C20H19O11 [M+H]+ 435.0927. Found:
435.0909.
4.3.12. 2-(30,40-Dihydroxyphenyl)-3-b-D-galactosyl-4H-
chromen-4-one (2f). In the same manner as described
for 1, 2f was prepared from 11 (R3 = OBz), yield 61%
(last step). Mp 156–158 ꢁC, 1H NMR (DMSO): d
3.30–3.7 (6H, m), 5.45 (1H, d), 6.86 (1H, d), 7.52 (1H,
t), 7.63 (1H, d), 7.74 (2H, d), 7.83 (1H, t), 8.10 (1H,
d); ESI-MS m/z: 433 [M+H]+. HRMS (ESI) m/z: Calcd
C21H21O10 [M+H]+ 433.1135. Found: 433.1175.
4.3.6.
2-(30,40-Dihydroxyphenyl)-5,7-dihydroxy-3-b-D-
glucosyl-4H-chromen-4-one (2d). In the same manner
as described for 1, 2d was prepared from 4 and acetob-
4.3.13.
2-Phenyl-3-b-D-galactosyl-4H-chromen-4-one
(2h). In the same manner as described for 1, 2h was pre-
pared from 11 (R3 = H), yield 64% (last step). Mp 175–

8304
L. Chen et al. / Bioorg. Med. Chem. 14 (2006) 8295–8306
1
177 ꢁC, H NMR (DMSO): d 3.30–3.8 (6H, m), 5.53
(1H, d), 7.57 (4H, m), 7.78 (1H, d), 7.86 (1H, t), 8.12
(1H, d), 8.23 (2H, d); ESI-MS m/z: 401 [M+H]+. HRMS
(ESI) m/z: Calcd C21H21O8 [M+H]+ 401.1236. Found:
401.1211.
4.6. Circular dichroism (CD) and fluorescence spectral
analyses
CD spectra of the wild and mutant type protease were
determined on a JASCO-810 spectropolarimeter collect-
ed with thermal controller according to Chen et al.16
The protein sample was prepared in 20 mM sodium
phosphate, pH 7.4, 100 mM NaCl at 25 ꢁC at a concen-
tration of 10 lM. Far-UV CD spectra from 195 to
250 nm were collected with 1 nm bandwidth using a
0.1-cm path length cuvette and normalized by subtract-
ing the baseline recorded for the buffer under identical
conditions. The fluorescent measurement was performed
on a HITACHI F-2500 fluorescence spectrophotometer
connected with a thermostat. Two proteases were pre-
pared in the same buffer and were diluted to the final
concentration of 10 lM. The emission fluorescence
intensity was monitored separately from 300 to 500 nm
wavelengths upon excitation at 280 nm (slit width
2.5 nm) at 25 ꢁC. The scan speed was 300 nm/min and
the PMT voltage was 400 V. Each measurement was
repeated three times, and the final result was the average
of the three independent scans.
4.4. Site-directed mutagenesis of SARS-CoV 3CLpro
Since Q189 was predicted from molecular modeling to
be a key residue for inhibitory activity, site-directed
mutagenesis was performed by a recombination PCR
method previously described to test out this hypothesis.
The plasmid, pQE30-SARS_3CLpro, was constructed
according to Sun et al.44 The plasmid carrying the gene
encoding the SARS-CoV 3CLpro Q189A mutant was
prepared with the mutagenesis kit using pQE30-
SARS_3CLpro as a template. A forward primer with
a sequence of 50-GGT CCA TTT GTT GAC AGA
GCA ACT GCA CAG GCT GCA GG-30 and a re-
verse primer 50-CC TGC AGC CTG TGC AGT
TGC TCT GTC AAC AAA TGG ACC-30 were de-
signed to amplify the DNA fragment encoding the en-
tire mutant protease. Subsequently, QuickChange site-
directed mutagenesis was performed in accordance with
manufacturer’s instructions. The mutation was verified
by DNA sequencing. After sequence analysis, the re-
combinant plasmid, designated pQE30-SARS_3CLpro
Q189A, was transformed into competent E. coli strain
M15 cells.
4.7. Enzyme activity assay
The activity of SARS-CoV 3CLpro or SARS-CoV
3CLpro Q189A was measured by cleavage of the fluor-
ogenic substrate Dabcyl-KNSTLQSGLRKE-Edans
based on the reported protocols.28 Briefly, the fluores-
cence intensity was monitored on a GENios microplate
reader (TECAN) and the enzyme activity was deter-
mined by the increase in fluorescence upon continuous
monitoring of the reactions in 96-well black micro-
plates (BMG LABTECH) with the wavelengths of
340 and 488 nm for excitation and emission, respective-
ly. Kinetic parameters (Km and kcat) of SARS-CoV
3CLpro or SARS-CoV 3CLpro Q189A were determined
by incubation of the fluorogenic substrate with varied
concentrations ranging from 5 to 200 lM, and 1 lM
SARS-CoV 3CLpro or SARS-CoV 3CLpro Q189A at
25 ꢁC. The reaction velocity (v0) for each substrate con-
centration was averaged from three assay results. Km
and kcat values were calculated by using a Linewe-
aver–Burk plot.
4.5. Protein preparation
Escherichia coli strain M15 cells containing pQE30-
SARS_3CLpro were grown in 20 mL LB medium con-
taining ampicillin (100 lg mLꢁ1
)
and kanamycin
(25 lg mLꢁ1) at 37 ꢁC overnight and then inoculated
into 1 L LB supplemented with both antibiotics.
The expression of SARS-CoV 3CLpro was induced
by the addition of 0.5 mM of isopropyl b-D-thioga-
lactoside (IPTG). The cells were harvested by centri-
fugation at 8000g, 4 ꢁC for 10 min after induction for
5 h at 22 ꢁC. The pellet was washed, frozen, and then
disrupted by sonication against the binding buffer
(20 mM Tris–HCl, 0.5 M NaCl, and 10 mM imidaz-
ole, pH 8.0). The lysate was centrifuged at 18,000g
for 30 min at 4 ꢁC to pellet the cellular debris. The
supernatant was then added to a HiTrap Ni2+ chelat-
ing column (1 mL) pre-equilibrated with the binding
buffer, followed by washing with 20 mL of washing
buffer (20 mM Tris–HCl, 0.5 M NaCl, and 60 mM
imidazole, pH 8.0). The protease of interest was elut-
ed with 10 mL elution buffer (20 mM Tris–HCl,
0.5 M NaCl, and 300 mM imidazole, pH 8.0). The
freshly prepared protease was dialyzed against the
appropriate buffer as required. The purified SARS-
CoV 3CLpro was concentrated by Amicon Ultra-4
centrifugal filter devices (Millipore) with molecular
cutoff at 10 kDa. The expression and purification of
SARS-CoV 3CLpro Q189A were performed using
the same procedure as for SARS-CoV 3CLpro. The
purity and molecular weights of SARS-CoV 3CLpro
and SARS-CoV 3CLpro Q189A were verified by
SDS–PAGE.
4.8. Inhibition activity assay
The inhibition activity of quercetin-3-b-galactoside or its
derivatives against SARS-CoV 3CLpro or SARS-CoV
3CLpro Q189A was measured by a quenched fluoresence
resonance energy transfer (FRET) assay with the fluor-
ogenic substrate Dabcyl-KNSTLQSGLRKE-Edans. A
stock solution of the substrate was dissolved in sterile
H2O and stored at ꢁ20 ꢁC. The SARS-CoV 3CLpro or
SARS-CoV 3CLpro Q189A (1 lM) and the test com-
pounds (from 0.1 to 500 lM) were incubated for 2 h at
4 ꢁC and the reaction was initiated by adding the sub-
strate to the desired final concentration (10 lM). The en-
zyme activity was determined by the increase in
fluorescence upon continuous monitoring of the
reactions in 96-well black microplates (BMG) using
wavelengths of 340 and 488 nm for excitation and emis-

L. Chen et al. / Bioorg. Med. Chem. 14 (2006) 8295–8306
8305
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G. M.; Ahuja, A.; Yung, M. Y.; Leung, C. B.; To, K. F.;
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monitoring of fluorescence for 60 min at 25 ꢁC on a
GENios (TECAN). The initial velocities of the inhibited
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4.9. Surface plasmon resonance (SPR) assay
Interaction studies between quercetin-3-b-galactoside
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were performed with the surface plasmon resonance
based biosensor instrument Biacore 3000 (Biacore AB,
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the baseline was completed by continuous flow of
HBS-EP running buffer (10 mM Hepes, 150 mM NaCl,
3 mM EDTA, and 0.005% (v/v) surfactant P20, pH
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flow cells was activated for 7 min by injecting a 1:1 fresh
mixture of 0.2 M N-ethyl-N0-dimethylaminopropyl car-
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(NHS) at 25 ꢁC. SARS-CoV 3CLpro was diluted with
10 mM sodium acetate buffer at pH 4.3 to a concentra-
tion of 25 lg mLꢁ1; SARS-CoV 3CLpro Q189A was
diluted with 10 mM sodium acetate buffer at pH 5.65
to a final concentration of 16 lg mLꢁ1 and immobilized
to the surface of sensor chip CM5, respectively. Finally,
unreacted protease was blocked by injecting 1 M etha-
nolamine–HCl at pH 8.5 for 7 min, resulting in immobi-
lized densities of 4000 RU. After stabilizing the baseline,
Biacore data were collected at 25 ꢁC with HBS–EP (con-
taining 0.4% DMSO) as the running buffer at a constant
flow of 30 lL/min. All the sensorgrams were processed
by using automatic correction for non-specific bulk
refractive index effects. The equilibrium constants
(KDs) evaluating the protein–inhibitor binding affinity
were determined by the 1:1 Langmuir binding fitting
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Acknowledgments
We gratefully acknowledge financial support from
Shanghai Key R&D Program (Grants 036505003 and
05JC14092), National Key R&D Program (Grant
2005BA711A04),
2004CB518901).
and
973
Program
(Grant
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