Molecular docking identifies the binding of 3-chloropyridine moieties specifically to the S1 pocket of SARS-CoV Mpro

Article abstract of DOI:10.1016/j.bmc.2007.09.034

The 3C-like main proteinase of the severe acute respiratory syndrome (SARS) coronavirus, SARS-CoV M^pro, is widely considered to be a major drug target for the development of anti-SARS treatment. Based on the chemical structure of a lead compound from a previous screening, we have designed and synthesized a number of non-peptidyl inhibitors, some of which have shown significantly improved inhibitory activity against SARS-CoV M^pro with IC₅₀ values of ～60 nM. In the absence of SARS-CoV M^pro crystal structures in complex with these synthetic inhibitors, molecular docking tools have been employed to study possible interactions between these inhibitors and SARS-CoV M^pro. The docking results suggest two major modes for the initial binding of these inhibitors to the active site of SARS-CoV M^pro. They also establish a structural basis for the 'core design' of these inhibitors by showing that the 3-chloropyridine functions common to all of the present inhibitors tend to cluster in the S1 specificity pocket. In addition, intrinsic flexibility in the S4 pocket allows for the accommodation of bulky groups such as benzene rings, suggesting that this structural plasticity can be further exploited for optimizing inhibitor-enzyme interactions that should promote a tighter binding mode. Most importantly, our results provide the structural basis for rational design of wide-spectrum antiviral drugs targeting the chymotrypsin-like cysteine proteinases from coronaviruses and picornaviruses.

Full text of DOI:10.1016/j.bmc.2007.09.034

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 293–302
Molecular docking identifies the binding
of 3-chloropyridine moieties specifically to the S1
pocket of SARS-CoV Mpro
Chunying Niu,a Jiang Yin,a Jianmin Zhang,b
John C. Vederasb, and Michael N. G. Jamesa,c, ,à
*
aGroup in Protein Structure and Function, 431 Medical Science Building, Department of Biochemistry, University of Alberta,
Edmonton, Alta., Canada T6G 2H7
bDepartment of Chemistry, University of Alberta, Edmonton, Alta., Canada T6G 2G2
cAlberta Synchrotron Institute, University of Alberta, Edmonton, Alta., Canada T6G 2E1
Received 5 July 2007; revised 17 September 2007; accepted 19 September 2007
Available online 22 September 2007
Abstract—The 3C-like main proteinase of the severe acute respiratory syndrome (SARS) coronavirus, SARS-CoV Mpro, is widely
considered to be a major drug target for the development of anti-SARS treatment. Based on the chemical structure of a lead com-
pound from a previous screening, we have designed and synthesized a number of non-peptidyl inhibitors, some of which have shown
significantly improved inhibitory activity against SARS-CoV Mpro with IC50 values of ꢀ60 nM. In the absence of SARS-CoV Mpro
crystal structures in complex with these synthetic inhibitors, molecular docking tools have been employed to study possible inter-
actions between these inhibitors and SARS-CoV Mpro. The docking results suggest two major modes for the initial binding of these
inhibitors to the active site of SARS-CoV Mpro. They also establish a structural basis for the ‘core design’ of these inhibitors by
showing that the 3-chloropyridine functions common to all of the present inhibitors tend to cluster in the S1 specificity pocket.
In addition, intrinsic flexibility in the S4 pocket allows for the accommodation of bulky groups such as benzene rings, suggesting
that this structural plasticity can be further exploited for optimizing inhibitor–enzyme interactions that should promote a tighter
binding mode. Most importantly, our results provide the structural basis for rational design of wide-spectrum antiviral drugs tar-
geting the chymotrypsin-like cysteine proteinases from coronaviruses and picornaviruses.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
viral proteinases, one of which is the main proteinase
(Mpro). Mpro, a highly conserved proteinase among cor-
onaviruses, is responsible for the majority of the prote-
olytic processing of two large viral polyproteins,
replicases pp1a and pp1ab. All of the cleavage products
from these replicases are required for the assembly of
membranous complexes in infected cells where the viral
RNA genome is replicated. Interruption of either viral
RNA translation or polyprotein proteolytic processing
abolishes viral RNA replication.6,7 Because the indis-
pensable involvement in polyprotein processing of
SARS-CoV Mpro is upstream of many replicative events
in the viral life cycle, reducing or blocking the peptidase
function of Mpro could have a profound impact on low-
ering progeny virus yield and enhancing cell survival.
Therefore, SARS-CoV Mpro is an attractive target for
the development of anti-SARS drugs.8
In the winter of 2002, a form of atypical pneumonia,
later termed the severe acute respiratory syndrome
(SARS), broke out in southern China and rapidly
spread to 32 countries and regions on five continents.
Approximately 8098 people were infected and of these,
774 deaths were reported in the ensuing months.1
The causative agent of SARS is a novel human corona-
virus (SARS-CoV).2–5 Similar to other coronaviruses,
the RNA genome of SARS-CoV encodes at least two
Keywords: SARS coronavirus; 3C-like proteinase; Small molecule
docking; Non-peptidyl proteinase inhibitor; Drug design;
Chloropyridine.
*
Corresponding author. Tel.: +1 780 492 4550; fax: +1 780 492
0886; e-mail: michael.james@ualberta.ca
The functional quaternary assembly of SARS-CoV
Mpro is believed to be a dimer.9 The two protomers
Canada Research Chair in Bioorganic and Medicinal Chemistry.
Canada Research Chair in Protein Structure and Function.
à
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.09.034

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C. Niu et al. / Bioorg. Med. Chem. 16 (2008) 293–302
are oriented almost perpendicular to one another and
each protomer consists of three domains: I (residues
1–101), II (residues 102–184), and III (residues 201–
306). Domains I and II are made of b-barrel motifs
similar to the domains in the chymotrypsin-type serine
proteases and in the 3C proteinases of picornaviruses.
The substrate binding sites, and the nearby catalytic
dyad consisting of residues Cys145 and His41, are
located in a cleft between domains I and II. Domain
III contains five a-helices connected to domain II via
an extended loop (residues 185–200); it plays an essen-
tial role for the proteolytic activity of SARS-CoV Mpro
by maintaining the proper conformation of the
dimer.9–11
2. Results
Prior to the virtual docking, the compounds were sorted
into two groups based on their chemical characteristics.
Both groups contain the ester function and the chloro-
pyridine moiety inherited from the parent compound.
In addition, group I compounds have a furan ring con-
nected to a derivatized benzene moiety (Table 1);
whereas the group II compounds have a derivatized,
six-membered cyclic aromatic system that is linked to
the central ester function (Table 2).
2.1. In vitro inhibition of SARS-CoV Mpro by non-
peptidyl inhibitors.
The substrate binding sites of SARS-CoV Mpro have
been probed using several classes of inhibitors.9,12,13
Chloromethyl ketones, epoxides, and Michael accep-
tors are among the functional groups known to form
a covalent bond with the nucleophilic sulfur atom of
Cys145, thereby leading to the inactivation of SARS-
CoV Mpro’s enzymatic activity. Similar to the main
proteinases of transmissible gastroenteritis virus
(TGEV) and human coronavirus strain 229E, SARS-
CoV Mpro exhibits well-defined S1, S2, and S4 pockets
that seem to be highly structurally conserved among
coronaviruses.8,12,13 Both in vitro and in vivo studies
show that the residues at the P1, P2, and P4 positions
of the substrate are involved in substrate binding, rec-
ognition, and cleavage.8,14,15 Consequently, the S pock-
ets of SARS-CoV Mpro have been the principal
targeting sites for anti-SARS (or wide-spectrum anti-
coronavirus) drugs.
In general, group I compounds are better inhibitors of
SARS-CoV Mpro than group II compounds as shown
by their lower IC50 values (Tables 1 and 2). Interest-
ingly, the positions of the electron-withdrawing substit-
uents on their terminal aromatic rings seem to influence
the inhibitory efficacy of these compounds dramatically.
For example, the difference in the position of the nitro-
gen atom of the pyridine moiety relative to the carbonyl
function in compounds 6 and 7 is associated with a four-
fold disparity in their IC50 values. The best group I
inhibitors, compounds 1–3, contain either a chloride
or a nitro group at the para position relative to the furan
function. Moving the nitro group of compound 2 to
either the ortho (compound 4) or the meta (compound
5) position led to a threefold and eightfold increase in
their IC50 values, respectively. In a similar manner to
compounds 2, 4, and 5, compounds 8–10 of group II
bear a chloro- or a nitro-substituent on the benzene ring
directly connected to the central ester function at the
para, ortho, and meta position relative to the ester bond,
respectively. The position of the substituents in the latter
three compounds has much less of an impact on the
inhibitory activity against SARS-CoV Mpro, although
the meta-substituted compound 10 is a markedly weaker
inhibitor than either of compounds 8 and 9. Compounds
11 and 12, having terminal conjugated rings, showed
inhibitory activities roughly equivalent to those of the
best group I compounds.
Various experimental techniques and modeling meth-
ods, such as structure-based discovery,16–20 experimen-
tal screening,21–25 and virtual screening,26–33 have been
employed in the search for effective anti-SARS inhibi-
tors. Although the efficacies of some compounds were
verified and characterized by biochemical and/or cell
culture-based assays, the inhibition mechanism remains
unclear for some of them primarily due to the lack of
structural information.
Recently, a large-scale high-throughput screening of
anti-SARS compounds identified 2-(5-chloropyridin-
2.2. Docked conformations of group I compounds
3-yl)-1-(thiophen-2-yl)ethanone (MAC-5576) as
a
Two possible binding modes for compounds 1–5, which
have similar chemical structures except for the different
substituents on the benzene ring, were clearly identified.
The first binding mode (the S2–S1 mode) of the group I
compounds occupies primarily the volume extending
from the S2 to the S1 substrate binding sites (Fig. 1a). Fig-
ure 1b shows the predicted interactions between the en-
zyme and compound 1 in the active site as an example.
The chloropyridine moieties of these inhibitors bind in-
side the S1 pocket in an orientation wherein the pyridine
plane is parallel to the two ‘walls’ of the S1 sites formed
by residues Phe140, Leu141, Asn142, Ser144, and residues
Glu166, His172, respectively. In the docked complexes of
compounds 1, 2, and 5, the common nitrogen atom of the
chloropyridine moieties receives a hydrogen bond from
potent inhibitor for SARS-CoV Mpro with an IC50
value of 0.5 0.3 lM.22 In order to derive even
better anti-SARS-CoV Mpro compounds, we then
synthesized a number of non-peptidic compounds
based on the chemical structure of MAC-5576. Some
of these compounds showed significant improvement
over MAC-5576 in their inhibitory activity.34 In this
paper, we report the in vitro inhibition of SARS-
CoV Mpro by a series of non-peptidic compounds de-
signed on the basis of MAC-5576 (Tables 1 and 2)
and analyze the possible modes of interaction be-
tween this new class of non-peptidyl inhibitors and
SARS-CoV Mpro using molecular docking tools.
Our docking results also allow us to put forward
constructive suggestions for the future design of
anti-SARS Mpro inhibitors.
N
e2 of His163, which is the P1 specificity-determining res-
idue (Fig. 1b). Similarly, the furan moieties of group I

C. Niu et al. / Bioorg. Med. Chem. 16 (2008) 293–302
295
Table 1. Structures and inhibitory activity of a set of non-peptidyl compounds (group I) based on the chemical structure of MAC-5576
c
d
e
f
No. Compound
IC50
DG 1sta
(nM) (kcal/mol) (kcal/mol) (2A5K) (A) (1UK4) (A) (2A5K) (A) (1UK4) (A)
DG 2ndb
D 1stc–s
D 1stc–s
D 2ndc–s
D 2ndc–s
˚
˚
˚
˚
1
63
60
À9.33
À9.34
À9.82
À8.73
À9.33
À9.88
À9.80
À9.84
À9.47
À10.19
4.41
4.51
3.80
3.56
4.66
3.70
3.82
3.10
2.80
3.80
6.88
6.65
6.54
7.12
6.92
5.62
5.48
5.30
5.88
5.68
2
3
4
5
122
208
500
a The binding free energies predicted in the S2–S1 binding mode.
b The binding free energies predicted in the S4–S1 binding mode.
c Distances between the carbonyl carbon of the compounds and the sulfur atom of Cys145 (PDB 2A5K) in the S2–S1 binding mode.
d Distances between the carbonyl carbon of the compounds and the sulfur atom of Cys145 (PDB 1UK4) in the S2–S1 binding mode.
e Distances between the carbonyl carbon of the compounds and the sulfur atom of Cys145 (PDB 2A5K) in the S4–S1 binding mode.
f Distances between the carbonyl carbon of the compounds and the sulfur atom of Cys145(PDB 1UK4) in the S4–S1 binding mode.
inhibitors occupy the same volume between the S1 and S2
pockets and form a few van der Waals interactions with
Met165 (not shown). In contrast, the benzene rings of
the group I inhibitors in the S2–S1 mode show relatively
larger conformational variations. Compounds 1 and 2,
which contain para-substituted benzene rings, can fit into
the S2 pocket well. Compounds 3–5 all contain ortho- or
meta-orienting groups on their benzene rings. This causes
the benzene moieties in these compounds to be pushed
outward from S2 site so as to avoid steric hindrance be-
tween the benzyl substituents and the side chains of
His41 and Met49. The docking models suggest that
hydrophobic interactions are formed between residues
His41, Met49, and Gln189 and the benzene ring moieties,
whereas the main chain NH of Gln189 forms a hydrogen
bond with the ortho-orienting nitro substituent on the
benzene rings.
catalytic residue Cys145 (Table 1 column 6); the car-
bonyl oxygen atoms of the inhibitors point into the oxy-
anion hole of SARS-CoV Mpro such that compounds 1,
3, and 4 receive hydrogen bonds from the main chain
NH of Gly143. Esters are known substrates for chymo-
trypsin-type serine proteases, enzymes that are structur-
ally similar to the catalytic domains of SARS-CoV
M
pro. This suggests that the ester bonds of group I
inhibitors bound in this conformation could be attacked
by the nucleophilic sulfur of Cys145, leading to the for-
mation of an acylated enzyme.
The second binding mode of group I compounds ex-
tends from the S4 to the S1 specificity pockets (the S4–
S1 mode) (Fig. 2a). In a similar fashion to the S2–S1
binding mode, the chloropyridine moieties of group I
inhibitors are also located in the S1 pocket. However,
the orientations of the chloropyridine rings are roughly
perpendicular to those of the chloropyridine rings in the
S2–S1 binding mode. The planes of the rings are parallel
In the S2–S1 binding mode, the ester functions of the
c
˚
inhibitors are approximately 3.5–4.7 A from S of the

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C. Niu et al. / Bioorg. Med. Chem. 16 (2008) 293–302
Table 2. Structures and inhibitory activity of a set of non-peptidyl compounds (group II) based on the chemical structure of MAC-5576
DGa (kcal/mol)
b
Dc–s (2A5K) (A)
c
Dc–s (1UK4) (A)
˚
˚
No.
Compound
IC50 (nM)
6
164
À6.92
4.50
3.03
7
8
697
434
À6.93
À7.49
4.49
4.45
3.02
3.01
9
333
684
À6.79
À7.23
4.43
4.60
3.10
3.15
10
11
12
124
108
À8.41
À8.58
4.14
3.74
2.85
3.38
a The binding free energies predicted in the Cys–S1 binding mode.
b Distances between the carbonyl carbon of the acyclic ester bond and the sulfur atom of Cys145 (PDB 2A5K) in the Cys–S1 binding mode.
c Distances between the carbonyl carbon of the acyclic ester bond and the sulfur atom of Cys145 (PDB 1UK4) in the Cys–S1 binding mode.
Figure 1. (a) The S2–S1 binding mode of group I compounds. The side chain of Met49, which forms the outer lid of the S2 pocket, was removed to
allow clearer visualization of the S2 pocket. (b) Interactions between SARS-CoV Mpro (white ribbon) and compound 1 (yellow carbon sticks) in the
S2–S1 binding mode. Hydrogen bonds are shown as green dashed lines.

C. Niu et al. / Bioorg. Med. Chem. 16 (2008) 293–302
297
Figure 2. (a) The S4–S1 binding mode of group I compounds. (b) Interactions between SARS-CoV Mpro (white ribbon) and compound 5 (yellow
carton sticks) in the S4–S1 binding mode. Hydrogen bonds are shown as green dashed lines.
to the ‘floor’ of the S1 site formed by the side chains of
His163 and His172. The m-chlorine on the pyridine ring
points into the solvent channel at the back side of the S1
pocket, occupying a volume very close to that of a chlo-
ride ion observed in the crystal structure of an unli-
tions of the inhibitors and Sc of Cys145 range from
˚
6.5 to 7.2 A (Table 1 column 8). The inhibitors bound
in this mode are very unlikely to be attacked by the sul-
fur nucleophile of Cys145 due to this long distance con-
straint. Therefore, the S4–S1 binding mode likely
represents a mechanism of competitive inhibition in
which the inhibitors block the activity of SARS-CoV
Mpro by denying entry of substrates into the active site.
Most significantly, this binding hypothesis suggests a
way to improve the design of more potent non-covalent
SARS-CoV Mpro inhibitors that promote tighter S4–S1
binding (discussed in later section).
ganded SARS-CoV Mpro (PDB code 2A5A). In the
35
docked complexes containing compounds 1, 4, and 5,
this chlorine is hydrogen bonded to Oc of Ser144
(Fig. 2b). Residues Cys145 and Met165 form hydropho-
bic interactions with the pyridine rings of these inhibi-
tors. The furan rings in the S4–S1 binding mode are
situated on top of Met165 and Glu166, making a few
van der Waals interactions with these residues. The S4
pocket is large enough to accommodate the benzene
rings of the group I inhibitors despite the presence of
various substituents at different positions on the rings.
Residues Met165, Glu166, Leu167, and Gln189 contrib-
ute mostly hydrophobic interactions with the benzene
rings. The main chain NH of Glu166 forms two hydro-
gen bonds with the oxygen atom on the furan and the
carbonyl oxygen at the b-position of the central ester
bond of group I compounds. In addition, the nitro func-
tion of compound 5 forms a hydrogen bond with Ne2 of
Gln192 (Fig. 2b). In the S4–S1 binding mode, the dis-
tances between the carbonyl carbons of the ester func-
2.3. Docked conformations of group II compounds
The extended end-to-end lengths of group II compounds
are shorter than those of the group I compounds. Con-
sequently, they do not span more than one substrate
binding sites. The most commonly observed binding
mode has an orientation that extended from the cata-
lytic dyad to the S1 pocket (Cys–S1 mode). As with
the S2–S1 binding mode of group I compounds, the
m-chloropyridine rings of the group II inhibitors are
reliably docked inside the S1 site with the m-chlorine
atom pointing to solvent (Fig. 3a). With the derivatized
Figure 3. (a) The Cys–S1 binding mode of group II compounds. (b) Interactions between SARS-CoV Mpro (white ribbon) and compound 12 (yellow
carbon sticks) in the Cys–S1 binding mode. Hydrogen bonds are shown as green dashed lines.

298
C. Niu et al. / Bioorg. Med. Chem. 16 (2008) 293–302
benzene functions of group II compounds docked near
the catalytic residues, the Cys–S1 mode can be viewed
as a partial S2–S1 binding mode (akin to fragment bind-
ing). The aromatic systems on the carbonyl side of the
ester bond form mostly van der Waals contacts with res-
idues Phe140, Leu141, Cys145, Met165, and Glu166.
However, their orientations with regard to the rest of
the compound vary substantially (Fig. 3a).
3. Discussion
The inhibitors used in this study bind to the active site of
SARS-CoV Mpro primarily through hydrophobic con-
tacts. Our docking results clearly show that the 3-chlo-
ropyridine moieties of the ester-based non-peptidyl
inhibitors have a strong propensity to enter the S1 spec-
ificity pocket of SARS-CoV Mpro. Accordingly, the res-
idues forming the S1 pocket play a major part in the
interactions between the inhibitors and SARS-CoV
The hydrogen bonds formed between group II com-
pounds and SARS-CoV Mpro also show polarized pat-
terns. The nitrogen atom of the pyridine ring of
compound 12 is hydrogen bonded to Ne2 of His163,
which also receives a hydrogen bond from the b-car-
bonyl oxygen atoms of other group II compounds. Fur-
thermore, in compound 12, the main chain N of Gly143
forms a bifurcated hydrogen bond to the carbonyl oxy-
gen of the ester bond and to the carbonyl oxygen of the
coumarin moiety, respectively (Fig. 3b).
M
pro. This is significant, as the chloropyridine function
does not resemble the cognate P1-Gln residue in terms
of chemical properties. Consequently, some interactions
between SARS-CoV Mpro and chloropyridine moiety
likely differ from those between P1-Gln and SARS-
CoV Mpro. Further derivatization of the chloropyridine
group has yielded only marginal improvement on the
efficacy of the resultant inhibitors, indicating that our
design may have its maximal potential concerning the
S1 pocket of SARS-CoV Mpro. Since the S1 pockets of
all coronaviral Mpro are structurally conserved and are
similar to those of the picornaviral 3Cpro, the inhibitors
described in this study or at least their basic designs
should prove useful in developing wide-spectrum antivi-
ral compounds. An early indication of that came from
the observation that the parent compound MAC-5576
showed very good inhibitory activity against both the
SARS-CoV Mpro and the HAV 3Cpro with correspond-
ing IC50 values in the high nanomolar range.22
In the Cys–S1 binding mode of the group II inhibitors,
the average distance between the carbonyl carbon
atoms of the central ester bonds and the sulfur atom
˚
of Cys145 is about 3.7–4.6 A. Compared to those
inhibitors of group I in the S4–S1 binding mode, the
ester bonds in the group II inhibitors are placed much
closer to the catalytic dyad; the carbonyl oxygen atoms
of the central ester point into the oxyanion hole.
Therefore, the group II compounds bound in the
Cys–S1 mode are potentially exposed to higher risk
of enzymatic hydrolysis.
A second ‘hotspot’ that could be targeted by anti-SARS-
CoV Mpro compounds is residue His41. His41 plays the
dual role of activating Sc of Cys145 during the catalytic
cycle as a general base as well as forming part of the S2
specificity pocket. In the S2–S1 binding mode, His41,
together with Met165 and Glu166, forms more than half
of the total hydrophobic interactions with the group I
inhibitors (Table 3). Met165 and Glu166 form the ‘wall’
of the S2 pocket opposite to that of His41; these residues
are also major contributors of hydrophobic interactions
in the S4–S1 and the Cys-S1 binding modes (Tables 4
and 5).
2.4. Binding free energy and inhibitor efficacy
The estimated free energies of binding (DG) between the
enzyme and the 12 inhibitors were predicted by Auto-
Dock based on the new scoring function, which was
developed using the AMBER force field and a set of
coefficients.36 Although AutoDock cannot provide an
absolute binding free energy, these predicted DGs can
be used for approximate ranking of the inhibitors
according to their predicted binding affinities. For group
I compounds, there are no significant differences in the
binding free energies between the S2–S1 mode (DG 1st)
and the S4–S1 binding mode (DG 2nd) (Table 1). This
suggests that group I compounds have comparable ten-
dencies to adopt either the S4–S1 or the S2–S1 confor-
mation prior to their possible covalent modification of
SARS-CoV Mpro enzyme. The binding free energies
for group II compounds are significantly higher than
those of group I compounds, with the exception of com-
pounds 11 and 12. This is in agreement with the general
trend that SARS-CoV Mpro is more strongly inhibited
by group I than group II compounds. Compounds 11
and 12 show binding free energies closer to those exhib-
ited by the group I compounds, probably because the
larger aromatic stabilization effects of the naphthalene
moiety of compound 11 and the coumarin moiety of
compound 12 make their central ester bonds less
susceptible to nucleophilic attack by Sc of Cys145.
Correspondingly, these two compounds exhibit better
anti-SARS-CoV Mpro activity than other group II
inhibitors.
The distances between Sc of Cys145 and the carbonyl
carbon atoms in the ester functions of group II inhibi-
Table 3. Numbers of hydrophobic contacts between residues of
SARS-CoV Mpro and group I compounds in the S2–S1 binding mode
Residues
Number of hydrophobic contacts
1
2
3
4
5
His41
10
5
9
6
3
4
2
1
4
7
8
5
3
3
3
2
3
3
4
4
3
3
4
2
3
3
5
1
3
4
1
1
3
7
Met49
Phe140
Leu141
Cys145
His163
Met165
Glu166
His172
Arg188
Gln189
2
2
3
2
4
7
1
2
6
2
Total
36
29
30
28
33

C. Niu et al. / Bioorg. Med. Chem. 16 (2008) 293–302
299
Table 4. Numbers of hydrophobic contacts between residues of
SARS-CoV Mpro and group I compounds in the S4–S1 binding mode
Residues
Number of hydrophobic contacts
1
2
3
4
5
Leu141
Asn142
Cys145
Met165
Glu166
Leu167
Pro168
Gln189
Gln192
1
1
1
2
1
1
12
5
10
2
11
4
10
2
13
3
5
7
5
4
5
1
1
1
1
3
3
2
4
3
2
1
Total
27
26
28
21
25
Table 5. Numbers of hydrophobic contacts between residues of
SARS-CoV Mpro and group II compounds in the Cys–S1 binding
mode
Residues
Number of hydrophobic contacts
6
7
8
9
10
11
12
His41
2
2
2
2
6
3
2
3
1
3
1
Met49
Phe140
Leu141
Asn142
Cys145
His163
His164
Met165
Glu166
His172
2
3
2
2
2
2
2
2
2
2
2
3
3
1
2
3
6
1
3
1
2
3
6
1
3
1
1
3
6
1
3
1
2
1
7
6
4
7
8
6
1
3
Figure 4. (a) Alignment of SARS-CoV Mpro crystal structures: 1UK4
(pink) and 2A5K (white). APE and the catalytic residues of His41 and
Cys145 are shown in the stick mode. Residues Met49 and Gln192-
Gly195 are shown in lines to illustrate the structural plasticity in the S2
and S4 pockets. The structural changes in these pockets involve both
the side chain and main chain atoms in the residues involved. (b) The
binding conformation of APE in the active site of SARS-CoV Mpro
(2A5K). APE is shown in the ball and stick mode. Oxygen, nitrogen
and carbon atoms are shown in red, blue, and gray, respectively. Color
of surface shows the cavity depth from the exterior (blue) to the
interior (yellow) of protein.
Total
23
22
21
17
18
26
22
tors are significantly shorter than those between the
nucleophilic sulfur and the corresponding atoms of
group I inhibitors in the S4–S1 binding mode (Table 1
column 8 and Table 2 column 5). It is worth mentioning
that the model structure used in docking is that of a
SARS-CoV Mpro covalently modified at Sc of Cys145.
The position of the nucleophilic sulfur atom, relative
to the other active site residues, has shifted significantly
from that of Sc in the unliganded enzyme structures
(PDB codes, e.g., 1UK4 or 2A5A) (Fig. 4a). Our recent
X-ray crystallographic analyses of SARS-CoV Mpro in
complex with some peptidyl inhibitors indicate that
the formation of tetrahedral intermediates during sub-
strate hydrolysis can take place without the requirement
of the previously observed conformational changes in
Cys145.37 Consequently, we aligned the 1UK4 structure
onto the docking molecule 2A5K to obtain the alternate
coordinates of the nucleophilic sulfur, which should rep-
resent the position of Sc in its resting state and when the
substrate/inhibitor first approaches. The distances
involving Sc of Cys145 were recalculated. It is clear that
the carbonyl carbon atoms of the group II compounds
are closer to Sc of Cys145 than those of the group I com-
pounds (Table 2 column 6). Note that some of the calcu-
lated distances (for group II compounds and for
compounds 3 and 4 in the S2–S1 binding mode) are
shorter than the sum of van der Waals radii of carbonyl
oxygen and thiol sulfur atoms, suggesting a strong pro-
pensity for the covalent attachment of these inhibitors to
Sc of Cys145.
In summary, our docking study provides a reasonable
structural interpretation for the in vitro activities of a
new class of non-peptidyl anti-SARS-CoV Mpro inhibi-
tors. It suggests that a chloropyridine function in the
context of an ester compound specifically binds to the
S1 substrate binding site of SARS-CoV Mpro. The inter-
actions between the S1 site of SARS-CoV Mpro and the
chloropyridine functions likely play an important role in
the initial binding of our inhibitors to the enzyme
in vitro, whereas the rate of subsequent water-mediated
hydrolysis of the resultant acyl enzyme depends on the
chemical properties of the leaving groups on the car-
bonyl side of the ester bond. It is worth mentioning that
additional docking studies indicate that the chloropyri-
dine function of these compounds likely targets the S1
site of HAV 3Cpro as well (data not shown). This corre-
lates well with the high structural similarity in the active
sites of SARS-CoV Mpro and HAV 3Cpro
.

300
C. Niu et al. / Bioorg. Med. Chem. 16 (2008) 293–302
The information obtained through our structural analy-
ses may be incorporated in the future design of more
potent anti-SARS-CoV Mpro inhibitors. Due to the
hydrolyzability of the ester bonds in these compounds,
their inhibitory effect on SARS-CoV Mpro is likely to in-
volve a covalent attachment of the inhibitors to the
nucleophilic cysteine Cys145. Further modifications
(individually or in combination) on the basis of com-
pound 1 may improve its efficacy of inhibition against
SARS-CoV Mpro and/or other structurally similar viral
proteinases.
crystal structures of SARS-CoV Mpro deposited in Pro-
tein Data Bank (PDB)38 were first aligned by a least-
square fit algorithm in the Sybyl 7.1 package (Tripos
Inc., St. Louis, USA). The conformations of the S2
and S4 pockets clearly showed structural variations
among these structures (Fig. 4a, for clarity, only two
models were overlaid). The depths of the S2 and S4
pockets could affect docking results dramatically sim-
ply because larger inhibitors will not be docked into
the smaller pockets. The crystal structure of SARS-
CoV Mpro in complex with an aza-peptide epoxide
(APE)35 (PDB code: 2A5K, chain A) was chosen to
construct the predictive model for this series of inhibi-
tors. The benzene rings in the P2 and P4 positions of
APE were accommodated cozily in the S2 and S4
pockets as a result of the relatively larger size of these
pockets in available crystal structures of SARS-CoV
Mpro (Fig. 4a and b). Therefore, using this structure
of the enzyme is, in principle, to allow the inhibitors
to adopt conformations that would maximize the inter-
actions between the inhibitors and the S2 and S4 sites
of the enzyme. The coordinates of the inhibitor, APE
and the solvent molecules were first removed from
the corresponding PDB file. Then essential hydrogen
atoms and Kollman united atom charges were added
to the protein structure using the molecular modeling
software, Sybyl version 7.1 (Tripos Inc., St. Louis,
USA). Fragmental volumes and atomic solvation
parameters that are important for computing the bind-
ing free energies were assigned using the ADDSOL
module of the AutoDock36 program.
1. The ester bond could be replaced by other functional
groups that react with Cys145. This should enhance
the inhibitory effect of inhibitors more prone to take
on the S2–S1 or Cys–S1 conformations. Possible can-
didates for the reactive function are: epoxides,
Michael acceptors, and azides.
2. Side chain improvement aimed at increasing the inter-
actions between the S2 or S4 sites and the inhibitor
would create a mode of inhibition by virtually deny-
ing the entry of peptidyl substrates. A bifurcated
modification diverging at the furan ring could, in
principle, involve both the S2 and S4 sites in the bind-
ing of the inhibitor to the enzyme, thereby maximiz-
ing their interactions.
4. Materials and methods
4.1. Syntheses of compounds and in vitro characterization
of their inhibitory effects on SARS-CoV Mpro
4.3. Preparation of inhibitor structures
The chemical syntheses and in vitro characterization of
the compounds described in this study were done essen-
tially as reported previously.34 In summary, the pyridinyl
esters were prepared through parallel synthesis, by the
coupling reactions between 5-chloro-3-pyridinol and
commercially available carboxylic acids using method A
or B. Briefly, in method A, EDCI (0.5 mmol, 1.0 equiv),
HOBt (0.5 mmol, 1.0 equiv), DIPEA (0.5 mmol,
1.0 equiv), and 5-chloro-3-pyridinol (0.5 mmol, 1.0 equiv)
were added to a solution of carboxylic acid (0.5 mmol,
1.0 equiv) in DMF (2 mL) at RT. After 24 h of stirring,
the solvent was removed in vacuo to afford the crude
mixture. In method B, to a solution of carboxylic acid
(1 mmol, 1.0 equiv) in DCM (5 mL) at RT was added
thionyl chloride (2.6 mmol, 2.6 equiv) dropwise and a
catalytic amount of DMF (2 drops). After overnight
stirring, the solvent was removed in vacuo to afford
the acyl chloride product. A solution of the acyl chloride
in DCM (5 mL) was then added dropwise to a solution
of 5-chloro-3-pyridinol (1 mmol, 1.0 equiv) and pyridine
(0.09 mL, 1.1 equiv) in DCM (5 mL) at 0 ꢁC. After 3 h
of stirring at RT, the solvent was removed in vacuo to
afford the crude mixture. The crude mixtures obtained
from both methods A and B were purified by HPLC–
MS automated instrument.
The 3D structures of the studied compounds were con-
structed using Sybyl 7.1 (Tripos Inc., St. Louis, USA).
Hydrogen atoms were added to the inhibitor structures
and partial atomic charges were calculated using the
Gasteiger–Marsili method.39 A geometry optimization
was performed by applying the Tripos force field in Syb-
yl 7.1 (Tripos Inc., St. Louis, USA). All possible rotat-
able bonds in the compounds were assigned using the
program AutoTors in AutoDock.36 This allowed search-
ing for flexible conformations of the compounds during
the docking process.
4.4. Molecular docking
The docking program AutoDock 3.0.536 was used to
perform the automated molecular docking. The
Lamarckian genetic algorithm (LGA) was applied to
deal with the inhibitor–enzyme interactions. The grid
˚
map with 60 · 60 · 60 points spaced equally at 0.375 A
was generated using the AUTOGRID program to eval-
uate the binding energies between the compounds and
the protein. Docking parameters were set to default val-
˚
ues except for the step size for translation (0.2 A) for the
orientation and torsion angles (5ꢁ), the number of
generations (37,000), and the energy evaluations
(1,500,000). The docked inhibitor–enzyme complexes
were ranked according to the predicted binding energies
and to the conformity to ideal geometry of the docked
structures.
4.2. Choice and preparation of protein structures
To choose rationally a conformation of the SARS-CoV
Mpro enzyme for the docking experiments, several

C. Niu et al. / Bioorg. Med. Chem. 16 (2008) 293–302
301
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Acknowledgments
We thank Ting-Wai Lee (University of Alberta) for
helpful discussions. This work has been supported by
the Alberta Heritage Foundation for Medical Research
(AHFMR), the Canadian Institutes of Health Research
(CIHR) and the Natural Sciences and Engineering
Research Council of Canada and the Canada Research
Chairs Program. J.Y. is a recipient of the postdoctoral
fellowships from Izaak Walton Killam Foundation
and AHFMR.
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