Peptide aldehyde inhibitors challenge the substrate specificity of the SARS-coronavirus main protease

Article abstract of DOI:10.1016/j.antiviral.2011.08.001

SARS coronavirus main protease (SARS-CoV M^pro) is essential for the replication of the virus and regarded as a major antiviral drug target. The enzyme is a cysteine protease, with a catalytic dyad (Cys-145/His-41) in the active site. Aldehyde i

Full text of DOI:10.1016/j.antiviral.2011.08.001

Antiviral Research 92 (2011) 204–212
Contents lists available at SciVerse ScienceDirect
Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral
Peptide aldehyde inhibitors challenge the substrate specificity
of the SARS-coronavirus main protease
Lili Zhu a, Shyla George a,1, Marco F. Schmidt b,2, Samer I. Al-Gharabli c, Jörg Rademann b,d
,
Rolf Hilgenfeld a,e,f,
⇑
a Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany
b Leibniz Institute for Molecular Pharmacology (FMP), Robert-Rössle-Strasse 10, 13125 Berlin, Germany
c German-Jordanian University, Chemical Pharmaceutical Engineering Department, P.O. Box 35247, Amman 11180, Jordan
d Institute of Pharmacy, Leipzig University, Brüderstraße 34, 04103 Leipzig, Germany
e Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China
f Laboratory for Structural Biology of Infection and Inflammation, c/o DESY, Building 22a, Notkestr. 85, 22603 Hamburg, Germany
a r t i c l e i n f o
a b s t r a c t
SARS coronavirus main protease (SARS-CoV Mpro) is essential for the replication of the virus and regarded
as a major antiviral drug target. The enzyme is a cysteine protease, with a catalytic dyad (Cys-145/His-41)
Article history:
Received 1 July 2011
Accepted 3 August 2011
Available online 11 August 2011
in the active site. Aldehyde inhibitors can bind reversibly to the active-site sulfhydryl of SARS-CoV Mpro
.
Previous studies using peptidic substrates and inhibitors showed that the substrate specificity of SARS-
CoV Mpro requires glutamine in the P1 position and a large hydrophobic residue in the P2 position. We
determined four crystal structures of SARS-CoV Mpro in complex with pentapeptide aldehydes (Ac-EST-
LQ-H, Ac-NSFSQ-H, Ac-DSFDQ-H, and Ac-NSTSQ-H). Kinetic data showed that all of these aldehydes exhi-
Keywords:
Aldehyde inhibitor
Antiviral drug design
Cysteine protease
bit inhibitory activity towards SARS-CoV Mpro, with Ki values in the
lM range. Surprisingly, the X-ray
structures revealed that the hydrophobic S2 pocket of the enzyme can accommodate serine and even
aspartic-acid side-chains in the P2 positions of the inhibitors. Consequently, we reassessed the substrate
specificity of the enzyme by testing the cleavage of 20 different tetradecapeptide substrates with varying
amino-acid residues in the P2 position. The cleavage efficiency for the substrate with serine in the P2
position was 160-times lower than that for the original substrate (P2 = Leu); furthermore, the substrate
with aspartic acid in the P2 position was not cleaved at all. We also determined a crystal structure of
SARS-CoV Mpro in complex with aldehyde Cm-FF-H, which has its P1-phenylalanine residue bound to
the relatively hydrophilic S1 pocket of the enzyme and yet exhibits a high inhibitory activity against
Methionine–aspartic acid interaction
X-ray crystallography
SARS-CoV Mpro, with Ki = 2.24 0.58
lM. These results show that the stringent substrate specificity of
the SARS-CoV Mpro with respect to the P1 and P2 positions can be overruled by the highly electrophilic
character of the aldehyde warhead, thereby constituting a deviation from the dogma that peptidic inhib-
itors need to correspond to the observed cleavage specificity of the target protease.
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
SARS coronavirus main protease (SARS-CoV Mpro) is essential
Abbreviations: SARS-CoV Mpro
, SARS coronavirus main protease; Cm-FF-H,
for the replication of the virus and regarded as a major antiviral
drug target (Anand et al., 2003; Yang et al., 2003; Tan et al.,
2005; Steuber and Hilgenfeld, 2010). Peptide aldehyde inhibitors
are widely used as research tools to characterize the substrate
specificity of serine and cysteine proteases. In complex with the
crystalline target enzyme, they provide a wealth of information
on the specificity-defining subsites of the substrate-binding site.
For this purpose, we have previously described peptide aldehydes
as inhibitors of the SARS-coronavirus main protease (SARS-CoV
Mpro) (Al-Gharabli et al., 2006). A major advantage of aldehydes
over other peptide electrophiles is their reversible binding to the
Cinnamoyl-Phe-Phe-H; Boc, tert-butyl oxycarbonyl; IBX, 2-iodoxybenzoic acid;
FRET, fluorescence resonance energy transfer; PEG, polyethylene glycol; MPD, 2-
methyl-2,4-pentanediol; MES, 2-(N-morpholino)ethanesulfonic acid; DMSO,
dimethylsulfoxide.
Corresponding author at: Institute of Biochemistry, University of Lübeck,
Ratzeburger Allee 160, 23538 Lübeck, Germany. Tel.: +49 451 500 4060; fax: +49
451 500 4068.
E-mail address: hilgenfeld@biochem.uni-luebeck.de (R. Hilgenfeld).
Present address: Aurigene Discovery Technologies Ltd., #39&40, KIADB Industrial
Area, Electronic City Phase-2, Hosur Road, Bangalore 560100, India.
⇑
1
2
Present address: University Chemical Laboratory, University of Cambridge,
Lensfield Road, Cambridge CB2 1EW, UK.
0166-3542/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.antiviral.2011.08.001

L. Zhu et al. / Antiviral Research 92 (2011) 204–212
205
active-site sulfhydryl of cysteine proteases. We have previously
product with cinnamoyl chloride to provide N-cinnamoyl-
L-phenyl-
used this property to screen a small library of non-peptide nucleo-
philes (mostly amines) for molecules that would efficiently en-
hance the inhibition of the aldehyde by formation of a stronger
binding aldehyde-nucleophile ligation product. In a second step
of this process that we named ‘‘Dynamic Ligation Screening’’
(Schmidt et al., 2008), the most efficient compound in this process
had its amino group replaced by an aldehyde moiety and was re-
acted with the target enzyme, and the resulting covalent adduct
was subsequently used to screen the same library of nucleophiles
for those that would react with the aldehyde. This way, starting
alanyl-L-phenylalanine methyl ester. Cm-FF-H was then obtained
by reduction of the ester with NaBH4/CaCl2 and alcohol oxidation
with IBX.
2.3. Enzyme kinetics
The recombinant production and purification of SARS-CoV Mpro
with authentic N and C termini were performed as described pre-
viously (Xue et al., 2007; Verschueren et al., 2008). The substrate
Dabcyl-KTSAVLQ;SGFRKME-(Edans)-amide (95% purity; Biosyntan
GmbH, Berlin, Germany), which contains a main-protease cleavage
site (indicated by the arrow), was used as the substrate in the fluo-
rescence resonance energy transfer (FRET)-based cleavage assay.
The substrate stock was prepared by dissolving 1 mM of the pep-
tide in DMSO. The dequenching of the Dabcyl fluorescence due to
the cleavage of the substrate as catalyzed by the SARS-CoV Mpro
was monitored at 490 nm with excitation at 340 nm, using a Cary
Eclipse fluorescence spectrophotometer. The experiments were
performed in the buffer consisting of 20 mM Tris-HCl (pH 7.3),
100 mM NaCl, and 1 mM EDTA. The reaction was initiated by add-
from a substrate-like peptide aldehyde, we obtained a low-lM
non-peptidic, reversible inhibitor from two fragments which by
themselves had no or only very low inhibitory activity (Schmidt
et al., 2008).
Interestingly, it turned out that some peptide aldehydes with an
amino-acid sequence deviating from the consensus sequence
embracing the cleavage sites of polyprotein substrates were sur-
prisingly efficient as inhibitors. In particular, peptide aldehydes
with P2 = Asp or Ser inhibited the main protease with a relatively
low IC50 (Al-Gharabli et al., 2006), although it is generally agreed
that the S2 specificity subsite of the enzyme has a strong prefer-
ence for large hydrophobic side-chains such as Leu, Phe, or Met
(Fan et al., 2004, 2005; Lai et al., 2006). We therefore assumed that
the hydrophilic P2 side-chain of these inhibitors would be oriented
towards the solvent, rather than occupy the S2 pocket, and that the
hydrophobic P3 residue would be binding to that pocket
(Al-Gharabli et al., 2006; Schmidt et al., 2008). This model was
based on a crystal structure of a peptidyl chloromethyl ketone
bound to the SARS-CoV main protease, in which this arrangement
had been observed (Yang et al., 2003). Here we show by X-ray crys-
tallography of a number of peptide aldehyde complexes with the
protein that this is not the case. Instead, the hydrophilic Ser or
Asp residues in the P2 position bind to the hydrophobic S2 subsite.
In order to understand these binding modes, the atomic interac-
tions were analyzed in detail. Also, we report the inhibition kinet-
ics of these compounds and re-evaluate the cleavage specificity of
the enzyme as far as the P2 position is concerned. In addition, we
determined a crystal structure of SARS-CoV Mpro in complex with
the aldehyde Cm-FF-H (cinnamoyl-Phe-Phe-H), which has a phen-
ylalanine residue in the P1 position and exhibits high inhibitory
ing different final concentrations of the FRET peptide (10–50
l
M)
solution containing SARS-CoV Mpro (final concentration
M). Kinetic constants (Vmax and Km) were derived by fitting
to
a
0.5
l
the data to the Michaelis–Menten equation, V = Vmax ꢀ [S]/
(Km + [S]). Then kcat was calculated according to the equation,
kcat = Vmax/[E]. In order to estimate the population of catalytically
active Mpro dimers, the respective monomer and dimer concentra-
tions were calculated according to Graziano et al. (2006). The
dimerization of the SARS-CoV Mpro follows the scheme
M þ MꢀD
The equilibrium dissociation constant, KD, is defined by
2
½Mꢁ
KD
¼
;
½Dꢁ
where [M] and [D] are the molar concentrations of the monomer
and dimer, respectively. The total protein concentration [MT] ex-
pressed in terms of molar monomer equivalents is
½MꢁT ¼ ½Mꢁ þ 2½Dꢁ:
Since
activity against SARS-CoV Mpro, with Ki = 2.24 0.58
lM. These re-
sults show that the stringent substrate specificity of the SARS-CoV
Mpro with respect to the P1 and P2 positions can be overruled by
the highly electrophilic character of the aldehyde warhead.
½MꢁT ꢂ ½Mꢁ
½Dꢁ ¼
;
2
substituting the above equation into the expression for the KD yields
2. Materials and methods
2
2½Mꢁ
KD
¼
:
½MꢁT ꢂ ½Mꢁ
2.1. Synthesis of aldehydes with various P2 residues
Solving the above quadratic equation for [M] gives
Chemical synthesis of peptide aldehydes Ac-ESTLQ-H,
Ac-NSFSQ-H, Ac-DSFDQ-H, and Ac-NSTSQ-H was performed
employing a solid-state method (Al-Gharabli et al., 2006). Briefly,
protected glutamine aldehyde obtained by racemization-free oxi-
dation of the corresponding amino alcohol with Dess–Martin peri-
odinane was immobilized on a threonyl resin as oxazolidine.
Following N-tert-butyl oxycarbonyl (Boc)-protection of the ring
nitrogen to yield the N-protected oxazolidine linker, peptide syn-
thesis was performed on the resin.
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ꢂKD
þ
K2D þ 8½Mꢁ KD
T
½Mꢁ ¼
:
4
It then follows that
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
KD þ 4½Mꢁ ꢂ K2D þ 8½Mꢁ KD
T
T
½Dꢁ ¼
:
8
2.4. Aldehyde inhibition assay
2.2. Synthesis of Cm-FF-H (Scheme 1)
Aldehyde stock solutions were prepared by dissolving the com-
pounds in DMSO at 1 mM. All aldehydes form covalent bonds be-
tween the aldehyde (–CHO) group of the inhibitor and the
sulfhydryl (–SH) group of Cys145 of SARS-CoV Mpro. The binding
Synthesis of Cm-FF-H was carried out by amidation of
Boc-L-phenylalanine with L-phenylalanine methyl ester followed
by deprotection of the Boc group and acylation of the corresponding

206
L. Zhu et al. / Antiviral Research 92 (2011) 204–212
Scheme 1. Synthesis of Cm-FF-H. Reagents and conditions: (a) EDCI, HOBt, Boc-Phe-OH, DMF, 0–25 °C, 18 h, 78%; (b) (i) TFA, CH2Cl2, 0–25 °C, 2 h; (ii) cinnamoyl chloride, N-
methylmorpholine, THF, DMF, 0–25 °C, 12 h, 50%; (c) NaBH4, CaCl2, EtOH, THF (1:1), 0–25 °C, 14 h, 35%; (d) IBX, DMSO, 0–25 °C, 5 h, 47%.
of these inhibitors is reversible (Schmidt et al., 2008), although it is
strong. Therefore, they were treated as reversible tight-binding
inhibitors. For the measurement of the inhibition constant Ki,
SARS-CoV Mpro was incubated with the aldehyde inhibitor in reac-
tion buffer at room temperature for 30 min. Then the FRET peptide
substrate, Dabcyl-KTSAVLQ;SGFRKME-Edans, was added and the
reaction velocity was calculated according to substrate cleavage.
10 mM. Crystals of the aldehyde complexes of SARS-CoV Mpro were
obtained either by adding a 4- l aliquot of aldehyde solution to the
l
drop and soaking of the crystals for 12 h, or by incubating the
enzyme for 2 h at 20 °C with a 7-fold excess of the aldehyde solu-
tion and subsequent cocrystallizing at 20 °C against a reservoir
containing 8% PEG 6000, 0.1 M MES (pH 6.0), 3% MPD, and 3%
DMSO. In the latter case, nucleation was initiated by microseeding
using crushed monoclinic (space group C2) crystals of the SARS-
CoV Mpro. Prior to diffraction data collection, the aldehyde complex
crystals obtained from soaking and cocrystallization were trans-
ferred for a few seconds to a cryoprotectant solution containing
the crystallization ingredients and 20% MPD.
Values of the intrinsic (V0i ) and apparent (Vapp, Kai pp) catalytic
i
parameters for SARS-CoV Mpro catalyzing the hydrolysis of peptide
substrate were determined in the absence and presence of
aldehyde, respectively. The apparent inhibition constants (Kiapp
)
for aldehyde binding to SARS-CoV Mpro were obtained from the
dependence of Vai pp on the inhibitor concentration ([I]) at fixed
substrate concentration ([S]), according to the equation Viapp
¼
2.7. Crystallographic data collection and processing, and structure
elucidation and refinement
Vai pp ꢀ ½IꢁÞ=Kai pp þ ½IꢁÞ (Ascenzi et al., 1987; Copeland, 2000). Values
of the intrinsic inhibition constant (Ki) for aldehyde binding to
SARS-CoV Mpro were calculated according to the equation Kiapp = Ki
ꢀ (1 + [S]/Km) (Ascenzi et al., 1987; Copeland, 2000).
All diffraction data were collected at 100 K at the Joint EMBL/
University of Hamburg/University of Lübeck synchrotron beamline
X13 at DESY (Hamburg, Germany), using a 165-mm MAR CCD
detector (Mar Research, Hamburg, Germany), or at synchrotron
beamline BL14.1 at BESSY (Berlin, Germany), using an MX225
CCD detector (Rayonics, Evanston, IL). Statistics of data collection,
processing, and refinement are summarized in Table 1. Data were
processed with MOSFLM (Leslie, 1992), and scaled using the SCALA
program from the CCP4 suite (Collaborative Computational Project,
1994; Evans, 2006).
Structure elucidation and refinement were carried out using the
CCP4 suite of programs (Collaborative Computational Project,
1994; Potterton et al., 2003). Crystal structures were determined
by molecular replacement, using the original X-ray structure of
the SARS-CoV Mpro with authentic chain termini (PDB ID: 2H2Z)
(Xue et al., 2007) as the initial model. REFMAC (Murshudov et al.,
1997) was employed for structure refinement. The computer
graphics program Coot (Emsley and Cowtan, 2004) was used for
interpretation of electron density maps and model building. Gap
volumes between inhibitor and protein in the subsites of the
enzyme were calculated using UCSF-Chimera (Pettersen et al.,
2004). The molecular graphics package PyMOL (DeLano, 2002)
was used to generate the figures.
2.5. Peptide cleavage assay
Twenty peptide substrates harboring alternative amino-acid
residues in P2 (=X; SWTSAVXQ;SGFRKWA) were purchased from
GL Biochemistry Ltd. (Shanghai, China). These peptides correspond
to the N-terminal autocleavage site of the SARS-CoV Mpro, with
the exception of the P7 Ile which had been replaced by Trp, and
the P60 Met which had also been replaced by Trp. All peptide sub-
strate stocks were dissolved in DMSO at 10 mM. To determine the
kcat/Km for the substrate, 0.2 mM substrate peptide was incubated
with 10 lM SARS-CoV Mpro in 40 mM Tris-HCl buffer, pH 7.3. Ali-
quots of reactions were removed at different times, stopped by the
addition of 1% trichloroacetic acid. Separation of products and sub-
strate was carried out using a reverse-phase (RP) HPLC and a linear
gradient (1–90%) of acetonitrile in 0.1% trifluoroacetic acid. The
absorbance was determined at 280 nm, and peak areas were calcu-
lated by integration. The (kcat/Km)app ratio was determined by plot-
ting the substrate peak area using the equation lnPA = C ꢂ (kcat
/
Km)app ꢀ CE ꢀ t, where PA is the peak area of the substrate peptide,
CE is the total concentration of SARS-CoV Mpro, and C is an experi-
mental constant (Fan et al., 2004, 2005).
3. Results and discussion
2.6. Crystallization of the complexes
3.1. Inhibition of SARS-CoV Mpro by peptide aldehydes
SARS-CoV Mpro with authentic chain termini was concentrated
to 10 mg/ml and crystallized by vapor diffusion using sitting drops
(Xue et al., 2007). The crystals grew overnight at 20 °C by equilibra-
tion against a reservoir containing 6–8% polyethylene glycol (PEG)
6000, 0.1 M MES (pH 6.0), 3% 2-methyl-2,4-pentanediol (MPD),
and 3% DMSO. All aldehydes were dissolved in 8% PEG 6000, 0.1
M MES (pH 6.0), 3% MPD, and 10% DMSO to a concentration of
The kinetic parameters of SARS-CoV Mpro with authentic chain
termini were determined using the FRET substrate Dabcyl-
KTSAVLQ;SGFRKME-Edans amide. The Km value for this substrate
was determined as 24.5 lM and the apparent kcat/Km value was
2359 Mꢂ1ꢃ19 sꢂ1. However, the dissociation of the catalytically
active SARS-CoV Mpro dimer into inactive monomers (Fan et al.,

L. Zhu et al. / Antiviral Research 92 (2011) 204–212
207
Table 1
Data collection and refinement statistics.
Inhibitor
Ac-ESTLQ-H
Ac-DSFDQ-H
Ac-NSTSQ-H
Ac-NSFSQ-H
Ac-ESTLQ-H
Cm-FF-H
Data collection statistics
Wavelenth (Å)
Method
0.8123
Soaking
C2
0.8123
Soaking
C2
0.8123
Soaking
C2
0.8123
Soaking
C2
0.8123
Cocrystallization
P21
0.9184
Soaking
C2
Space group
Unit cell dimensions (Å, °)
108.91,
81.36,
53.40
b = 104.35
1
108.74,
81.86,
53.21
b = 104.26
1
107.83,
82.46,
53.32
b = 104.65
1
107.87,
82.09,
53.08
b = 104.30
1
52.37,
96.79,
68.07
b = 102.49
2
107.75,
82.88,
53.50
b = 104.63
1
NPa
Resolution range (Å)
Number of reflections
Redundancyb
32.21–2.60
13.957
3.8(3.8)
99.9(100.0)
10.2(45.6)
8.7(2.4)
32.32–2.40
16.009
3.4(2.8)
91.3(85.7)
10.4(25.7)
5.8(2.3)
26.58–2.58
13.938
3.0(2.7)
96.7(82.5)
6.0(32.0)
10.1(2.8)
64.56–3.05
8643
3.4(3.4)
99.9(100.0)
8.3(47.2)
10.2(2.3)
27.29–1.89
50.901
2.8(2.6)
94.5(78.3)
6.7(33.6)
10.3(2.7)
32.82–1.99
31.388
3.6(3.4)
99.7(98.1)
17.0(36.6)
4.6(2.2)
Completeness (%)b
Rmerge (%)b
I/r
(I)b
Refinement statistics
R/Rfree (%)
18.5/22.5
21.2/23.9
19.8/26.0
19.0/25.5
18.7/23.8
21.7/26.9
r.m.s deviation from idea geometry
Bonds (Å)
Angles (°)
0.010
1.535
0.009
1.162
0.017
1.820
0.015
1.661
0.009
1.172
0.018
1.689
Ramachandran plot
Most favored (%)
Allowed (%)
Generously allowed (%)
Disallowed (%)
88.0
10.9
0.4
90.3
8.6
0.7
83.9
14.2
1.1
80.6
18.3
0.4
90.6
8.3
0.4
89.8
9.1
0.8
0.7
0.4
0.7
0.8
0.8
0.4
PDB ID
3SNE
3SNB
3SNC
3SNA
3SND
3SN8
a
Number of protein molecules per asymmetric unit.
Numbers in parentheses are for the outermost resolution shell.
b
2004) has to be taken into account. A wide range of KD values has
been reported for Mpro dimer dissociation (see Grum-Tokars et al.
(2008) for an overview). For the enzyme with authentic chain ter-
of the crystal. The four pentapeptide aldehydes Ac-ESTLQ-H, Ac-
NSTSQ-H, Ac-DSFDQ-H, and Ac-NSFSQ-H are bound in extended
conformations in the S6–S1 specificity subsites of SARS-CoV Mpro
.
mini, the latter authors reported a KD of 0.25 to 1.0
l
M. As the Mpro
Cm-FF-H occupies sites S3–S1. Remarkably, the P1 phenylalanine
side chain of this inhibitor is bound deeply in the S1 pocket, which
is generally considered to be specific for glutamine. 2Fo ꢂ Fc elec-
tron density maps of these aldehyde inhibitors are shown in
Fig. 1. In all complexes, continuous electron density between the
dimer tends to be stabilized by the presence of substrate (Cheng
et al., 2010), we used the lower limit of this range for estimation
of the necessary corrections and obtained a kcat/Km value of
7863 Mꢂ1 sꢂ1. The Km value determined in our study is of the same
magnitude as other values derived from data not corrected for dis-
aldehyde carbonyl C-atom of the inhibitor and Cys145-S
c of
sociation, which are typically in the range from 10 to 50
lM
SARS-CoV Mpro (Fig. 1) indicates the formation of a thiohemiacetal,
as a result of the nucleophilic attack of the catalytic cysteine onto
the C-terminal aldehyde of the inhibitor. The main-chain confor-
mations of SARS-CoV Mpro in the five complexes are basically iden-
tical, with overall root mean-square deviations (RMSD) of
(Grum-Tokars et al., 2008). This suggests that much higher values
reported previously (e.g., Verschueren et al., 2008) should probably
be reconsidered.
Initially, four pentapeptide substrate-analogous aldehydes con-
taining the canonical P1 residue, glutamine, namely Ac-ESTLQ-H,
Ac-NSTSQ-H, Ac-DSFDQ-H, and Ac-NSFSQ-H, were tested for inhi-
bition of SARS-CoV Mpro. Overall, the four analogues were found
to be reversible tight-binding inhibitors. Harboring the canonical
P2 Leu, aldehyde Ac-ESTLQ-H exhibited inhibition with a relatively
low Ki of 8.27 1.52 lM. Aldehydes Ac-NSTSQ-H, Ac-DSFDQ-H,
and Ac-NSFSQ-H, all with a non-canonical P2 residue, exhibited
moderate inhibition with Ki values of 40.98 2.63, 41.24 2.25,
and 72.73 3.60 lM. Surprisingly, aldehyde CmFF-H, carrying a
0.16 ꢂ 0.36 Å for Ca atoms. In addition to the crystal soaking
experiments, we also tried to cocrystallize SARS-CoV Mpro with
the aldehydes. The crystals obtained were predominantly of space
group P21, with the exception of the complex with Ac-NSFSQ-H,
which still displayed space group C2. However, in most of the
P21 crystals, no electron density for the aldehyde could be
observed; thus, while the presence of the inhibitors induced a
change of space group in most cases, the compounds themselves
were not detected in the crystals. An exception was Ac-ESTLQ-H.
The crystals of its complex with the Mpro diffracted to 1.89 Å, but
in both enzyme monomers in the asymmetric unit, electron den-
sity could only be seen for residues P1 (Gln) and P2 (Leu) of the
inhibitor (Fig. 1E and F). Crystallographic data and refinement
statistics for all six crystal structures are summarized in Table 1.
cinnamoyl group in the P3 and a Phe residue in the P1 position,
had an even higher inhibitory activity against SARS-CoV Mpro than
the four pentapeptide aldehydes, with a Ki of 2.24 0.58 lM.
3.2. Overall structures of the aldehyde complexes
The aldehydes Ac-ESTLQ-H, Ac-NSTSQ-H, Ac-DSFDQ-H,
Ac-NSFSQ-H, and Cm-FF-H were separately soaked into crystals
of SARS-CoV Mpro. The crystals were all of space group C2, which
is often observed for SARS-CoV Mpro (Lee et al., 2005; Xue et al.,
2007; Verschueren et al., 2008). These crystals contain one SARS-
CoV Mpro monomer per asymmetric unit and the dimer (which is
the enzymatically active species) is formed through the symmetry
3.3. Dual configurations of the thiohemiacetal in the complex with Ac-
NSFSQ-H
In the SARS-CoV Mpro complex with aldehyde inhibitor
Ac-NSFSQ-H, the thiohemiacetal adopts alternative configurations
(Fig. 2A and B). This is likely the result of nucleophilic attack by
Cys145 onto the planar carbonyl from either side. In the (R)-isomer,

208
L. Zhu et al. / Antiviral Research 92 (2011) 204–212
Fig. 1. Inhibitor binding at the active site of SARS-CoV Mpro. 2Fo ꢂ Fc electron-density maps are shown for inhibitors (A) Ac-ESTLQ-H, (B) Ac-NSTSQ-H, (C) Ac-DSFDQ-H, (D)
Ac-NSFSQ-H, (E) the visible portion of Ac-ESTLQ-H cocrystallized with the Mpro, molecule A, and (F) molecule B, (G) Cm-FF-H. The maps are contoured at a level of 1
catalytic Cys145 of SARS-CoV Mpro forms a thiohemiacetal with the aldehyde group of the inhibitors.
r. The
the thiohemiacetal oxygen forms a hydrogen bond with the imidaz-
ole of His41 (3.30 Å) of the catalytic dyad (Fig. 2C), whereas in the
(S)-isomer, the oxygen atom points away from His41 and into the
oxyanion hole, forming H-bonds with the main-chain amides of

L. Zhu et al. / Antiviral Research 92 (2011) 204–212
209
Fig. 2. Dual configurations of the thiohemiacetal in the complex with Ac-NSFSQ-H. (A) and (B): Ac-NSFSQ-H binding at the active site in two alternative configurations.
Hydrogen bonding interactions are represented by broken lines. Distances between atoms are shown in Å. (C) and (D): Schematic diagram of the aldehyde inhibitor Ac-
NSFSQ-H attacked by the catalytic cysteine 145, leading to the formation of two possible diastereomeric products.
Gly143 (2.71 Å) and Cys 145 (3.15 Å) (Fig. 2D). Previous X-ray and
NMR studies of aldehyde binding to various cysteine and serine
proteases demonstrated that one or both configurations were pres-
ent in the structures of the complexes (Delbaere and Brayer, 1985;
Webber et al., 1998; Robin et al., 2009).
of a well-ordered water in the S2 pocket, although disordered
water is probably present in the pocket on both sides of the serine
side-chain, where free spaces with a total volume of >70 Å3 are
present between the substrate and the protein. It should be noted
that the side-chain of Gln189, which contributes to one wall of the
S2 pocket, is quite flexible and adopts different conformations in
the various inhibitor complexes, allowing or preventing the access
of water to the pocket.
3.4. The S2 subsite
The S2 subsite is a large pocket lined by residues His41, Met49,
Cys145, His164, Met165, Asp187, Arg188mc (mc: contribution
from main-chain atoms only), and Gln 189 (Fig. 1). At the cleavage
sites of the SARS-CoV Mpro in the viral polyproteins, the P2 position
is mostly found to be occupied by Leu or Phe. Accordingly, almost
all inhibitors designed for the enzyme carry a large hydrophobic
group in the P2 position. We were therefore very surprised to find
the Ser or Asp side chains in the P2 position of the aldehydes bound
to this hydrophobic site (Fig. 1). In the SARS-CoV Mpro complexes
with Ac-NSTSQ-H or Ac-NSFSQ-H, there is no hydrogen-bonding
interaction between the side chain of P2-Ser and residues of the
S2 subsite. As the Ser side chain is small, there is no steric barrier
for Ser binding in the large S2 pocket. The serine does not quite
reach the ‘‘bottom’’ of the S2 pocket, but the distance between
In case of the SARS-CoV Mpro complex with Ac-DSFDQ-H, the P2
Asp side-chain is situated between the side chains of Met49 and
Met165, which form opposite walls of the S2 pocket. Most interest-
ingly, its carboxylate oxygens undergo close interactions with the
sulfur atoms of the two methionines. The distances (rS. . .O) between
the Sd atoms of Met49 and Met165 on the one hand and the Od2
and Od1 atoms, respectively, of P2-Asp on the other are 3.36 and
3.45 Å (Fig. 3). Then the relative distances (dS. . .O) can be calculated
according to dS. . .O = rS. . .O ꢂ vdw(S) ꢂ vdw(O), where values of 1.80
and 1.52 Å are used as van der Waals radii (vdw) for S and O atoms,
respectively. With dS. . .O = 0.04 and 0.13 Å, the distances between
the P2-Asp and the two S2-Met side-chains fulfill the condition
of dS. . .O 6 0.2 Å (Iwaoka et al., 2002) for a non-bonded S. . .O
contact. These interactions are not in the plane of the methionine
sulfides; the h values (h is the polar angle between the normal to
the sulfide plane and the S. . .O vector (Pal and Chakrabarti,
2001)) are 30.6° and 52.8°. Similar nonbonded interactions
its Oc atom and the carbonyl oxygen of residue Gln192 is 4.0 Å,
leaving no space for a water molecule to locate in between. Accord-
ingly, we did not find any electron density suggesting the presence

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L. Zhu et al. / Antiviral Research 92 (2011) 204–212
Fig. 4. Relative cleavage efficiencies of 20 peptide substrates harboring different
amino-acid residues in P2.
specificity results (Fan et al., 2004, 2005; Lai et al., 2006), P1 has
to be Gln. Moreover, in all SARS-CoV Mpro complex structures de-
scribed so far (Lee et al., 2005; Xue et al., 2007; Yang et al., 2005,
2003; Yin et al., 2007), the S1 subsite is occupied by a polar group,
i.e. the side chain of Gln or a five-membered lactam (used as a glu-
tamine surrogate). Shie et al. synthesized a series of
rated esters containing both P1 and P2 phenylalanine residues
a,b-unsatu-
Fig. 3. Interactions of P2-Asp in the S2 subsite in the complex structure SARS-CoV
Mpro: Ac-DSFDQ-H. P2-Asp carboxylate oxygens interact with the sulfur atoms of
Met49 and Met165. These non-bonded interactions are represented by dashed
lines. Distances between atoms are shown in Å.
which showed modest inhibitory activity against the SARS-CoV
Mpro (IC50 = 11–39
l
M) (Shie et al., 2005). The
ethyl ester of 4-(dimethylamino)cinnamoyl-Phe-Phe was reported
to be a potent inhibitor, with an inhibition constant of 0.52
a,b-unsaturated
l
M
between the methionine sulfur atoms and main-chain carbonyl
oxygens or carboxylate side-chains have been detected previously
in the hydrophobic cores of proteins and were proposed to stabi-
lize the protein fold (Pal and Chakrabarti, 2001). It has also been
suggested that S. . .O interactions should be taken into account in
protein engineering studies (Iwaoka et al., 2002; Pal and Chakrab-
arti, 2001), but to the best of our knowledge, we provide here the
first description of a methionine-carboxylate interaction in a pro-
tein-ligand complex. The unexpected finding of Ser and Asp bind-
ing in the S2 subsite constitutes a deviation from the dogma that
peptide inhibitors of proteases should contain amino-acid residues
corresponding to the sequence specificity of the target enzyme.
(Shie et al., 2005). The computer model for the complex of SARS-
CoV Mpro with the compound proposed that the P1 and P2 phenyl
groups occupy the S2 and S3 pockets, respectively (Shie et al.,
2005). However, in the crystal structure of the complex with the
aldehyde Cm-FF-H that we describe in this communication, the
side chain of P1-Phe is inserted into the S1 subsite (Fig. 1G). In or-
der to understand this unexpected deviation from the commonly
observed specificity, we compared the interaction with the S1 sub-
site observed in our structures of the complexes with Cm-FF-H and
Ac-ESTLQ-H, the latter of which corresponds exactly to the
3.5. Analysis of peptide substrates harboring different amino-acid
residues in P2
The unexpected observation of the P2-Asp residue of aldehyde
Ac-DSFDQ-H binding to the hydrophobic S2 pocket prompted us
to re-determine the cleavage specificity of the SARS-CoV Mpro with
respect to the P2 position. Within the context of the peptide sub-
strate SWTSAVXQ;SGFRKWA, all 20 proteinogenic amino acids
were tested in position P2 (=X). In the HPLC-based assay, the pro-
tease concentration was kept constant at 0.5 lM. The canonical
substrate with Leu in the P2 position was completely cleaved with-
in two minutes and was therefore used as the standard to compare
the proteolytic activities with other substrates. The results are
listed in Fig. 4. Our study confirmed that the most favored residues
in the P2 position of the substrate (after Leu) are the hydrophobic
amino acids Phe, Met, Val, and Ile. Substrates with polar amino
acids in P2 are poorly cleaved. The cleavage efficiency for the sub-
strate with P2 = Ser was 160-times lower than for the best sub-
strate (P2 = Leu); furthermore, the peptide with P2 = Asp was not
cleaved at all after incubation with SARS-CoV Mpro for 16 h.
3.6. S1 subsite
Fig. 5. Superimposition of the structures of SARS-CoV Mpro complexes with Cm-FF-
H (red) and Ac-ESTLQ-H (green) showing the conformational changes in the S1
subsite. (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of this article.)
The S1 subsite is lined by residues Phe140, Asn142, Gly143,
Ser144, Cys145, His163, Glu166, and His172. From the substrate

L. Zhu et al. / Antiviral Research 92 (2011) 204–212
211
described cleavage specificity of the Mpro. The predominant S1
specificity of the enzyme for Gln is determined primarily by
His163. In the complex formed with Ac-ESTLQ-H, N 2 of His163
donates a 2.83-Å hydrogen bond to the side-chain carbonyl oxygen
(O 1) of P1-Gln (Fig. 1). In the complex with Cm-FF-H, the side
chain of Asn142 undergoes an 83.8° rotation about its 1 torsion
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Acknowledgments
We thank Sebastian Schwarz for technical assistance during the
re-determination of the cleavage rates of SARS-CoV Mpro with re-
spect to the P2 position of the substrate, and Stefan Anemüller,
Qingjun Ma, Jeroen R. Mesters as well as Jiajie Zhang for discussion.
This work was initially supported by the Deutsche Forschungs-
gemeinschaft (Grants Hi611/4, Ra895/2), the Sino-German Center
for the Promotion of Research, Beijing, and the Sino-European Pro-
ject on SARS Diagnostics and Antivirals (SEPSDA) of the European
Commission (contract number SP22-CT-2004-003831), and subse-
quently, by the SILVER project of the European Commission (con-
tract number HEALTH-F3-2010-260644). RH and JR acknowledge
continuous support by the Fonds der Chemischen Industrie. RH is
also supported by a Chinese Academy of Sciences Visiting Profes-
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