Structure-based design, synthesis, and biological evaluation of peptidomimetic SARS-CoV 3CLpro inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.08.031

Structure-based design, synthesis, and biological evaluation of a series of peptidomimetic severe acute respiratory syndrome-coronavirus chymotrypsin-like protease inhibitors are described. These inhibitors were designed and synthesized based upon our X-r

Full text of DOI:10.1016/j.bmcl.2007.08.031

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5876–5880
Structure-based design, synthesis, and biological evaluation
of peptidomimetic SARS-CoV 3CLpro inhibitors
a,
a
b
a
*
Arun K. Ghosh, Kai Xi, Valerie Grum-Tokars, Xiaoming Xu,
b
b
c
c
Kiira Ratia, Wentao Fu, Katherine V. Houser, Susan C. Baker,
b
b
Michael E. Johnson and Andrew D. Mesecar
a
Departments of Chemistry and Medicinal Chemistry, Purdue University, West Lafayette, IN 47907, USA
b
Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,
University of Illinois at Chicago, 900 S. Ashland, IL 60607, USA
Department of Microbiology and Immunology, Loyola University of Chicago, Stritch School of Medicine, Maywood, IL, USA
c
Received 28 June 2007; revised 31 July 2007; accepted 1 August 2007
Available online 19 August 2007
Abstract—Structure-based design, synthesis, and biological evaluation of a series of peptidomimetic severe acute respiratory syn-
drome-coronavirus chymotrypsin-like protease inhibitors are described. These inhibitors were designed and synthesized based upon
our X-ray crystal structure of inhibitor 1 bound to SARS-CoV 3CLpro. Incorporation of Boc-Ser as the P -ligand resulted in
4
enhanced SARS-CoV 3CLpro inhibitory activity. Structural analysis of the inhibitor-bound X-ray structure revealed high binding
affinity toward the enzyme.
Ó 2007 Elsevier Ltd. All rights reserved.
Severe acute respiratory syndrome (SARS), a conta-
gious and fatal respiratory illness, was first reported in
Guangdong province, China, in November 2002. Its ra-
The SARS-CoV is a positive-strand RNA virus whose
genome sequence reveals only moderate relation to
other known coronaviruses. During viral replication,
1
5
pid and unexpected spread to other Asian countries,
North America, and Europe alarmed both the public
and the World Health Organization. SARS is caused
viral replicase polyproteins undergo extensive process-
ing by two viral proteases, namely chymotrypsin-like
protease (3CLpro) and papain-like protease (PLpro),
to generate a functional viral replication complex.
Therefore, both SARS-CoV 3CLpro and SARS-CoV
PLpro are recognized as logical targets for therapeutic
2
,3
6,7
by a novel coronavirus, SARS-CoV. Its emergence af-
fected more than 8000 individuals and caused more than
4
8
00 deaths within a few months. This signifies just how
rapidly a contagious illness can spread in the more mo-
bile and highly interconnected world in the 21st century.
Remarkably, the world has witnessed how concerted
international cooperation allowed health experts to
identify the novel etiologic agent and contain the SARS
epidemic just months after its emergence. Currently,
there is no known SARS transmission anywhere in the
world. At the same time, there is no guarantee that this
outbreak will not strike again in an even more severe
form. Thus far, no effective therapy exists for this viral
infection.
intervention
interactions.
against
SARS-CoV
and
related
8
The structure and activity of the SARS-CoV 3CLpro
9
have been investigated. The enzyme active site contains
a catalytic dyad where a cysteine residue acts as a nucle-
ophile and a histidine residue acts as the general acid
base. SARS-CoV 3CLpro is functionally analogous to
the main picornaviral protease 3Cpro and there have
been significant drug-design efforts against human rhi-
noviral 3C protease which is also a cysteine protease.
We recently described structure-based design of a num-
ber of potent SARS-CoV 3CLpro inhibitors. These
inhibitors were designed based upon modification of
AG7088, a potent inhibitor of the human rhinoviral
1
0
1
1
Keywords: Design; Synthesis; SARS 3CLpro; Inhibitor; X-ray
structure.
1
2
*
Corresponding author. Tel.: +1 765 494 5323; fax: +1 765 496
612; e-mail: akghosh@purdue.edu
3C protease. While AG7088 does not exhibit inhibi-
tory activity against SARS-CoV 3CLpro, the modified
1
0
960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.08.031

A. K. Ghosh et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5876–5880
5877
inhibitors are active in enzymatic assays as well as in cell
1
H
N
O
3
culture assays. The X-ray structure of inhibitor-bound
SARS-3CLpro revealed important molecular insight
O
1
1
into the ligand-binding sites of enzyme. The inhibitors
bind to SARS-CoV 3CLpro through covalent bonding
with the active site cysteine 145 residue. Based upon this
molecular insight, we have now modified our initial
inhibitors to accommodate specific ligand-binding site
BocHN
N
H
CO Et
2
O
Si
6
interactions in the S -subsite of the SARS-CoV 3CLpro
4
active site. Herein we describe the synthesis, biological
evaluation, and X-ray structures of inhibitor-bound
SARS-CoV 3CLpro for two potent peptide and
peptidomimetic inhibitors (Fig. 1).
a, b
H
N
O
O
BocHN
N
H
CO2Et
As described previously, inhibitor 2 makes a number of
1
1
O
important interactions in the enzyme active site. These
include, the covalent bond formed by the Cys-145 thiol
c, d
3
7
with the a,b-unsaturated ester at the S -subsite. His-172
1
makes hydrogen bond with the P -lactam-NH, and the
Glu-166 nitrogen makes a hydrogen bond with the ketone
of the inhibitor. The P -oxazole group appears to fill in
4
1
Scheme 1. Reagents: (a) TBAF, THF; (b) Dess–Martin periodinane,
CH Cl , 73% (two steps); (c) TFA, CH Cl ; (d) EDC, HOBt, DIPEA,
Boc-(L)-Serine, CH Cl , 72%.
2
2
2
2
1
1
2
2
the S -hydrophobic pocket. Based upon this X-ray struc-
4
ture, we subsequently speculated that the replacement of
P -oxazole with a Boc-Serine P -ligand would promote
additional hydrogen-bonding interactions in the S -site.
followed by coupling of the resulting amine with Boc-
(L)-Serine to provide 3 in 72% yield.
4
4
4
This will result in further enhancement of inhibitory
potency. We have also evaluated the corresponding
peptide-derived inhibitors for our studies.
The syntheses of peptide-based inhibitors are summa-
rized in Scheme 2. General peptide coupling reactions
between (S)-valine and (S)-leucine or (S)-phenylalanine
followed by ester hydrolysis gave the carboxylic acid
intermediates 10 and 11 in 81% and 90% yields, respec-
The synthesis of inhibitor 3 is outlined in Scheme 1. The
synthetic procedure of silyl ether derivative 6 was de-
tively. Coupling of these acids with lactam fragment
11,14
1
1
scribed by us previously. Removal of the silyl protect-
ing group with tetrabutylammonium fluoride in THF
provided a secondary alcohol, which was oxidized to ke-
tone 7 with the Dess–Martin periodinane in 73% yield
over 2 steps. The ketone 7 was converted to inhibitor
12
afforded Boc-derivatives 13 and 14 in 89% and
86% yields, respectively. Exposure of these compounds
to TFA effected the removal of the Boc-group to provide
the corresponding free amines. Coupling of these amines
with Boc-(L)-serine furnished inhibitors 4 and 5 in 55%
and 95% yields, respectively. Furthermore, reaction of
3
by exposure to trifluoroacetic acid (20% TFA/CH Cl )
2 2
O
R
O
O
N
O
O
H
H
N
H
N
Me
H N
CO Me
2
2
a, b
N
CO2H
Boc N
H
O
O
R
R
H
O
8
R =
i-Pr
10 R =
i-Pr
1
2
R = Ph
N
H
9 R = Ph
11 R = Ph
c
R = CH=CMe2
O
H
N
H
N
O
O
HO
O
H
O
H
N
N
Boc
N
H
O
H
O
O
N
H N
2
Boc N
H
N
H
CO Et
2
3
H
O
O
R
N
H
CO Et
12
2
O
13 R = i-Pr
14 R = Ph
HO
R
O
O
H
O
N
H
N
Me
N
N
CO H
Boc
N
H
2
d, e
d, f
H
15
4, 5
16, 17
O
O
H
O
4
5
R = i-Pr
R = Ph
Scheme 2. Reagents: (a) EDC, HOBt, DIPEA, Boc-(L)-valine,
CH Cl ; (b) LiOH (1 M in H O), THF; (c) EDC, HOBt, DIPEA,
2, CH Cl ; (d) TFA, CH Cl ; (e) EDC, HOBt, DIPEA, Boc-(L)-
Serine, CH Cl ; (f) EDC, HOBt, DIPEA, 15, CH Cl
N
H
2
2
2
1
2
2
2
2
Figure 1. Structures of SARS-3CLpro inhibitors.
2
2
2
2
.

5878
A. K. Ghosh et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5876–5880
1
7
the 13 or 14-derived amines with 5-methylisoxazole-3-
1
a,b-unsaturated esters. The assays were performed in
5
carboxylic acid 15 afforded inhibitors 16 (75%) and
7 (81%) in very good yields.
96-well microplates using a reaction volume of 100 lL
which contained 50 mM Hepes, (pH 7.5), 500 nM
SARS-CoV 3CLpro enzyme, and varying concentra-
tions of synthetic inhibitors. The reaction components,
with the exception of substrate, were incubated for
20 min and the reaction was initiated by the addition
of FRET-substrate [AlexaFluor 488-cys]-Glu-Ser-Ala-
Thr-Leu-Gln-Ser-Gly-Leu-Ala-[Lys-QSY -7]-Ser, giving
a final substrate concentration of 1 lM.
1
The inhibitory activities of the synthetic inhibitors are
shown in Table 1. The enzyme inhibitory activity of
the compounds against SARS-CoV 3CLpro was deter-
mined in a FRET-based, microplate assay described
Ò
1
1,16
Ò
by Grum-Tokars et al. This protocol has previously
been used for the determination of IC50 values for
1
1,16
The IC50
Table 1. Structures and activity of inhibitors
Entry
Inhibitor structure
3CLpro, IC50 (lM)
Antiviral, IC50 (lM)
Ph
O
O
CO2Et
N
N
O
O
H
N
H
N
Me
Me
1.
2.
3.
4.
5.
6.
7.
870
45
O
O
H
O
1
2
N
H
O
CO2Et
H
N
H
N
800
70
O
H
N
H
O
HO
CO2Et
O
H
H
N
N
Boc N
H
80
75
O
O
O
H
O
3
(GRL-0155S)
N
H
HO
CO2Et
O
O
H
N
H
N
Boc N
H
N
H
10
100
100
nd
O
O
H
O
4
5
N
H
HO
Ph
O
CO Et
2
H
N
H
N
Boc N
H
N
H
15
H
O
N
H
Ph
O
CO2Et
N
O
H
H
Me
Me
N
N
N
H
300
O
O
O
O
H
O
1
1
6
N
H
O
CO2Et
N
O
H
N
H
N
N
H
200
nd
H
7
N
H
O

A. K. Ghosh et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5876–5880
Table 2. Summary of X-ray data processing and refinement statistics
5879
˚
Resolution (A)
b
˚
˚
˚
c
d
R
merge (%) % Complete a (A)
b (A) c (A) b (deg)
R
cryst (%)
R
free (%)
R
free after TLS
Rfree after TLS
a
0.0–1.9 (1.97–1.90) 3.1 (21.7)
2
91.5 (53.0) 108.32 82.22 53.68 104.67 23.6 (33)
27.5 (36) 19.6 (28)
22.8 (35)
a
Last shell resolution statistics are shown within parentheses.
P
P
b
R
reflection. 118,202 measured reflections. 31,114 unique reflections.
merge
=
jI ꢀ hIij/ I, where I is the observed intensity and hIi is the average intensity of multiple symmetry-related observations of that
P
P
c
R
cryst
=
reflection.
jjFobsj ꢀ jFcalcjj/ jFobsj, where jFobsj and jFcalcj are the observed and calculated, respectively, structure factor amplitudes for each
d
R
cases).
free is the same as Rcryst, except it is calculated over a set of reflections not used in structural refinement at any stage (5% of the observed data in all
Figure 2. The X-ray crystal structure of 3 (thick stick with gray carbon) with SARS-CoV 3CLpro. Hydrogen bonds between inhibitor and 3CLpro
are shown in white dotted lines.
values for inhibitors were determined by measuring the
rates of reaction with increasing inhibitor concentra-
tions. As shown in Table 1, our structure-based incorpo-
ration of P -Boc-Ser in place of P -oxazole in inhibitor 2
ing distance of Glu 166. This may explain its enhanced
binding affinity over the oxazole-bearing inhibitor 2.
In conclusion, our structure-based design strategies
resulted in significant improvement of enzyme inhibitory
activity against SARS-CoV 3CLpro. An X-ray structure
of the 3-bound enzyme provided critical molecular insight
into the ligand-binding site interactions in the active site.
4
4
resulted in (inhibitor 3) significant enhancement of enzy-
matic inhibitory potency, while its antiviral activity was
not improved. It exhibited antiviral activity similar to
inhibitors 1 and 2. This difference of activity may be
due to the presence of peptide-like features in the inhib-
itors. Peptide-like molecules often exhibit poor passive
transcellular diffusion across cell membranes. Antiviral
It appears that the P -Boc-Ser is involved in important
4
0
hydrogen-bonding interactions in the S -subsite. The
4
present molecular insight will facilitate the design of more
effective inhibitors. Further investigations are in progress.
1
1,18
assays followed our previously published protocol.
We have compared the binding affinity of the corre-
sponding peptide-based inhibitors incorporating both
P -oxazole and P -Boc Ser. Peptide-based inhibitors (4
Acknowledgment
4
4
and 5) with P -Leu and P -Phe have displayed 20-fold
2
2
enzymatic potency enhancement over the respective
inhibitors (16 and 17) containing P -oxazole.
The financial support of this work is provided by the
National Institute of Health (NIAID, P01 A1060915).
4
To obtain molecular insight into the binding properties
of inhibitor 3, we determined the X-ray structure of 3-
bound to SARS-CoV 3CLpro to a resolution of
References and notes
˚
19
1
.9 A. The refinement statistics and data collection
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˚
to the enzyme via a C–S bond (1.77 A) to the active site
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0
1
proximity to Thr-26 oxygen (3.38A). The Leu-27 side
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˚
0
1
1
with the imidazole ring of His-163. The P -carbonyl
3
group of 3 forms a hydrogen bond with the NH of
Glu-166. The P -Ser side chain is within hydrogen-bond-
4

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combination of ion-exchange, ammonium sulfate frac-
tionation, and separation by hydrophobic interaction
1
6
chromatography according to our published procedures.
The refinement statistics and data collection are summa-
rized in Table 2. Purifed SARS-CoV 3CLpro was
exchanged into buffer (20 mM Tris, pH 7.5, 10% glycerol,
and 10 mM 2-mercaptoethanol) and concentrated to
35 mg/mL using an Amicon Centrifugal Filter, molecular
weight cutoff 10,000 Da. The enzyme, at a concentration
of 35 mg/mL, was incubated with 1 mM inhibitor on ice
for 1 h prior to crystallization which was conducted by
mixing 5 lL enzyme and 1 lL of mother liquor solution
and allowing the mixture to undergo vapor diffusion
against 11% PEG 20,000, 50 mM NaCl, 50 mM Na-
cacodylate, pH 6.5. Single crystals grew within 3 h and
were cryoprotected and flash-frozen in liquid nitrogen.
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SER-CAT beamline 22-BM at the Advanced Photon
Source, Argonne National Laboratory. Data was indexed
with HKL2000 and scaled and merged with SCALE-
1779.
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1. Ghosh, A. K.; Xi, K.; Ratia, K.; Santarsiero, B. D.;
Fu, W.; Harcourt, B. H.; Rota, P. A.; Baker, S. C.;
Johnson, M. E.; Mesecar, A. D. J. Med. Chem. 2005,
7
8
2
0
PACK (Otwinowski and Minor). The structure was
determined via molecular replacement using the coordi-
1
1
nates 2ALV from the PDB (Ghosh et al.) and refined
with REFMAC from the CCP4 suite (Collaborative,
1994). R-Factors were minimized by an additional 4–5%
by using 10 cycles of TLS refinement on 19 segments. The
3CLpro-compound 4 complex crystallized in space group
C121 with one molecule in the asymmetric unit with unit
9
1
˚
˚
˚
1
cell parameters of a = 108.32 A, b = 82.22 A, c = 53.68 A
and b = 104.7°. The structure was determined by molec-
ular replacement using as a search model. The final Rcryst
and Rfree values were 19.6% and 22.8%, respectively. The
final model includes the inhibitor covalently linked to
Cys145.
48, 6767.
1
2. (a) Patick, A. K.; Binford, S. L.; Brothers, M. A.; Jackson,
R. L.; Ford, C. E.; Diem, M. D.; Maldonado, F.;
Dragovich, P. S.; Zhou, R.; Prins, T. J.; Fuhrman, S. A.;
Meador, J. W.; Zalman, L. S.; Matthews, D. A.; Worland,
20. Otwinowski, Z.; Minor, W. Macromol. Crystallogr. 1997,
276, 307.