Discovery of Potent Anilide Inhibitors against the Severe Acute Respiratory
Syndrome 3CL Protease
Jiun-Jie Shie,
†Jim-Min Fang,*
,†,‡Chih-Jung Kuo,
§Tun-Hsun Kuo,
§Po-Huang Liang,*
,§Hung-Jyun Huang,
†Wen-Bin Yang,
‡Chun-Hung Lin,
§Jiun-Ling Chen,
†Yin-Ta Wu,
‡and Chi-Huey Wong
‡,|Department of Chemistry, National Taiwan University, Taipei, 106, Taiwan, Genomics Research Center, Academia Sinica, Taipei, 115, Taiwan, Institute of Biological Chemistry, Academia Sinica, Taipei, 115, Taiwan, and Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037
Received February 28, 2005
A diversified library of peptide anilides was prepared, and their inhibition activities against
the SARS-CoV 3CL protease were examined by a fluorogenic tetradecapeptide substrate. The
most potent inhibitor is an anilide derived from 2-chloro-4-nitroaniline,
L-phenylalanine and
4-(dimethylamino)benzoic acid. This anilide is a competitive inhibitor of the SARS-CoV 3CL
protease with K
i) 0.03 µM. The molecular docking experiment indicates that the P1 residue
of this anilide inhibitor is distant from the nucleophilic SH of Cys145 in the active site.
Introduction
Severe Acute Respiratory Syndrome (SARS) first
occurred in Guandong (China) in November of 2002 and
spread through many countries in 2003. This illness is
caused by infection with a novel human coronavirus
(SARS-CoV).
1The fatality rate of SARS-CoV infection
is rather high, estimated to be 14-15%. In only a few
months, nearly 1000 lives were claimed.
2The natural
source of SARS-CoV is unclear, though studies on the
molecular evolution of SARS-CoV indicate that the virus
may have emerged from wild animals.
3At present, no
efficacious therapy for SARS is available. Therefore, a
search for effective antivirals for the SARS-CoV is of
current interest.
SARS coronavirus is a positive-strand RNA virus,
4that encodes two replicase polyproteins pp1a and pp1b.
Extensive proteolytic processing of these nonstructural
polyproteins is required to provide the functional
pro-teins for viral propagation. These processes are
medi-ated primarily by the main protease (M
pro), which is also
known as dimeric chymotrypsin-like protease (3CL
pro).
5The active site of SARS-CoV 3CL protease contains
Cys145 and His41 to constitute a catalytic dyad, in
which cysteine functions as the common nucleophile in
the proteolytic process.
5The 3CL protease cleaves pp1a
at no less than 11 conserved sites with a sequence of
(Leu,Met,Phe)-GlnV(Ser,Ala,Gly), in which a P1 glutamine
residue invariably occupies the S1 site.
5,6The 3CL
protease is essential for the propagation of the virus and
thus serves as a key target for the discovery of
anti-SARS drugs.
So far, only a few inhibitors have been validated by
in vitro protease assays. These protease inhibitors
include C2 symmetric peptidomimetic compounds,
7zinc-conjugated compounds,
8bifunctional aryl boronic acids,
9a quinolinecarboxylate derivative,
10a
thiophenecar-boxylate,
11and
phthalhydrazide-substituted
keto-glutamine analogues.
12As a part of our efforts directed toward the
develop-ment of anti-SARS agents, we prepared several
chro-mogenic peptides (AA)x-Gln-pNA, e.g.
QTSITSAVLQ-pNA containing a p-nitroaniline moiety at the C-terminal
glutamine, to test their activities as substrates for the
SARS-CoV 3CL protease. Unlike typical
nitroanilide-based peptides which are readily hydrolyzed by serine
and cysteine proteases,
13these (AA)x-Gln-pNA peptides
were not efficiently cleaved by the SARS-CoV 3CL
protease but displayed weak inhibition against the
enzyme. This observation, along with an earlier
cell-based assay showing the inhibitory activity of
N-(2-chloro-4-nitrophenyl)-5-chloro-2-hydroxybenzamide
(Niclosamide) against the replication of SARS-CoV,
14led
us to explore peptide nitroanilides as inhibitors of the
SARS-CoV 3CL protease.
Results and Discussion
We chose to prepare a series of peptide anilides
having
L-phenylalanine as the P1 residue, on the basis
* To whom correspondence should be addressed. Tel: 8862-23637812. Fax: 8862-23636359. E-mail: jmfang@ntu.edu.tw.
†National Taiwan University.
‡Genomics Research Center, Academia Sinica. §Institute of Biological Chemistry, Academia Sinica. |The Scripps Research Institute.
10.1021/jm050184y CCC: $30.25 © 2005 American Chemical Society Published on Web 06/04/2005
of another study on AG7088 analogues that the
inhibi-tory activity can be improved by using
L-phenylalanine
to replace
L-glutamine or its γ-lactam isostere (see Table
3 in Supporting Information). Anilide 1 was prepared
by condensation of 2-chloro-4-nitroaniline with the acyl
chloride derivative of Boc-Phe-OH. Using the previously
reported amide formation in a microtiter plate,
15the
coupling reactions of a 60-member library of carboxylic
acids with the amine generated by removal of the Boc
group from anilide 1 afforded a 60-member library of
anilide 2. Tripeptide anilides 3a-x (24 members) and
tetrapeptide anilides 4a-x (24 members) were also
created by coupling of 1 with appropriate peptides. The
dimeric peptide anilides 5a-c and 6a-c were prepared
by using a diacid, e.g. succinic acid, S-malic acid, and
(2R,3R)-tartaric acid, as the core structure to link with
appropriate amino acids or peptides.
On the basis of the previously reported synthesis of
AG7088,
16we also devised an expedient method for the
synthesis of anilides 7a-x, the ketomethylene isosteres
of tripeptides 3a-x (Scheme 1).
These anilide products 2-7 were characterized by
mass analyses and directly subjected, without isolation,
to the inhibition assay against the SARS-CoV 3CL
protease according to the previously described
fluoro-metric method.
7,17The initial velocities of the inhibited
reactions using 50 nM of SARS-CoV 3CL protease and
6 µM of the fluorogenic substrate were plotted against
the different inhibitor concentrations to obtain the IC
50values. On the basis of the preliminary results of assays,
some of the most promising inhibitor candidates were
selected for the scale-up synthesis. The IC
50values and
inhibition constants (K
i) of the pure samples were then
measured to validate their activities.
The results of preliminary assays indicated that the
2-chloro-4-nitroanilides 2-6 generally possessed good
inhibitory activities against the SARS-CoV 3CL
pro-tease, whereas the ketomethylene isosteres 7 were less
potent than the tripeptide counterpart 3. Surprisingly,
Niclosamide showed no inhibitory activity at a
concen-tration of 50 µM.
8The IC
50
data for some anilide
inhibitors, either prepared in microtiter plate without
isolation or in pure form, are listed in Table 1. Anilide
2a (JMF1507) derived from 2-chloro-4-nitroaniline,
L-phenylalanine, and 4-(dimethylamino)benzoic acid is the
most potent inhibitor, showing an IC
50of 0.06 µM and
K
i) 0.030 µM.
Lineweaver-Burk plots of kinetic data were fitted
with the computer program KinetAsyst II
(IntelliKinet-ics, PA) by nonlinear regression to obtain the K
ivalues.
The double reciprocal plot of the initial rate vs substrate
concentration indicates that all these compounds are
competitive inhibitors. A representative example for
inhibition of the SARS-CoV 3CL protease by anilide 2a
is shown in Figure 1. It is worth to note that anilide 2a
functions as a potent inhibitor with K
i) 0.03 µM, rather
than a substrate for the SARS-CoV 3CL protease. The
HPLC and absorption spectral analyses indicated that
no decomposition of anilide 2a occurred under the
enzymatic conditions for a period over 16 h (see the
Supporting Information). In hydrolysis of
N-nitrophen-ylamide, alignment of n,π-orbitals is required for the
facile leaving of nitroaniline. Due to the steric effect of
the chlorine atom at the ortho-position, the
2-chloro-4-nitrophenyl ring and amido group cannot be in a
coplanar conformation, thus making hydrolysis
unfavor-able. This speculation is in agreement with the
com-puter modeling shown in Figure 2.
To know the structure-activity relationship, a series
of the 2a analogues was synthesized and the inhibitory
activity was examined. None of the analogues 8a-f
showed adequate activity (IC
50> 10 µM). Deletion of
Scheme 1. Synthesis of Ketomethylene Isosteres of Tripeptides
aaReagents and conditions: (i) NaH, THF, 0 °C to room temperature, 24 h. (ii) CF
3CO2H, CH2Cl2, rt, 24 h. (iii) H2, Pd/C, Boc2O, MeOH, rt, 10 h. (iv) allyl iodide, Cs2CO3, DMF, 45 °C, 5 h. (v) HCl, 1,4-dioxane, rt, 2 h. (vi) N-methylmorpholine, CH2Cl2, 0-25 °C, 2 h. (vii) Pd(PPh3)4, morpholine, THF, 25 °C, 3 h. (viii) HOBt, EDCI, (i-Pr)2NEt, CH2Cl2, 0 °C to room temperature, 20 h.
Figure 1. Lineweaver-Burk plot for inhibition of the SARS-CoV 3CL protease by anilide 2a (Ki) 0.03 µM).
the chloro, nitro, or dimethylamino substituents from
anilide 2a significantly deteriorated potency as did
replacing the dimethylamino group in 2a with a nitro
group.
The crystal structure of SARS-CoV 3CL protease in
complex with a specific inhibitor of hexapeptidyl
chlo-romethyl ketone, Cbz-Val-Asn-Ser-Thr-Leu-Gln-CH
2Cl,
has been reported (coded 1uk4 in the Protein Data Bank
deposition).
5bOn this basis, the docking experiment
(Autodock version 3.0.5)
18using p-nitroaniline as the
core structure formed three main clusters (RMSD ) 2
Å). The clusters with lowest binding free energy occupies
Table 1. IC50Values for Some 2-Chloro-4-nitroanilide Inhibitors against the SARS-CoV 3CL Protease
structure type compd R R′ IC50(µM) Ki(µM)a
niclosamide >50a anilide 1 t-BuO >50a 2a Me2NC6H4 0.06a 0.03 ( 0.001 2b C14H29CH(Br) 3b 2c 3,4-(NH2)2C6H3 2b 2d (indol-3-yl)-CHdCH 3b 2e (2-NH2-1,3-thiazol-4-yl)-C(dNOCH3) 7b
tripeptide anilide 3a i-Bu Et 7b
3c i-Bu Ph 4b 3d i-Bu t-BuO >10a 3f i-Bu morpholino 19b 3h i-Bu thien-2-yl 5a 2.29 ( 1.01 3o PhCH2 5-Me-isoxazol-3-yl 7a 2.90 ( 1.27 3p PhCH2 Thien-2-yl 5a 4.3 ( 1.9
tetrapeptide anilide 4a i-Bu Et 7b
4f i-Bu morpholino 16b 4g PhCH2 t-Bu 2b 4j PhCH2 5-Me-isoxazol-3-yl 5a 1.61 ( 1.03 4k PhCH2 PhCH2O 6a 1.51 ( 0.95 4q 4-FC6H4CH2 Et 5b 4s 4-FC6H4CH2 Ph 2b dimeric anilide 5a H H >50b 5b (S)-OH H 4a 3.1 ( 0.4 5c (R)-OH (R)-OH 5b 6a H H 2b 6b (S)-OH H 2b 6c (R)-OH (R)-OH 2b
ketomethylene anilide 7a i-Bu Et 27b
7c i-Bu Ph 21b 7d i-Bu t-BuO 19b 7f i-Bu morpholino 29b 7h i-Bu thien-2-yl 22b 7o PhCH2 5-Me-isoxazol-3-yl 6b 7p PhCH2 Thien-2-yl 16b
aThe sample is in pure form.bThe sample is synthesized in a microtiter plate and assayed in situ without isolation.
Figure 2. Computer modeling of compound 2a binding to SARS-CoV 3CL protease (1uk4). Compound 2a is colored in yellow. The van der Waals filling space is generated by 1.0 scale and colored according to atom type. Clusters of possible docking modes were sorted by computed binding free energy and the docking mode with lowest docking energy (-9.1 kcal/ mol in this case) is generated by MGLTOOLS.
the pocket formed by Cys145, Ser144, His163, and
Phe140 or the pocket formed by Thr25, His41, Cys44,
Thr45, and Ala46. The 2-chloro-4-nitroanilide moiety of
2a was found to occupy the second favorite pocket
described above (Figure 2). The nitro group in 2a is
predicted to be hydrogen bonded with the HN of Ala46,
while the chlorine atom is within 3 Å from γ-S atom of
Cys145 and -N2 atom of His41, providing a possible
key interaction with the catalytic dyad. The
(dimethyl-amino)phenyl group is fitted to the cleft formed by
Gln189-Gln192 and Met165-Pro68. The P1 phenyl
residue in 2a is positioned in the S1 pocket, which may
be modified to increase the interactions with Phe140,
His163, and Glu166.
The docking study showed that anilide 2a has the
lowest binding free energy of -9.1 kcal/mol in
compari-son with the anilides 8a-e (∆G* ) -7.5 to -8.7 kcal/
mol, see the Supporting Information). This docking
experiment supports the observation in the enzymatic
assay, which reveals the important roles of the
2-chloro-4-nitroanilide and (dimethylamino)phenyl moieties in
inhibition of the SARS-CoV 3CL protease. The docking
model also shows that the P1 site of anilide 2a is distant
(
∼4.95 Å) from the nucleophilic SH of Cys145. This
model is in agreement with the observation that anilide
2a is stable in the SARS 3CL protease.
Under the assay conditions similar to that for the
SARS-CoV 3CL protease, the IC
50values of anilide 2a
against trypsin, chymotrypsin, and papain were
mea-sured to be 110, 200, and 220 µM, respectively. In
comparison, anilide 2a is a potent inhibitor for the
SARS-CoV 3CL protease with an IC
50value of 0.06 µM.
Experimental Section
Materials and Methods. SARS-CoV 3CL protease was prepared according to the previously described procedure.17
Reactions requiring dry conditions were carried out under an inert atmosphere using standard techniques. All the reagents and solvents were reagent grade and were used without further purification unless otherwise specified. THF was distilled from sodium benzophenone ketyl under N2.
Representative Procedure of Coupling Reactions. A solution of N-Boc phenylalanine (2.65 g, 10 mmol) and 2-chloro-4-nitroaniline (1.73 g, 10 mmol) in pyridine (30 mL) was cooled to -15 °C, and phosphorus oxychloride (1 mL, 11 mmol) was added dropwise with vigorous stirring. After the mixture was stirred for 1.5 h at -15 °C, the reaction was quenched by pouring into ice-water (100 mL). The mixture was extracted with EtOAc (1× 50 mL and 3 × 30 mL). The combined organic phase was washed with saturated NaHCO3(2× 50 mL) and
brine (30 mL). The organic phase was dried over MgSO4and
filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel column chromatography with elution of EtOAc/hexane (5:95) to give N-Boc phenylalanine 2-chloro-4-nitroanilide (1, 3.25 g, 78%) as white solids.
A solution of N-Boc phenylalanine 2-chloro-4-nitroanilide (0.42 g, 1 mmol) in 1,4-dioxane (3 mL) was treated with a solution of HCl in 1,4-dioxane (4 M, 2 mL) at 25 °C. The mixture was stirred for 1.5 h and concentrated under reduced pressure to give a crude aminium chloride salt. The material was dissolved in CH2Cl2(5 mL), cooled to 0 °C, and treated
with 4-methylmorpholine (0.3 mL, 2.5 mmol) and 4-(dimethyl-amino)benzoyl chloride (0.22 g, 1.2 mmol) sequentially. The ice bath was removed, and the mixture was stirred for 2 h at 25 °C. The reaction was quenched by addition of brine (15 mL). The mixture was extracted with CH2Cl2 (2 × 20 mL). The
organic phase was dried over Na2SO4and filtered, and the
filtrate was concentrated under reduced pressure. The residue
was purified by flash column chromatography with elution of EtOAc/hexane (1:9) to provide N-[4-(dimethylamino)phenyl]-phenylalanine 2-chloro-4-nitroanilide (2a, 0.29 g, 61%) as light yellow solids.
4-(Dimethylamino)benzoyl-L -Phe-(2-chloro-4-nitro-anilide) (2a). Light yellow solid; mp 205-207 °C; λmax) 320
nm; TLC (EtOAc/hexane, 1:1) Rf) 0.5; IR (KBr) 3304, 2926, 1710, 1607, 1512, 1344, 1279, 1185 cm-1;1H NMR (CDCl 3, 400 MHz) δ 9.17 (1 H, s), 8.67 (1 H, d, J ) 9.2 Hz), 8.24 (1 H, d, J ) 2.4 Hz), 8.14 (1 H, dd, J ) 9.2, 2.4 Hz), 7.60 (2 H, d, J ) 8.9 Hz), 7.35-7.31 (3 H, m), 7.30-7.27 (2 H, m), 6.65 (2 H, d, J ) 8.9 Hz), 6.41 (1 H, d, J ) 7.1 Hz), 5.08-5.06 (1 H, m), 3.34 (2 H, d, J ) 7.1 Hz), 3.04 (6 H, s);13C NMR (CDCl 3, 100 MHz) δ 170.5 (C), 168.1 (C), 152.9 (C), 143.0 (C), 140.4 (C), 136.2 (CH), 129.3 (C), 129.0 (CH, 2× ), 128.7 (CH, 2 × ), 127.4 (CH, 2 × ), 124.7 (CH), 123.3 (CH), 122.9 (C), 120.4 (C), 119.2 (CH), 111.0 (CH, 2× ), 55.6 (CH), 40.0 (CH3, 2× ), 36.7 (CH2); FAB MS m/z 467.1 (M+ + H); HRMS calcd for C24H24ClN4O4:
467.1486 (M++ H); found: 467.1488; Anal. Calcd for C 24H23
-ClN4O4: C 61.74, H 4.97, N 12.00; found: C 61.71, H 5.01, N
11.96.
Inhibition Assay against the SARS-CoV 3CL Protease. A fluorometric assay17was utilized to determine the inhibition
constants of the prepared samples. Briefly, a fluorogenic peptide Dabcyl-KTSAVLQSGFRKME-Edans is used as the substrate, and the enhanced fluorescence due to cleavage of this substrate catalyzed by the protease was monitored at 538 nm with excitation at 355 nm. The IC50value of individual
sample was measured in a reaction mixture containing 50 nM of the SARS 3CL protease and 6 µM of the fluorogenic substrate in 20 mM Bis-Tris (pH 7.0). The enzyme stock solution was kept in 12 mM Tris-HCl (pH 7.5) containing 120 mM NaCl, 0.1 mM EDTA, and 1 mM DTT plus 7.5 mM β-ME before adding to the assay solution. The Kimeasurements were
performed at two fixed inhibitor concentrations and various substrate concentrations.
Acknowledgment. We thank National Science
Coun-cil (Taiwan) for financial support, and Shih-Jia Shiao
and Su-Lung Tang for preparation of some anilide
inhibitors.
Supporting Information Available: Physical and spec-troscopic properties of new compounds, inhibition assay, HPLC and UV-vis analyses, molecular modeling, and NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org.
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