Diarylheptanoids from Alnus japonica inhibit papain-like protease of severe acute respiratory syndrome coronavirus

Article abstract of DOI:10.1248/bpb.b12-00623

The papain-like protease (PL^pro), which controls replication of the severe acute respiratory syndrome coronavirus (SARS-CoV), has been identified as a potential drug target for the treatment of SARS. An intensive hunt for effective anti-SARS dr

Full text of DOI:10.1248/bpb.b12-00623

2
036
Regular Article
Biol. Pharm. Bull. 35(11) 2036–2042 (2012)
Vol. 35, No. 11
Diarylheptanoids from Alnus japonica Inhibit Papain-Like Protease of
Severe Acute Respiratory Syndrome Coronavirus
a,b
a
a
a
a
Ji-Young Park, Hyung Jae Jeong, Jang Hoon Kim, Young Min Kim, Su-Jin Park,
b
c
,a
,a
Doman Kim, Ki Hun Park, Woo Song Lee,* and Young Bae Ryu*
a
Infection Control Material Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB);
b
Jeongeup 580–185, Republic of Korea: School of Biological Science and Biotechnology, Chonnam National
University; Gwangju 500–757, Republic of Korea: and Division of Applied Life Science (BK 21 Program, IALS),
c
Graduate School of Gyeongsang National University; Jinju 660–701, Republic of Korea.
Received July 17, 2012; accepted August 28, 2012; advance publication released online September 3, 2012
pro
The papain-like protease (PL ), which controls replication of the severe acute respiratory syndrome
coronavirus (SARS-CoV), has been identified as a potential drug target for the treatment of SARS. An in-
tensive hunt for effective anti-SARS drugs has been undertaken by screening for natural product inhibitors
pro
that target SARS-CoV PL . In this study, diarylheptanoids 1–9 were isolated from Alnus japonica, and the
pro
inhibitory activities of these compounds against PL were determined. Of the isolated diarylheptanoids,
pro
hirsutenone (2) showed the most potent PL inhibitory activity, with an inhibitory concentration (IC )
5
0
value of 4.1µM. Structure–activity analysis showed that catechol and α,β-unsaturated carbonyl moiety in the
molecule were the key requirement for SARS-CoV cysteine protease inhibition.
Key words diarylheptanoid; Alnus japonica; severe acute respiratory syndrome; papain-like protease;
cysteine protease
Severe acute respiratory syndrome (SARS), a contagious of natural products based on 1,7-diphenylheptane skeleton.
and often fatal respiratory illness, was first reported in Alnus japonica and its constituents exhibit various biological
Guandong province, China, in November 2002. Its rapid and properties, including anti-inflammatory, anticancer, and anti-
16–18)
unexpected spread to other Asian countries, North America, influenza activities.
To the best of our knowledge, the
and Europe alarmed both the public and the World Health Or- study of this plant with respect to SARS-CoV papain-like
ganization (WHO). SARS is a life-threatening form atypical cysteine protease inhibition has never been reported. In this
pneumonia caused by infection with a novel human corona- study, we expressed and purified in Escherichia coli (E. coli)
1–3)
pro
virus (SARS-CoV).
SARS-CoV is a plus-strand RNA the full length PL as well as truncated forms containing
virus that encodes four structural proteins, 16 nonstructural only the catalytic domains.
2
,4)
proteins, and 8 accessory proteins.
The 16 nonstructural
proteins are the cleavage products of the 2 large polypeptides MATERIALS AND METHODS
pp1a and pp1ab and are generated by the virally encodes pro-
pro
1
13
teases 3-chymotrypsin-like protease (3CL ) and papain-like
protease (PL , EC 3.4.22.46).
General H- and C-NMR along with 2D-NMR data
pro
5–8)
These proteases are consid- were obtained on JNM-EX 400 (Jeol, Japan) spectrometers in
ered the most prominent and effective drug targets in antiviral methaol-d and tetramethylsilane (TMS) as internal standards.
6
therapies. Because proteolytic processing is essential for the Electrospray ionization (ESI) mass spectra were scanned
pro
generation of a functional replication complex, 3CL and using ESI in negative or positive mode with bruker esquire
pro
PL are potentially effective targets for anti-SARS drugs. 6000 mass spectrometer. All of the reagent grade chemicals
Most efforts reported to date have focused on the development were purchased from Sigma-Aldrich Chemical Co. (St. Louis,
pro
of 3CL inhibitors, which have been identified in both syn- MO, U.S.A.). Chromatographic separations were carried out
9
–11)
12,13)
thetic peptidyl libraries
and natural derived libraries.
by thin-layer chromatography (TLC) (E. Merck Co., Dar-
pro
Fewer inhibitors of PL have been studied, and those that mastdt, Germany), using commercially available glass plates
have been studied include thiopurine analogs and benzamide precoated with silica gel and visualized under UV at 254
14,15)
derivatives.
No naturally derived inhibitor of this protease and 366nm. Column chromatography was carried out using
has been reported previously. In the study described in this 230–400 mesh silicagel (Kieselgel 60, Merck, Germany).
letter, an intensive hunt for effective anti-SARS drug was un- RP-18 (ODS-A, 12µm, S-150Å, YMC) and Sephadex LH-20
dertaken by screening natural products for inhibitory activity (Amersham Biosciences) were used for column chromatogra-
pro
against SARS-CoV PL
.
phy.
Plant Material and Preparation of Extracts The air-
Natural products have been found to be an important
source of drug and drug leads. As part of our screening of dried bark of Alnus japonica (3.0kg) was extracted with
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SARS-CoV PL inhibitors from natural sources, we found 95% ethanol (2×10L) at room temperature for a week. The
that an ethanol extract of the stem bark of Alnus japonica ex- combined extract was concentrated in vacuo to give a dark
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hibited PL inhibitory activity. Alnus japonica (Betulaceae) residue (122g). The crude extract was suspended in water
has led to the identification of numerous diarylheptanoids. Di- and successively partitioned with organic solvents (hexane
arylheptanoids, such as curcumin, belong to a phenolic class and ethyl acetate) of the different polarities to obtain frac-
tions of hexane (13g), ethyl acetate (36g) and H O (69g). The
2
The authors declare no conflict of interest.
ethyl acetate fraction (36g) was fractionated over a silica gel
*
To whom correspondence should be addressed. e-mail: wslee@kribb.re.kr; ybryu@kribb.re.kr
© 2012 The Pharmaceutical Society of Japan

November 2012
2037
Fraction 3 (1.6g) was chromatographed by Sephadex LH-20
to give five fractions (fr. 31–35). Fraction 32 was subjected
to RP-C18 chromatography column chromatography (mobile
phase: 85% acetonitile) yielding five fractions (fr. 321–325).
Fractions 321–323 were identical to compound 2 (350mg). Fi-
nally, fraction 8 (1g) was purified via Sephadex LH20 column
chromatography (mobile phase: MeOH) to give six fractions
(fr. 81–86). Then, 62mg of compound 9 were gained from fr.
8
4. The physical and spectroscopic data of compounds 1–9
14,16)
agree with those previously published
for platyphyllenone
(1), hirsutenone (2), platyphyllone (3), platyphyllonol-5-xylo-
pyranoside (4), hirsutanonol (5), oregonin (6), rubranol (7),
rubranoside B (8) and rubranoside A (9) (Fig. 1).
Synthesis of Compounds 10 and 11 The keto-enol deriv-
atives (10, 11) were synthesized from curcumin, as shown in
Chart 1. A solution of curcumin (200mg, 0.54mmol) in dry
CH Cl2 (30mL) was stirred at −50°C for 10min and BBr3
2
(
2.5mL, 17% in CH Cl ) was slowly added. On completion of
2 2
the reaction, water was added and the mixture was extracted
with EtOAc. The combined organic phase was washed with
H O, dried over anhydrous MgSO . The solvent was evapo-
2
4
rated, and then the crude product was subjected to purification
through silica gel column chromatography (n-hexane:ethyl
acetate=1:1) to afford 10 in 20% yields. For selective reduc-
tion of α,β-carbonyl group in curcumin, platinum on activated
charcoal (25mg, 10% Pt basis) was added methanol solution
Fig. 1. Chemical Structures of Isolated Diarylheptanoids from A. ja-
ponica (xyl: β-D-Xylopyranosyl and glc: β-D-Glucopyranosyl)
(
50mL) containing curcumin (1.0g, 2.7mmol) at room tem-
perature and the reaction mixture was stirred at room temper-
column (10×30cm, 230–400 mesh; Merk) using a gradient of ature for 8h at H atmosphere. On completion of the reaction,
2
chloroform–acetone (30:1→1:1, v/v) to yield nine fractions the suspended material was removed by filtration and filtrate
(fr. 1–fr. 9) based on TLC profile. Fraction 6 (1.0g) was chro- was evaporated, and then the crude product was subjected to
matographed over a Sephadex LH-20 column (2×90cm) using purification through silica gel column chromatography (n-hex-
MeOH as eluting solvent to give four fractions (fr. 61–fr. 64); ane:ethyl acetate=1:1) to afford tetrahydrocurcumin in 40%
fr. 62 and fr. 63 (235.7mg) was resubjected to RP-18 (ODS-A, yields. Tetrahydrocurcumin was subjected to demethylation in
1
2nm, S-150mM, YMC) chromatography to yield give similar manner to that of compound 10 to give compound 11
compound 6 (200mg). The fraction 7 (7.2g) was separated (25%).
through chromatography on Sephadex LH-20 column (mo- Preparations of SARS-CoV 3CL
pro
pro
and PL
The
bile phase: MeOH), a RP-C18 chromatography column and two genes encoding the 3CL protease and PL protease of
preparative-HPLC to yield compounds 1 (27mg), 3 (49mg) SARS-CoV were designed based on the sequence reported
19)
20)
and compound 4 (54mg). Next, compounds 7 (8mg) and 8 by Sun et al. and Han et al. The synthesized genes were
pro
pro
(
38mg) were isolated from fraction 5 (1.6g) using a Sepha- inserted into protein expression vector so that 3CL and PL
dex LH-20 column and a RP-C18 chromatography column. are tagged with 6× histidine at the C-terminus. Expression
Reagents and conditions: a) H /Pt-C, MeOH; b) BBr , CH Cl , −50°C.
2
3
2
2
Chart 1. Catalytic Hydrogenation and Demethylation of Curcumin

2
038
Vol. 35, No. 11
pro
pro
Fig. 2. (A) Purification of SARS-CoV PL ; (B) Lineweaver–Burk Plot for the Determination of the PL Km Value
(
A) M, protein molecular-weight markers (kDa); CL, cell lysate; FT, flow-through; W1, 20mM imidazole wash; W2 and W3, 50mM imidazole wash; E1, E2 and E3,
00–300-mM imidazole elution; 1, concentrated protein using an Amicon Ultra 10,000 MW cut-off. (B) The reaction was done at various substrate concentrations to obtain
Km value of the enzyme. SigmaPlot was used to fit the kinetic data using Lineweaver–Burk double reciprocal plots.
1
pro
pro
of recombinant 3CL and PL were induced with 0.5mM of inhibitor to obtain the IC50 values by properly fitting the
1
3)
isopropyl β-D-thiogalactopyranoside in E. coli BL21 (DE3) data according to the analysis method previously reported.
pro
pro
CodonPlus-RIL (Stratagene, La Jolla, CA, U.S.A.) during mid-
SARS-CoV PL Inhibition Assay The PL inhibition
2
2)
log phase. The cells were grown at 16°C for 12h, followed assay applied report by Ratia et al. The inhibition assay was
by sonication in buffer A (20mM sodium phosphate, 300mM optimized in a 96-well plate format to establish suitable assay
NaCl, 10mM imidazole, 0.2% Triton X-100, pH 7.5), and cen- conditions and incubation times. The fluorogenic peptide sub-
trifuged at 15000×g for 30min. The supernatant was loaded strate, Arg-Leu-Arg-Gly-Gly-AMC (RLRGG-AMC), was pur-
on a Ni-Sepharose column equilibrated with buffer A, and the chased from Bachem Bioscience. The enhanced fluorescence
recombinant protein was eluted by buffer A with 100–200mM emission upon substrate cleavage was monitored at excitation
imidazole. The purified proteases ran approximately at the cal- and emission wavelengths of 360 and 460nm, respectively, in
pro
pro
2e
culated size of 33kDa (3CL ) and 36.5kDa (PL , Fig. 2A) a SpectraMax M Multimode Reader (Molecular Devices Co.,
pro
on sodium dodecyl sulfate-polyacrylamide gel electrophoresis U.S.A.). The reaction mixture contained 54nM PL in 20mM
(SDS-PAGE). The elution buffer was then changed to 20mM Tris–HCl buffer (pH 6.8) in a total volume of 200µL. After
Tris–HCl buffer (pH 7.5) with 1mM dithiothreitol, and the the addition of 30µM substrate to the reaction mixture, the in-
protein was concentrated using an Amicon Ultra filter with crease in fluorescence at 460nm was continuously monitored
pro
a 10000 molecular weight cut-off (Millipore, Billerica, MA, at 37°C. For the inhibition studies, 54nM PL and 0–200µM
U.S.A.). The enzyme concentration was determined from its of the individual compounds were mixed with the substrate
absorbance at 280nm. To calculate the kinetic parameters of (30µM) at 37°C, and the fluorescence intensity was monitored.
pro
pro
the purified 3CL and PL , enzyme activity was analyzed For the determination of enzyme activity (fitting experimental
with fluorescent substrate from 1.25 to 100µM. The K value data to the logistic curve by (Eq. 1), used time-drive protocol
m
pro
pro
were 32.0± 5.2µM (3CL ) and 38.3± 1.8µM (PL , Fig. 2B). with initial velocity was recorded over a range of concentra-
The purified proteins were stored at −80°C before use in any tions and the data were analyzed using a nonlinear regression
of the assays.
SARS-CoV 3CL
the inhibitory effects of each compound on the enzymatic
activities of 3CL was evaluated using purified enzyme and
program [Sigma Plot 10.0 (SPCC Inc., Chicago, IL, U.S.A.)].
pro
21)
Inhibition Assay As described,
Activity (%)=100[1/ (1+([I] / IC50))]
(1)
pro
The inhibition mechanism was determined, and the appar-
pro
fluorogenic substrate peptide. In this assay, the 12-mer fluoro- ent inhibition constants (K ) for the respective PL enzymes
i
genic peptide Dabcyl-KNSTLQSGLRKE-Edans (Anygen Co., were performed on the test compounds, for which the IC50
Republic of Korea) was used as a substrate, and the enhanced values were below 200µM. In general, the test compounds
fluorescence due to cleavage of this substrate catalyzed by the were studied at three different concentrations that were chosen
pro
protease was measured at 590/40nm with excitation 360nm based on the IC50 values obtained with each PL enzyme
using a fluorescence plate reader (BioTeck Instrument Inc., (approximately 1/2×IC , IC , 2×IC ). The concentrations of
5
0
50
50
U.S.A.). The inhibitory concentration (IC ) values of isolated marker substrates were chosen (approximately 1/4 K , 1/2 K ,
5
0
m
m
compounds were measured in a reaction mixture containing K ) with regard to their Michaelis–Menten kinetics (Km and
m
pro
1
0µg/mL of the 3CL (final concentration, 2.5µg), the test Vmax). The K values were calculated by nonlinear regression
i
compounds (from 0 to 200µM), and 10µM of fluorogenic 12- analysis by fitting different models of enzyme inhibition to
mer peptide substrate in 20mM Bis-Tris buffer (pH 7.5). The the kinetic data using SigmaPlot Enzyme Kinetics Module 1.3
reactions were run for 60min at 37°C while continuously (SPSS Inc., Chicago, IL, U.S.A.). The inhibition mechanism
monitoring the fluorescence. The initial velocities of the inhib- of compounds were determined by comparing the statistical
ited reactions were plotted against the different concentrations results including the Akaike’s information criterion values of

November 2012
2039
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Fig. 3. (A) Effects of Diarylheptanoids (2, 5–9) in the Activity of SARS-CoV PL and (B) Relationship of the Hydrolytic Activity of SARS-CoV
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PL Concentrations at Different Concentrations of Compound 2
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Table 1. SARS-CoV PL Inhibitory Activity of Diarylheptanoid Derivatives (1–11) and Curcumin
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a)
Compound
SARS-CoV PL , IC50 (µM)
Inhibition type
Km
Ki
b)
1
2
3
4
5
6
7
8
9
>200
4.1± 0.3
>200
NT
NT
14.9± 0.9
NT
NT
10.1± 0.4
NT
Noncompetitive
NT
>200
NT
NT
NT
7.8± 1.7
20.1± 2.2
12.3± 0.9
8.0± 0.2
9.1± 1.0
6.2± 2.2
59.8± 1.3
5.7± 0.3
Mixed-type
Noncompetitive
Uncompetitive
Noncompetitive
Noncompetitive
NT
16.8± 2.7
9.8± 6.7
20.2± 2.0
18.6± 3.3
23.2± 4.6
NT
13.2± 3.6
21.5± 3.5
9.2± 0.6
18.0± 1.9
39.3± 0.4
NT
1
1
0
1
NT
NT
NT
Curcumin
NT
NT
NT
a) All compounds were examined in a set of experiments repeated three times; IC50 values of compounds represent the concentration that caused 50% enzyme activity loss.
b) Not tested.
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different inhibition models and selecting the one with the best Table 2. SARS-CoV 3CL and Ubiquitinase Inhibitory Activity of Iso-
2
3)
lated Diarylheptanoids (1–9)
fit.
SARS-CoV PL
pro
Deubiquitination Assay For the
SARS-CoV 3CLpro, IC50
Deubiquitination
activity, IC50 (µM)
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deubiquitination assay, purified PL (54nM) was mixed with
fixed compound concentration of of 0–200µM before the
substrate, Ubiquitin-AMC (Enzo Life sciences Inc., U.S.A.),
was added in 20mM Tris–HCl buffer (pH 6.8). All assays
were performed at 37°C in 96-well plate format. The enzyme
activities were determined by monitoring the enhanced fluo-
rescence emission upon substrate cleavage at excitation and
emission wavelengths of 360 and 460nm, respectively, in a
Compound
a)
(µM)
1
2
3
4
5
6
7
8
9
>200
36.2± 2.0
>200
>200
3.0± 1.1
>200
>200
>200
105.6± 6.5
129.5± 3.1
144.6± 4.8
105.3± 1.7
102.1± 2.5
24.1± 2.0
44.5± 5.3
35.2± 1.7
7.2± 2.2
14.4± 3.0
2
e
SpectraMax M Multimode Reader (Molecular Devices Co.,
U.S.A.). The substrate concentration was 100nM, and release
of AMC was measured in the same manner as for the IC
5
0
2
2)
a) All compounds were examined in a set of experiments repeated three times; IC50
values of compounds represent the concentration that caused 50% enzyme activity
loss.
measurements described above.
RESULTS AND DISCUSSION
(
2), platyphyllone (3), platyphyllonol-5-xylopyranoside (4),
pro
SARS-CoV PL inhibitory activity-guided fractionation of hirsutanonol (5), oregonin (6), rubranol (7), rubranoside B (8)
14,16)
the ethanol extract led to the isolation of nine diarylheptanoids and rubranoside A (9), respectively.
1–9) (Fig. 1). The isolated diarylheptanoids 1–9 were identi- 1–9 were tested against SARS-CoV PL using a continu-
fied by spectroscopic data as platyphyllenone (1), hirsutenone ous fluorometric assay. Of the isolated compounds (1–9), six
Isolated compounds
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(

2
040
Vol. 35, No. 11
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Fig. 4. Lineweaver–Burk Plots for the Inhibition of the Proteolysis of SARS-CoV PL by Compounds [2 (A), 5 (B), 6 (C), 7 (D), 8 (E), 9 (F), Re-
spectively]
compounds (2, 5–9) of isolated diarylheptanoids showed a a β-hydroxyl carbonyl, and only a hydroxyl moiety in the C7
pro
dose-dependent inhibitory effect against the PL (Fig. 3A). aliphatic molecule are compared, the α,β-unsaturated carbonyl
The diarylheptanoids were found to be reversible inhibitors moiety (2, IC =4.1µM) proved to be 2-fold more effective
5
0
because an increase in concentration rapidly reduced enzyme than the other moieties (5, 7.8µM, 7, 12.3µM, respectively). In
pro
activity. Inhibitors with PL inhibitory activity showed a the commonly adopted mechanism of inhibition of cysteine
similar relationship between enzyme activity and enzyme protease, a covalent bond is formed between the carbonyl
concentration [e.g., see Fig. 3B for hirsutenone (2)]. The in- group located at the warhead of the inhibitor and the active
hibitory activities of the first series of naturally derived di- site cysteine residue in the enzyme. Improving the electro-
pro
arylheptanoids against SARS-CoV PL are reported in Table negativity at this warhead carbonyl moiety could accelerate
1. The inhibitory activities are expressed as IC50 values of the the covalent bond formation. The α,β-unsaturated carbonyl
pure compounds 1–9. The known viral protease inhibitor cur- is a good Michael acceptor and undergoes nucleophilic ad-
cumin was used as a reference inhibitor (IC50 value of 5.7µM). dition. Thus, the above finding suggests that the α,β-unsat-
As shown in Table 1, the bioactivity of these compounds urated carbonyl and catechol groups may play a pivotal role
pro
pro
depended on the substituted groups in the diaryheptanoid in SARS-CoV PL inhibition by interacting with the PL
scaffold. Of the tested diarylheptanoids 1–9, compound 2, nucleophiles.
containing an α,β-unsaturated carbonyl group with a catechol
To determine the importance of the α,β-unsaturated carbo-
moiety in the backbone, was the most effective inhibitor, with nyl moiety in diarylheptanoid to the inhibition of SARS-CoV
pro
an IC50 value of 4.1µM. Mono hydroxyl substitution on the PL , we synthesized the corresponding keto-enol derivatives
2
4)
terminal aromatic ring had a substantial effect on the inhib- (10, 11) from curcumin, as shown in Chart 1. As shown in
itory activity [compare, for example, 1 (IC50 >200µM), 2]. Table 1, the demethylated curcumin 10, with two α,β-unsatu-
All mono hydroxyl-substituted diarylheptanoids (1, 3, 4) are rated carbonyls in the molecule, exhibits potency comparable
pro
poorer inhibitors of PL than the corresponding compounds to those of the corresponding compound 2 and curcumin.
(2, 5, 6) with the catechol moiety. When the three classes of However, the incorporation of a keto-enol into tetrahydro-
diarylheptanoids (2, 5, 7) having an α,β-unsaturated carbonyl, curcumin afforded a less potent derivative (compound 11,

November 2012
2041
IC =59.8µM).
finding demonstrate that the catechol and α,β-unsaturated car-
5
0
When we compared diarylheptanoids with diarylheptanoid bonyl moieties in diarylheptanoid are critical factors affecting
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glycosides, we found that the activities of diarylheptanoids SARS-CoV PL inhibition activity. The IC50 value of this
were higher than were those of their corresponding glycosides. inhibitor, although higher than those of synthetic PL inhib-
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Diarylheptnoids with glucopyranoside and xylopyranoside itors, is nonetheless in the low micromolar range. We believe
moieties, 4 (IC50 >200µM) and 6 (IC =20.1µM), respectively, that this lead structure will facilitate the design of more effec-
50
had higher IC50 values than other diarylheptanoids (1–3, 5). tive inhibitors against SARS-CoV infection.
Bulky substituent like sugar moieties in the frame of the
diarylheptanoid resulted in higher IC50 values. However, the
Acknowledgements This research was supported by
substitution of the glycosides affecting the IC50 of the only National Research Foundation Grant founded by Korea gov-
hydroxyl group substituted in the C7 frame (7–9) cannot be ernment (MEST) (No. 2012-0001110) and KRIBB Research
ruled out. Compounds 8 and 9 exhibited modest activity, with Initiative Program, Republic of Korea.
IC50 of values 8.0 and 9.1µM, similar to that of the correspond-
ing inhibitor 7 (IC =12.3µM).
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5
0
To determine if the isolated compounds described above
pro
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pro
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8)
deubiquitinating (DUB) enzymes. It has become clear that
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we observed the DUB activity for in vitro characterization.
Table 2 lists the in vitro activity of compounds 1–9 against
DUB. The IC50 values demonstrated that the presence of
naphthalene in 2 (IC =3.0µM) and 8 (IC =7.2µM) provide
4)
5
)
5
0
50
a greater inhibitory effect than the other diarylheptanoid de-
rivatives. In a similar vein, compounds 1, 3, and 4, bearing
mono hydroxyl substitution on the terminal aromatic ring,
showed any inhibitory activity (IC50 >200µM). To date, there
is no potent and selective chemical compound available for the
inhibition of DUB. Thus, further investigation is necessary to
determine the specifics of the inhibition with the diarylhepta-
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noids and the PL -DUB interaction
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2
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