Discovery and SAR studies of methionine-proline anilides as dengue virus NS2B-NS3 protease inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.10.071

A series of methionine-proline dipeptide derivatives and their analogues were designed, synthesized and assayed against the serotype 2 dengue virus NS2B-NS3 protease, and methionine-proline anilides 1 and 2 were found to be the most active DENV 2 NS2B-NS3 competitive inhibitors with K_i values of 4.9 and 10.5 μM. The structure and activity relationship and the molecular docking revealed that l-proline, l-methionine and p-nitroaniline in 1 and 2 are the important characters in blocking the active site of NS2B-NS3 protease. Our current results suggest that the title dipeptidic scaffold represents a promising structural core to discover a new class of active NS2B-NS3 competitive inhibitors.

Full text of DOI:10.1016/j.bmcl.2013.10.071

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6549–6554
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and SAR studies of methionine–proline anilides as dengue
virus NS2B-NS3 protease inhibitors
a
b
c
a
d
a
Guo-Chun Zhou a, , Zhibing Weng , Xiaoxia Shao , Fang Liu , Xin Nie , Jinsong Liu , Decai Wang ,
⇑
Chunguang Wang b, Kai Guo a,
⇑
a School of Pharmaceutical Sciences, and College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, PR China
b Institute of Protein Research, Tongji University, Shanghai 200092, PR China
c School of Life Sciences, Lanzhou University, Lanzhou 730000, PR China
d Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of methionine–proline dipeptide derivatives and their analogues were designed, synthesized and
assayed against the serotype 2 dengue virus NS2B-NS3 protease, and methionine–proline anilides 1 and 2
were found to be the most active DENV 2 NS2B-NS3 competitive inhibitors with Ki values of 4.9 and
Received 15 August 2013
Revised 10 October 2013
Accepted 31 October 2013
Available online 8 November 2013
10.5 lM. The structure and activity relationship and the molecular docking revealed that L-proline, L-
methionine and p-nitroaniline in 1 and 2 are the important characters in blocking the active site of
NS2B-NS3 protease. Our current results suggest that the title dipeptidic scaffold represents a promising
structural core to discover a new class of active NS2B-NS3 competitive inhibitors.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Dengue virus NS2B-NS3 protease
Methionine–proline anilides
Proline
Methionine
4-Nitroaniline
Dengue viruses (DENVs) are classified as the Flavivirus genus1
and are causative pathogens of mild to severe epidemic diseases
like dengue fever (DF), dengue hemorrhagic fever (DHF), and den-
gue shock syndrome (DSS).2,3 These diseases are transmitted by
Aedes aegypti mosquitoes and are threatening up to 2.5 billion peo-
ple in more than 100 countries in tropical and subtropical regions.
Until now, there is no specific treatment for dengue diseases.4,5
There are four distinctive, but closely related, serotypes of den-
gue viruses named as DENV-1, DENV-2, DENV-3 and DENV-4.
Recovery from infection by one serotype of DENV provides lifelong
immunity against that particular serotype, however, cross-immu-
nity to the other serotypes after recovery is only partial and tempo-
rary, and more dangerously, subsequent infection by any of the
other three serotypes will enhance the risk of developing severe
dengue.5,6 This antibody-dependent enhancement effect (ADE) of
DENVs makes the vaccine development extremely difficult, which
is the major reason for no effective vaccine against DENV/s up to
now.6 Therefore, targeting DENV NS2B-NS3 protease using small
molecule drugs is an urgent and important antiviral therapeutic
treatment.
DENV is an enveloped RNA virus with ꢀ11 kb positive strand
RNA genome encoding a single polypeptide, which is subsequently
processed by the virus-encoded trypsin-like NS2B-NS3 protease to
generate three structural proteins (capsid protein, precursor-
membrane protein, and envelope protein) and seven nonstructural
proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).7,8 The
184-residue NS3pro is the enzymatic domain with
a serine
protease catalytic triad (His51, Asp75, and Ser135). Optimal
catalytic activity of NS3 depends on the presence of NS2B.9 Since
the NS2B-NS3 protease is essential for the maturation and
pathogenesis of dengue virus, it is considered to be a promising
target for antidengue virus drug development.10,11
Many efforts have been made to develop antidengue virus
NS2B-NS3 agents.10,12 In the light of the fact that the active site
of NS3-NS2B is shallow and prefers to cleave substrates with diba-
sic amino acids in vitro,9 we screened our lab’s chemical library
biased on compounds with multi-substituted and strained ring/s
and varied potential binding sites and some hits were discovered.
Among these hits, two modified dipeptides of methionine–proline
Abbreviations: SAR, structure and activity relationship; NS2B-NS3, nonstructural
protein 2B and 3; DENV, dengue virus; Ki, inhibition constant; TFA, trifluoroacetic
acid; EtOAc, ethyl acetate; DCM, dichloromethylene; EtOH, ethyl alcohol; DMAP, 4-
(N,N0-dimethyl)-aminopyridine; DCC, dicyclohexylcarbodiimide.
⇑
Corresponding authors.
E-mail addresses: gczhou@njut.edu.cn (G.-C. Zhou), kaiguo@njut.edu.cn (K. Guo).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.10.071

6550
G.-C. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6549–6554
Firstly, we investigated the structural requirements in 1 by the
H
N
NO2
replacement of p-nitro-aniline with different nitro-substituted
phenyl amines or benzyl amines. In order to assess the importance
of the nitro group and the effect of different nitro position at the
aniline on the inhibitory activity, a series of dipeptide compounds
(12b, 12c and 12d) were synthesized from commercially available
starting materials of o-nitro- and m-nitro-anilines (4 and 5) and
aniline (6) (Scheme 1). The dipeptide derivatives 12e and 12f of
p-nitro-benzylamine (7) and benzylamine (8) (Scheme 1), respec-
tively, were also prepared to evaluate the effect of elongation on
activity.
N
XHN
O
O
1, X = H
S
2, X = Boc
Figure 1. Structures of two dipeptide inhibitors.
anilides 113 and 213 (Fig. 1) were demonstrated as good NS2B-NS3
protease inhibitors with Ki values of 4.9 and 10.5 M, respectively,
l
Considering that the chirality of drug isomers sometimes plays
the important role in the recognition between the drug and its tar-
get/s and different stereochemistry probably results in distinctive
binding capacity, we studied the contribution and potential utility
of the stereochemistry of proline to the anti-NS2B-NS3 enzymatic
activity after the first run. Starting from 4-nitroaniline, D-proline
was used to synthesize the dipeptide 12g (Scheme 1) as a part of
our exploration.
and 1 with an N-terminal free amine is more active than 2 with an
N-terminal carbamate.
In our aims to search for small molecular entities targeting den-
gue virus NS2B-NS3 protease, we launched our preliminary SAR
studies on these dipeptides as the first step. In this Letter, we
describe the design, synthesis and evaluation of a series of com-
pounds based on lead dipeptide compound 1 and their structure–
activity relationship (SAR).
To probe the feature of methionine in 1, modifications on
methionine moiety of 1 were employed. Sulfoxide 14a and sul-
phone 16a derivatives of 1 were prepared by mCPBA oxidation
of 2 followed by HCl-deprotection of Boc group (Scheme 1).
Trifluoroacetyl amide 17a was generated by refluxing trifluoro-
acetic salt of 1 in toluene (Scheme 1). The N-alkylated deriva-
The title modified dipeptides were synthesized according to the
general methodology for polypeptide synthesis and the synthesis
of the desired compounds is outlined in Schemes 1 and 2. Boc-pro-
tected L- or D-proline was reacted with an amine to produce a Boc-
protected amide, followed by deprotection of the Boc group to
yield a free amine, later the free amine group was coupled with
Boc-protected methionine to generate the title Boc-protected
dipeptide, and at last the title amine of dipeptide was yielded after
the deprotection of Boc group. Their inhibitory activities were
tested against dengue virus type 2 NS2B-NS3 protease. The struc-
tures and their inhibitory activities were summarized in Tables 1
and 2.
tives (18a and 19a) of
amination of 1 (Scheme 1) using NaBH3CN. In addition, com-
pounds 21, 22, 24, 25 and 26 (Scheme 2), which -phenylala-
nine and -arginine substituted for -methionine, respectively,
1 were synthesized from reductive
L
L
L
were designed to explore the favor of methionine for the inhib-
itory activity of 1.
R
R
R
DCC
TFA
H
N Y
H
OH
in DCM
in DCM
+
H2N Y
N Y
N
N
0 oC, ~1h
rt, ~ 5h
~ 70%
N
Boc
O
H
Boc
O
3, 4-nitroaniline
4, 3-nitroaniline
5, 2-nitroaniline
6, aniline
7, 4-nitrobenzylamine
8, benzylamine
O
L-proline
or
D-proline
9a - 9g
10a - 10g
R
R
OH
BocHN
S
H
H
N Y
HCl (g) in EtOH
N Y
N
O
H2N
N
BocHN
S
rt, ~ 1h
~ 90%
O
O
O
O
DCC, DMAP
in DCM, rt, ~ 5h
~ 80%
1, 12b - 12g
S
2, 11b - 11g
4-Nitrobenzaldehyde (18) or
4-methoxybezaldehyde (19),
NaBH3CN/MeOH, rt
TFA in DCM
rt, ~ 2 h
mCPBA
13a from 2, 60%
15a from 2, 30%
H
N
NO2
H
N
N
NO2
XHN
N
XHN
S
O
O
O
O
Z
15a, Z = S(O2)
13a, Z = S(O)
X = Boc
HCl (g)
EtOAc
rt, ~ 1h
18a from 18,
1 from 2, X = H, 90%
X = Boc
85%
83%
X = 4-nitrobenzyl, 75%
19a from 19,
reflux in toluene
14a, Z = S(O)
16a, Z = S(O2)
X = 4-methoxybenzyl, 70%
X = H
17a from 1, X = COCF3, 20%
X = H
a, Y = empty, R = 4-nitro; b, Y = empty, R = 3-nitro; c, Y = empty, R = 2-nitro;
d, Y = empty, R = H; e, Y = CH2, R = 4-nitro; f, Y = CH2, R = H; g, Y = empty, R = 4-nitro
a -f, from L-proline; g, from D-proline
Scheme 1.

G.-C. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6549–6554
6551
OH
O
NHBoc
BocHN
H
H
H
N
N
OH
N
NO2
+
Pbf
+
N
H
NH
O
O
23, Boc-L-Arg (Pbf)-OH
20, Boc-L-Phe-OH
10a
DCC, DMAP
75%
DCC, DMAP
in DCM, rt, ~ 5h
80%
in DCM, rt, ~ 5h
X
H
H
N
N
NO2
NO2
HN
X
N
N
H
Z
HN
HN
HN
O
O
N
O
O
26
X = H
Z = H
25
X = H
Z = Pbf
24
X = Boc
Z = Pbf
21
22
X = H
X = Boc
85%
75%
80%
HCl in EtOH
rt, ~ 1h
TFA/DCM = 1/4
rt, ~ 1h
TFA/DCM = 1/1
rt, ~ 1h
Scheme 2.
Table 1
Chemical structures of dipeptides and their activities against DENV 2 NS2B-NS3 protease
(R) or (S)
R
H
X
HN
N Y
*
N
O
O
Z
Entry
Compd
R
X
Y
Z
(R) or (S)
Ki (
4.9
10.5
l
M)
Remaining activity of DENV 2 NS2B-NS3 (inhibitor concentration, lM)
1
2
3
4
5
6
7
8
9
10
11
12
13
1
2
4-NO2
4-NO2
3-NO2
2-NO2
H
H
Boc
H
H
H
H
H
H
Empty
Empty
Empty
Empty
Empty
CH2
S
S
S
S
S
S
S
S
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(R)
(S)
(S)
(S)
(S)
(S)
31% (15)
45% (15)
37% (20)
96% (20)
60% (200)
36% (200)
58% (15)
71% (20)
99% (20)
103% (20)
54% (15)
38% (20)
90% (20)
12b
12c
12d
12e
12f
12g
14a
16a
17a
18a
19a
4-NO2
H
CH2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
Empty
Empty
Empty
Empty
Empty
Empty
H
H
S(O)
S(O2)
S
S
S
CF3CO
40-NO2-Bn
40-MeO-Bn
With these compounds in hand, their inhibitory activities
against the serotype 2 DENV NS2B-NS3 protease were studied.
The serotype 2 DENV NS2B-NS3 protease was expressed with
the plasmid kindly provided by Dr. Siew Phengsuozeg Lim, Novar-
tis Institute for Tropical Diseases, Singapore.14 The inhibitory
activity of a compound against DENV2 NS2B-NS3 protease were
measured at 37 °C in the reaction buffer (50 mM Tris–HCl, pH
9.0, 10 mM NaCl, 20% glycerol, 1 mM CHAPS, and 0.04% NaN3)
and Benzoyl-Nle-Lys-Arg-Arg-AMC was used as a substrate.14,15
The protease was incubated with gradient concentrations of a
inhibitors, NS2B-NS3 with substrate, and NS2B-NS3 with substrate
and 2 or other inhibitors, respectively. From these data (Fig. S1 and
Supplementary data), we can conclude that there is no fluorescent
interference from these inhibitors.
As starting points, compounds 1 and 2 were firstly assayed and
discovered as good inhibitors against DENV 2 NS2B-NS3 protease
(Table 1). According to Dixon plot, it was found that both of
compounds 1 and 2 behave as competitive inhibitors for DENV 2
NS2B-NS3 (Fig. 2a and b). Unlike peptide NS2B-NS3 competitive
inhibitors derived from the substrates of NS2B-NS3,9,14,17 methio-
nine–proline anilide without basic residue is a new scaffold and
its binding to NS2B-NS3 should be in a distinctive mode. This find-
ing established a new direction to find NS2B-NS3 competitive
inhibitors. Based upon these results, the SAR studies of these
dipeptides were explored.
inhibitor at 37 °C for 3 min before the substrate (33 or 66 lM)
was added. The remaining enzyme activity was measured with
a fluorescence spectrophotometer using 356 and 438 nm as the
excitation and emission wavelengths, respectively. The inhibition
constant (Ki) value was determined with Dixon plot (1/v values
against concentrations of the inhibitor),16 using two different con-
As shown in Table 1,
exhibited much lower activity than 1 of
logue. This fact may result from the steric problem to the binding
to NS2B-NS3 after -proline incorporation and the result provides
D
-proline derivative 12g (Table 1, entry 8)
centrations of the substrate (33 and 66
l
M). The assay data were
L-proline counterpart ana-
averaged from triplicate measurements and are shown in Table 1
and Table 2.
D
In order to exclude the fluorescent interference from these
inhibitors, we also compared the fluorescence changes vs time
(Fig. S1) in three parallel experiments of NS2B-NS3 with 2 or other
us a clue that the chirality of proline is essential to the inhibitory
activity (Fig. 3). Because of this reason, our further studies in this
work focused on L-proline derivatives.

6552
G.-C. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6549–6554
Table 2
Chemical structures of dipeptides and their activities against DENV 2 NS2B-NS3 protease
X
HN
H
H
N
N
NO2
NO2
X
HN
N
N
H
N
Z
HN
O
O
O
O
HN
Entry
Compd
Residue
X
Z
Remaining activity of DENV 2 NS2B-NS3 (inhibitor concentration, lM)
1
2
3
4
5
21
22
24
25
26
Phe
Phe
Arg
Arg
Arg
Boc
H
Boc
H
H
H
Pbf
Pbf
H
25% (200)
31% (200)
90% (20)
106% (20)
93% (20)
H
Figure 2. Dixon plots of compounds 1 (a) and 2 (b) against DENV 2 NS2B-NS3 protease.
that the nitro group is important for the binding of inhibitors to
NS2B-NS3 and the p-position provides the most optimal binding
and the m-position is approximate to the optimal spatial binding
position but the o-position encounters some spatial restriction to
optimal binding position.
H
X
N Y
NO2
N
HN
O
O
It is interesting in understanding the linker Y’s influence on the
inhibitory activity (Scheme 1 and Table 1). Considering that p-nitro
group in 1 has priority to the inhibitory activity, benzyl (the linker
Y is methylene) derivatives 12e and 12f (Table 1, entries 6 and 7)
were assayed. Surprisingly and interestingly, compound 12e bear-
ing p-nitro group was inactive but compound 12f without p-nitro
group showed moderate activity compared to 1 and 2. This unex-
pected activity change suggests that the linker Y regulates the
binding to NS2B-NS3 in an unusual mode, which makes the p-nitro
group not favorable for the inhibitory activity (Fig. 3).
1, X = H, Y = empty, Ki = 4.9 µM
S
2, X = Boc, Y = empty, Ki = 10.5 µM
Red: o-substitution, (R)-configuration and
oxidation of thioether are forbidden
Green: some modifications are acceptable
Blue: occupation at m-position is acceptable
but less active than at p-position
Yellow: Y as methylene is not favorable
for p-nitro occupation
Figure 3. The SAR of the dipeptide NS2B-NS3 inhibitors.
Methionine residue-modified derivatives of 1 were examined
and revealed the well-established feature of 1. Oxidation of
thioether of 1 into sulfoxide (14a) and sulphone (16a) completely
destroyed the activity of 1 (Table 1, entries 9 and 10) and the
observation at least in part demonstrated the importance of
methionine in 1. The inhibitory activity of trifluoroacetyl amide
17a (Table 1, entry 11) was almost as potent as that of 2, suggest-
ing that 17a and 2 have the parallel variation trend of the activity
The relationship of the activity and R group (Scheme 1 and Ta-
ble 1) disclosed that the inhibitory activity of compound 12b with
m-nitro group (Table 1, entry 3) was lower than and still compara-
ble to compounds 1 and 2; however, the inhibitory activity of com-
pound 12c (Table 1, entry 4) with o-nitro group decreased sharply
and compound 12d (Table 1, entry 5) without nitro group showed
almost no activity (Fig. 3). These activity differences demonstrated

G.-C. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6549–6554
6553
Figure 4. Three-dimensional (3D) interaction diagram of putative binding pose of 1 (a) and superposition diagrams of 1 and 2 (b), 1 and 12g (c) in NS2B-NS3 active site
(His51, Asp75 and Ser135). The figures were generated from the chain A of 2FOM17 from the same view using Discovery Studio 3.5. Carbons and their bonds of 1 in (a), (b) and
(c) are in the green color, and carbons and their bonds of 2 in (b) or 12g in (c) are in the brick-red color. Hydrogen bonds are connected by the green dash lines. Critical amino
acid residues of the binding pocket are labeled.
compared to 1 and the binding capacity for the methionine termi-
nal amine and its modifications is flexible. Benzylation of the
methionine terminal amine led to two different results that 40-ni-
tro-benzylated analogue 18a (Table 1, entry 12) exhibited a good
inhibitory activity, which is close to the activity of Boc analogue
2; whereas, 40-methoxy-benzylated analogue 19a (Table 1, entry
13) lost most of the activity of 1. These results imply that 40-nitro
group exerts a positive influence on the binding and inhibitory
activity and it is possible that the binding mode of 18a and 19a
is quite different from that of 2 and 17a. On the other hand, com-
pounds 21–26 by replacement of methionine with phenylalanine
or arginine totally destroyed the inhibitory activity (Table 2, en-
tries 1–5). These data supported that the integrity of methionine
is indispensable to the inhibitory activity (Fig. 3), at least for this
title linear dipeptide.
To dicipher the structural basis for the above SARs, we com-
pared the putative binding poses of compounds 1, 2 and 12g to
NS2B-NS3 by the means of the molecular docking. The docking
model was generated from the chain A of 2FOM18 using Discovery
Studio 3.5 and the results are shown in Figures 4 and S3. In the fit-
ted low energy conformation, the most potent dipeptide 1 is bind-
ing close to the catalytic pocket (His51, Asp75 and Ser135) of
NS2B-NS3 protease (Fig. 4a and Fig. S3). The binding was stabilized
by hydrogen bonding network between the oxygen atom of proli-
nyl carbonyl group with Ser135 from the catalytic pocket, two
hydrogen atoms of methioninyl amine with Gly151 and Asn152
(Fig. 4a). As shown in Figure 4b and Figure S3c, compounds 1
and 2 are anchored in the similar binding pose but the N-Boc pro-
tection in 2 loses hydrogen bonding ability and also makes the
prolinyl carbonyl group far away from Ser135, which might ex-
plain the decrease of inhibitory activity observed in 2. In compar-
ison with 1, different configurations of prolinyl moieties make two
planes of the nitro groups in 1 and 12g be vertical and produce the
orientation discrepancy of two pyrrolyl rings and two methylthio
groups in 1 and 12g, and this predicted binding pose of 12g results
in loss of its binding and inhibitory activity.
ent data show in Table 1 and Table 2 that methionine–proline ani-
lides 1 and 2 are the most active DENV 2 NS2B-NS3 competitive
inhibitors with Ki values of 4.9 and 10.5
the molecular docking (Fig. 4 and Fig. S3) revealed that
-methionine and p-nitroaniline construct a solid structural scaf-
l
M. The SAR (Fig. 3) and
L-proline,
L
fold blocking the active site of NS2B-NS3 protease. These results
demonstrated that the current dipeptide scaffold represents a
promising structural core to discover a new class of active NS2B-
NS3 competitive inhibitors. We are still surprised by the structural
loyalty to the inhibitory activity and pursuing more analogues
against NS2B-NS3 protease.
Acknowledgments
We thank Dr. Siew Phengsuozeg Lim for kindly providing the
expression plasmid of DENV2 NS2B-NS3 protease. Authors thank
Dr. Qian Li for his kind help of the molecular docking. This work
was supported by Natural Science Foundation of China (Grant
U0632001).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
10.071. These data include MOL files and InChiKeys of the most
important compounds described in this article.
References and notes
1. Stevens, A. J.; Gahan, M. E.; Mahalingam, S.; Keller, P. A. J. Med. Chem. 2009, 52,
7911.
2. Rigau-Perez, J. G.; Clark, G. G.; Gubler, D. J.; Reiter, P.; Sanders, E. J.; Vorndam, A.
V. Lancet 1998, 352, 971.
3. Murgue, B. Microbes Infect. 2010, 12, 113.
4. http://www.who.int/mediacentre/factsheets/fs117/en/ (Fact sheet
January, 2012).
N 117,
5. Guzman, M. G.; Halstead, S. B.; Artsob, H.; Buchy, P.; Farrar, J.; Gubler, D. J.;
Hunsperger, E.; Kroeger, A.; Margolis, H. S.; Martinez, E.; Nathan, M. B.;
Pelegrino, J. L.; Simmons, C.; Yoksan, S.; Peeling, R. W. Nat. Rev. Microbiol. 2010,
8, S7.
6. Goncalvez, A. P.; Engle, R. E.; St Claire, M.; Purcell, R. H.; Lai, C. J. Proc. Natl. Acad.
Sci. U.S.A. 2007, 104, 9422.
In summary, a series of the dipeptide methionine–proline deriv-
atives and their analogues were designed, synthesized and assayed
against the serotype 2 dengue virus NS2B-NS3 protease. The pres-

6554
G.-C. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6549–6554
7. Rice, C. M.; Lenches, E. M.; Eddy, S. R.; Shin, S. J.; Sheets, R. L.; Strauss, J. H.
Science 1985, 229, 726.
8. Chambers, T. J.; Hahn, C. S.; Galler, R.; Rice, C. M. Annu. Rev. Microbiol. 1990, 44,
649.
9. Yusof, R.; Clum, S.; Wetzel, M.; Murthy, H. M.; Padmanabhan, R. J. Biol. Chem.
2000, 275, 9963.
14. Li, J.; Lim, S. P.; Beer, D.; Patel, V.; Wen, D.; Tumanut, C.; Tully, D. C.; Williams, J.
A.; Jiricek, J.; Priestle, J. P.; Harris, J. L.; Vasudevan, S. G. J. Biol. Chem. 2005, 280,
28766.
15. Xu, S.; Li, H.; Shao, X.; Fan, C.; Ericksen, B.; Liu, J.; Chi, C.; Wang, C. J. Med. Chem.
2012, 55, 6881.
16. Dixon, M. Biochem. J. 1953, 55, 170.
10. Lescar, J.; Luo, D.; Xu, T.; Sampath, A.; Lim, S. P.; Canard, B.; Vasudevan, S. G.
Antiviral Res. 2008, 80, 94.
11. Falgout, B.; Pethel, M.; Zhang, Y. M.; Lai, C. J. J. Virol. 1991, 65, 2467.
12. Noble, C. G.; Chen, Y. L.; Dong, H. P.; Gu, F.; Lim, S. P.; Schul, W.; Wang, Q. Y.;
Shi, P. Y. Antiviral Res. 2010, 85, 450.
17. Yin, Z.; Patel, S. J.; Wang, W.-L.; Chan, W.-L.; Rao, K. R. R.; Wang, G.; Ngew, X.;
Patel, V.; Beer, D.; Knox, J. E.; Ma, N. L.; Ehrhardt, C.; Lim, S. P.; Vasudevan, S. G.;
Keller, T. H. Bioorg Med. Chem. Lett. 2006, 16, 40.
18. Erbel, P.; Schiering, N.; D’Arcy, A.; Renatus, M.; Kroemer, M.; Lim, S. P.; Yin, Z.;
Keller, T. H.; Vasudevan, S. G.; Hommel, U. Nat. Struct. Mol. Biol. 2006, 13, 372.
13. Zhou, Y.; Guo, X.-C.; Yi, T.; Yoshimoto, T.; Pei, D. Anal. Biochem. 2000, 280,
159.