Synthesis, crystal structure, in vitro acetohydroxyacid synthase inhibition, in vivo herbicidal activity, and 3D-QSAR of new asymmetric aryl disulfides

Article abstract of DOI:10.1021/jf302206x

Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) is an important bioactive target for the design of environmentally benign herbicides. On the basis of previous virtual screening, 50 asymmetric aryl disulfides containing [1,2,4]triazole groups were synthesized

Full text of DOI:10.1021/jf302206x

Article
pubs.acs.org/JAFC
Synthesis, Crystal Structure, in Vitro Acetohydroxyacid Synthase
Inhibition, in Vivo Herbicidal Activity, and 3D-QSAR of New
Asymmetric Aryl Disulfides
Jun Shang, Wei-Min Wang, Yong-Hong Li, Hai-Bin Song, Zheng-Ming Li, and Jian-Guo Wang*
State Key Laboratory and Institute of Elemento-Organic Chemistry, Nankai University, Tianjin 300071, China
S
* Supporting Information
ABSTRACT: Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) is an important bioactive target for the design of environmentally
benign herbicides. On the basis of previous virtual screening, 50 asymmetric aryl disulfides containing [1,2,4]triazole groups were
1
synthesized and characterized by H NMR, HRMS, and crystal structure. Compounds I-a, I-b, and I-p show Ki values of 1.70,
4.69, and 5.57 μM, respectively, for wild type Arabidopsis thaliana AHAS (AtAHAS) and low resistance against mutant type
AtAHAS W574L. At 100 mg L−1 concentration, compounds I-a, II-a, and II-b exhibit 86.6, 81.7, and 87.5% in vivo rape root
growth inhibition. CoMFA steric and electrostatic contour maps were established, and a possible binding mode was suggested
from molecular docking, which provide valuable information to understand the key structural features of these disulfide
compounds. To the authors' knowledge, this is the first comprehensive case suggesting that asymmetric aryl disulfides are novel
AHAS inhibitors.
KEYWORDS: AHAS, molecular design, branched-chain amino acids, chemical synthesis, asymmetric aryl disulfides, crystal structure,
biological activity, herbicide, molecular docking, 3D-QSAR
INTRODUCTION
■
The branched-chain amino acids (BCAAs) biosynthetic
pathway exists only in plants and micro-organisms, not in the
bodies of mammals. For this reason, enzymes involved in the
BCAAs biosynthesis pathway are ideal targets for the design of
environmentally benign “green” herbicides.1 Acetohydroxyacid
synthase (AHAS; EC 2.2.1.6, also referred to as acetolactate
synthase, ALS) is the first enzyme in this biological event and
has been a successful herbicide target since the 1980s.2−4
Mainly there are four families of commercial herbicidal AHAS
inhibitors: sulfonylureas, imidazolinones, pyrimidinylthio (or oxo)
benzoates, and triazolopyrimidine sulfonanilides.5,6
It was not until the 2000s that the mode of action of AHAS
inhibitors became clear, when crystal structures of yeast AHAS
Figure 1. Some reported disulfides as AHAS inhibitors (A, B) or
anticancer agents (C).
and plant AHAS in complex with commercial herbicides were
continuously determined by Guddat et al.7−12 These studies
groups to test their inhibition of plant AHAS and to see if these
not only have added to the understanding of the catalysis and
compounds possess herbicidal activity.
inhibition mechanism of AHAS13−15 or helped to interpret the
Disulfide bonds play essential roles for bioactive proteins to
keep their correct folding.24 There are a few examples that
relationship of the enzyme residue mutations and herbicide
resistance16 but also have provided excellent models to ratio-
simple disulfide compounds such as diallyl disulfide and
dimethyl disulfide exhibit hypochlorous acid scavenging acti-
nally design novel inhibitors.17−20
vity25 and tyrosinase inhibitory activity.26 Some imidazolyl dis-
In 2007, we virtually screened the ACD-3D database and
successfully identified some novel plant AHAS inhibitors.21
ulfides (Figure 1C) were reported to display anticancer acti-
vity.27 However, there are no reports that disulfides have
Among the hits, a disulfide structure (Figure 1A) that shows
strong inhibition against Arabidopsis thaliana AHAS (AtAHAS)
attracted us. It is a coincidence that Yoon et al. carried out
high-throughput screening, and some disulfide compounds
(Figure 1B) were also discovered to show potent inhibition
against Mycobacterium tuberculosis AHAS and Haemophilus
influenzae AHAS.22,23 Here, we have designed and synthesized a
series of asymmetric aryl disulfides containing [1,2,4]triazole
herbicidal activity. This paper describes the first study on the
in vitro and in vivo activities of asymmetric aryl disulfides as plant
Received: May 21, 2012
Revised: July 27, 2012
Accepted: July 31, 2012
Published: August 20, 2012
© 2012 American Chemical Society
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Molecular Modeling. Comparative Molecular Field Analysis
(CoMFA) Analysis. The chemical structures of the compounds were
built within Sybyl 7.3 (Tripos Inc., St. Louis, MO, USA) based on the
crystal structure of compound I-a. All of the molecules were assigned
AHAS inhibitors. These disulfides are also potent inhibitors of
some resistant types of plant AHAS. The −S−S− bond was
observed in the crystal structure of the typical compound.
Computational three-dimensional quantitative structure−activity
relationship (3D-QSAR) analysis and molecular docking were
also undertaken, which provided valuable information to under-
stand the possible binding mode and to design more potent
inhibitors.
Gasteiger−Huckel charges and minimized by the Tripos force field
̈
when convergence reached 0.001 kcal mol−1 Å−1. I-a was used as the
template to superimpose all of the disulfides. CoMFA was used for the
3D-QSAR analysis.32 The cutoff was set to 30 kcal mol−1, and column
filtering was set to 2.0 kcal mol−1; all other default values in the setting
were used. The “leave-one-out” (LOO) cross-validation method was
applied to determine the optimum number of partial least-squares
(PLS) components. The biological activities were expressed in terms
of D using the following formula that has been explained elsewhere:33
MATERIALS AND METHODS
■
General Synthesis and Instruments. The starting chemical
materials were purchased from Tianjin Guangfu Fine Chemical
Research Institute, Alfa-Aesar, or Shanghai Aladdin Regeants. All sol-
vents and liquid reagents were dried by standard methods in advance
and distilled before use. Melting points were determined using an X-4
melting apparatus (Beijing Tech Instruments Co., Beijing, China) and
D = log10[a/(1 − a)] + log10 Mw
Molecular Docking. The molecular docking of the disulfides to
AtAHAS was performed by FlexX.34 The docking method is similar to
our published procedure, and all default values were used for the
molecular simulation.21 Compounds that have in vitro AtAHAS
percent inhibition >70% at 10 mg L−1 concentration were docked to
the AtAHAS binding site. The dominant conformations of the docked
molecules were chosen for further analysis. The figures were generated
by Sybyl 7.3 and LIGPLOT.35
1
were uncorrected. H NMR spectra were obtained using a 400 MHz
Varian Mercury Plus 400 spectrometer in DMSO-d6 with TMS as an
internal standard. HRMS data were obtained on an FTICR-MS
instrument (Ionspec 7.0T). The control herbicide monosulfuron ester
was synthesized in our laboratory.28 Molecular modeling studies were
performed using an SGI 350 server.
Procedures for the Synthesis of the Target Compounds I, II,
and III (Scheme 1). Example Compound I-a. The commercial
RESULTS AND DISCUSSION
Synthetic Chemistry of the Target Compounds. All of
the target compounds were characterized by H NMR and
■
Scheme 1. Synthetic Route of the Asymmetric Aryl Disulfide
Compounds
1
HRMS. Their molecular structures are listed in Table 1.
Our synthetic work dated back to early 2010. In 2011, we
noticed that Hahn et al. reported a novel method that asym-
metric disulfides can be prepared in a reaction similar to ours.36
In that case, moist tetrahydrofuran gave better yields than dried
benzene as the solvent for the reaction, so the authors added
5−10 M equivalent of water to the reaction mixture and
observed satisfactory yields. Thus, the presence of water was
thought to be preferable for the reaction. However, it is
surprising that these researchers did not notice an important
patent, which in 1983 reported that the reaction of sulfenyl
chloride with thiols can occur easily using various ethers as the
solvent.37 Here, we have synthesized 50 asymmetric aryl
disulfides (compounds I-a and I-g were previously coincidently
reported by Hahn et al. in the literature 36) using only ethyl
ether as solvent under very mild conditions, and most of the
yields for the reactions are >90%. Compared with Hahn’s method,
our synthetic method is more straightforward. Our results indicate
that this type of reaction does not require additional water as
essential reactant or catalyst to afford high yield.
Crystal Structure Analysis. Compound I-a was recrystal-
lized from ethanol/dichloromethane to give colorless crystals
suitable for X-ray single-crystal diffraction. In the range of 2.91°
≤ θ ≤ 27.89°, 2197 independent reflections were obtained. The
compound crystallized in the orthorhombic system, and the
space group is P212121. The crystallographic parameters are as
follows: a = 9.412(5) Å, b = 9.813(5) Å, c = 9.964(5) Å, α =
90°, β = 90°, γ = 90°, μ = 0.530 mm−1, V = 0.9202(9) nm3, Z =
4, Dc = 1.511 g cm−3, F(000) = 432. The final R factors are R1 =
0.0259 and ωR2 = 0.0518.
sources or the synthesis procedure of substituted [1,2,4]triazole-3-thiol
(A) and arenesulfenyl chloride (B) are described in the Supporting
Information. The synthesis route of the target compounds is shown in
Scheme 1. For the synthesis of compound I-a, [1,2,4]triazole-3-thiol
(0.50 g, 5 mmol) was added to a solution of freshly prepared phenyl-
sulfenyl chlorine a (0.72 g, 5 mmol) in 15 mL of anhydrous ethyl
ether at room temperature. The reaction mixture was stirred for 5 h
at the same temperature to give a white precipitate. The product
was filtered, washed with anhydrous ethyl ether, and further purified
by silica gel column chromatography, when pure I-a was obtained:
1
yield, 94%; mp, 85−87 °C; H NMR (DMSO-d6, 400 MHz) δ 8.61
(s, 1H, NHCH), 7.67 (d, J = 7.2 Hz, 2H, ArH), 7.41 (t, J = 7.4 Hz, 2H,
ArH), 7.34 (d, J = 7.1 Hz, 1H, ArH); HRMS (ESI), m/z [M + H]+
calcd for C8H7N3S2 210.0154, found 210.0152. The same method was
used to synthesize and purify compounds I-b−I-v, II-a−II-n, and
III-a−III-n.
Analytical data for compounds I-b−I-v, II-a−II-n, and III-a−III-n
are detailed in the Supporting Information.
X-ray Diffraction. The crystal structure of compound I-a (0.20 ×
0.18 × 0.10 mm in size) was determined, and X-ray intensity data
were recorded on a Rigaku Saturn 724 CCD diffraction meter using
graphite monochromated Mo KR radiation (λ = 0.71073 Å). The
Bruker software package SHELXTL was used to solve the structure
using “direct methods” techniques.29 All calculations were refined
anisotropically.
Biological Assays. Determination of Inhibition of Arabidopsis
thaliana AHAS (AtAHAS). The expression and purification of wild
type AtAHAS and mutant type AtAHAS W574L have been described
previously.30 AHAS activity was measured using the colorimetric assay
in a previous publication, and Ki values were estimated using the
following equation, where v0 is the uninhibited rate:31
The crystal structure of I-a is shown in Figure 2. The linkage
of the −S−S− bond between the phenyl ring and the triazole
ring can be seen. The whole molecule adopts a twist shape.
The bond lengths in the phenyl ring are similar, whereas in
the heterocycle, bonds C(2)−N(3), N(1)−C(1), and C(1)−N(2)
are shorter than bonds N(1)−C(2) and N(2)−N(3). It is
notable that the N(2) atom is attached with a hydrogen atom.
The dihedral angle between the triazole ring and the phenyl
v = v0/(1 + [I]/Ki)
In Vivo Inhibition of the Root Growth of Rape (Brassica campestris
L.). The in vivo rape root growth inhibition data were determined
using the previously published method.31
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Table 1. Chemical Structures of the Target Asymmetric Disulfides
ring is 63.5°. The selected bond lengths and torsion angles are
listed in the Supporting Information.
Biological Activity. The in vitro wild type AtAHAS inhibi-
tion and in vivo herbicidal activity of the 50 asymmetric aryl
disulfides are listed in Table 2. The in vitro activity was evaluated
at 100, 10, 3, and 1 mg L−1 concentrations. The in vivo herbicidal
activity was measured using the rape root growth inhibi-
tion method at 100 mg L−1 concentration. A commercialized
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Table 2. In Vitro AtAHAS Inhibition and in Vivo Herbicidal
Activity of the Asymmetric Aryl Disulfides
rape root
growth
inhibition (%)
AtAHAS inhibition (%)
compd
100 mg L−1 10 mg L−1 3 mg L−1 1 mg L−1 100 mg L−1
I-a
I-b
I-c
I-d
I-e
I-f
97
96
97
94
96
90
48
43
84
93
91
96
97
92
83
95
80
86
84
85
43
87
82
69
62
61
74
84
57
94
55
48
29
52
74
56
96
48
81
60
86
69
78
97
48
87
55
61
80
85
98
91
86
86
87
87
75
0
79
70
81
80
81
63
0
50
48
39
44
36
3
86.6
67.5
54.8
21.1
69.8
44.6
50.2
27.4
47.2
43.6
16.3
69.4
62.5
10.6
26.6
59.6
28.3
24.1
17.6
13
Figure 2. Molecular structure of compound I-a.
sulfonylurea herbicide, monosulfuron ester, was used as a
control.
I-g
I-h
I-i
0
0
0
0
For the in vitro biological activities, most of the compounds
exhibited desirable inhibition against wild type AtAHAS at
100 mg L−1 concentration, and nearly half of the compounds
have percent inhibition >80%. It is encouraging that at a low
concentration of 3 mg L−1, compounds I-a, I-b, I-c, I-d, I-e, I-f,
I-k, I-n, I-p, I-r, I-s, and I-t still have inhibition >60%. The
inhibition constants of compounds I-a, I-b, and I-p are 1.70
0.08, 4.69 0.39, and 5.57 0.61 μM (Table 3), which are
similar to the Ki values of imidazolinone herbicides (ranging
from 3 to 16 μM),30 but not as good as those of sulfonylurea
herbicides (ranging from 8 to 400 nM).30,31 The inhibition
curve of representative compound I-a is shown in Figure 3. On
the basis of the preliminary in vitro AHAS inhibition data, the
asymmetric disulfides appear to be promising new plant AHAS
inhibitors.
When subjected to W574L inhibition assay, compounds I-a,
I-b, and I-p still have Ki values of 24.5 2.4, 25.7 5.3, and
27.1 5.5 μM, respectively (Table 3). Similar to the definition
from a previous publication,30 resistance here means the ratio
of Ki for W574L compared with that of the wild type AtAHAS
for the same inhibitor. In this study, resistance data for I-a, I-b,
and I-p are 14.4, 5.5 and 4.9, which are low resistance levels.
However, for the same mutant type, Ki values are 85.3
19.2 μM (data in this study) for monosulfuron ester and 470
2 μM for imazaquin (data from the literature30). The corres-
ponding resistance values for monosulfuron ester and
imazaquin against W574L are 320 and 161, much higher than
those of compound I-a, I-b, and I-p. Usually, for W574L, the
sulfonylureas cover a resistance range of 130−2830 and the
imidazolinones have a resistance range of 155−186.30 As
summarized by Duggleby et al. in 2008,38 W574L mutation
even leads to strong resistance for pyrimidinylthio (or oxo)
benzoates and triazolopyrimidine sulfonanilides for cocklebur
AHAS (corresponding residue number W552L). It is exciting
that the current disulfide compounds have obvious advant-
ages over the sulfonyluea and imidazolinone herbicides against
W574L. This result will attract our interest to further investi-
gate the molecular basis of asymmetric aryl disulfides as novel
AHAS inhibitors.
70
80
76
86
84
80
69
81
66
66
67
72
0
59
59
65
54
47
63
17
76
56
60
61
60
0
7
I-j
3
I-k
I-l
19
0
I-m
I-n
I-o
I-p
I-q
I-r
0
46
0
48
11
26
15
11
0
I-s
I-t
I-u
I-v
II-a
II-b
II-c
36.8
0
79
66
58
43
39
52
18
0
52
54
0
19
0
81.7
87.5
52.8
34.3
0
0
0
0
II-d
II-e
0
0
0
0
II-f
0
0
29.4
37.5
54.1
0
II-g
0
0
II-h
II-i
83
15
0
16
0
0
0
II-j
0
0
44.9
0
II-k
15
37
6
0
0
II-l
0
0
0
II-m
II-n
III-a
III-b
III-c
III-d
III-e
III-f
III-g
III-h
III-i
III-j
III-k
III-l
III-m
III-n
0
0
11
0
0
0
24.1
70.2
6.9
86
30
60
49
72
0
34
0
0
0
12
28
16
0
0
30.5
0
0
0
0
0
0
54
92
26
70
30
38
53
65
95
13
54
0
0
0
9
66.2
0
0
40
0
0
0
0
0
31
20
29
92
0
27.4
14.3
0
The target compounds also displayed significant in vivo rape
root growth inhibition at 100 mg L−1 concentration. The
herbicidal activities for I-a, II-a, and II-b are 86.6, 81.7, and
87.5%, similar to the level of monosulfuron ester, which has an
inhibition of 88.5%. Moreover, compounds I-b, I-c, I-e, I-g, I-l,
I-m, I-p, II-c, II-h, III-a, and III-h showed >50% rape root
growth inhibition at the test condition. Thus, the asymmetric
disulfides not only exhibit strong in vitro AHAS inhibition but
also show good herbicidal activity. It should be noted that for
I-a, I-b, I-c, I-e, I-l, I-m, I-p, II-h, III-a, and III-h, the wild type
0
0
monosulfuron
ester
85
88.5
AtAHAS inhibition data at 100 mg L−1 are all >95% and the
inhibition data at 10 mg L−1 are all >80%. This indicates that
these compounds may undergo some loss after the absorp-
tion, distribution, metabolism, and excretion (ADME) process.
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Table 3. Ki Values of Representative Disulfides against Wild
Type AtAHAS and Mutant Type AtAHAS W547L
Ki (μM) for AtAHAS
compd
wild type
W574L
resistance
I-a
I-b
I-p
1.70 0.08
4.69 0.39
5.57 0.61
0.26 0.03
3.0 0.1
24.5 2.4
25.7 5.3
27.1 5.5
8.53 1.92
470 20
14.4
5.5
4.9
monosulfuron ester
320
161
a
imazaquin
a
Data from the literature.30
Figure 3. Inhibition curve of compound I-a against wild type AtAHAS.
However, for I-g, II-a, II-b, and II-c, their in vivo biological
activities at 100 mg L−1 show a good correlation with the
in vitro inhibition data, despite the fact that their in vitro inhibi-
tion data at 100 mg L−1 are <85% and at 10 mg L−1 are <66%,
meaning that these compounds may have high efficiency to
bind with the plant AHAS for in vivo herbicidal assay. Bearing
these facts in mind, compounds with enhanced herbicidal
activity can be further designed and evaluated.
Figure 4. Electrostatic contour map (A) and steric contour map (B)
for the CoMFA model. In map A, positive charge favored regions are
shown in blue and negative charge favored regions are shown in red. In
map B, sterically favored regions are shown in green and sterically
disfavored regions are shown in yellow.
Quantitative Structure−Activity Relationships. The
alignment of the compounds is shown in the Supporting
Information. The biological activity D values were calculated on
the basis of the in vitro data at 10 mg L−1 concentration.
Compounds I-g, I-h, I-u, II-g, II-j, II-n, and III-f were excluded
because their inhibition values are 0. For the CoMFA com-
putation, compound I-m was also removed from the training
set as an outlier. The leave-one-out q2 value is 0.603 when the
optimum number of components is 7, indicating that the model
is reliable. The non-cross-validated r2 value is 0.903, with a
standard error of estimate of 0.17 and an F value of 45.195. The
steric and electrostatic contributions are 61.6 and 38.4%, res-
pectively. The predictive biological activity versus the experi-
mental biological activity are plotted in the Supporting Information.
The 3D-CoMFA contour maps are shown in Figure 4.
Compound I-a was used as the template to illustrate the
structure−activity relationships. For the electrostatic contour
map (Figure 4A), the blue contour defines a region where an
increase in the positive charge will result in an increase of
activity, whereas the red contour defines a region of space
where negative charge is favorable. It can be observed that
the electrostatically favored region locates at ortho- or meta-
positions of the phenyl ring, whereas the electrostatically
disfavored region covered a large space around the phenyl ring.
For the steric contour map (Figure 4B), the green part displays
a position where a bulky group would be favorable for potent
inhibition and the yellow part represents a position where a
bulky group is likely to decrease the in vitro activity. The
sterically favored region covers a small space near the phenyl
ring; however, the sterically disfavored space locates in bigger
regions not only around the phenyl ring but also near the
triazole ring. The CoMFA contour map provides valuable infor-
mation for further structural optimization of this family of new
AHAS inhibitors.
Molecular Docking. In total 20 compounds were docked
to the herbicide binding site of AtAHAS. After investigation of
the resulting docked conformations, eight compounds (I-a, I-i,
I-j, I-l, I-t, II-h, III-a, and III-h) were found to overlay one
another quite well (figure in the Supporting Information). The
docked conformations for the other 12 compounds (I-b, I-c, I-
d, I-e, I-f, I-k, I-m, I-n, I-p, I-v, III-e, and III-j) were in
unreasonable binding space, and they failed to overlay well with
one another. Thus, the docked conformations of the eight com-
pounds were regarded as the possible binding conformations in
the study. Compound I-a was also chosen here to illustrate the
binding mode of the disulfide AHAS inhibitors. Figure 5A is a
two-dimensional representation of the interactions between I-a
and surrounding residues of AtAHAS drawn by LIGPLOT. The
herbicide binding site is at the interface of two subunits. The
inhibitor interacts with AtAHAS via multiple H-bonding
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Figure 5. FlexX docking of asymmetric disulfide I-a to AtAHAS. (A) 2D plot by LIGPLOT. The hydrogen bonds between the enzyme and the
inhibitor are shown as green dashed lines, and distances are in Å units. Amino acid residues that are within van der Waals contact of I-a are shown as
red arcs. An atom belonging to I-a that participates in van der Waals attractions to the enzyme is identified by a red star. Residues labeled with a
prime are from the alternate subunit. (B) Picture drawn in Sybyl. The green molecule represents the disulfide I-a, the magenta molecule represents
sulfonylurea herbicide chlorimuron ethyl, and the red part represents the W574 residue in AtAHAS.
contacts and hydrophobic contacts. R377(A), S653 (A), and
K256(B) form four H-bonds with the inhibitor, whereas
W574(A), G121(B), P197(B), F206(B), and Q207(B) form
hydrophobic interactions with the small molecule. Figure 5B is a
three-dimensional picture showing the binding mode of disulfide,
and it compares the modes of action between sulfonylurea chlo-
rimuron ethyl (magenta molecule CE) and I-a (green molecule
LIG1). It can be seen that there exists minor overlapping of the
two inhibitors. However, there is significant difference between
their binding modes with regard to an important residue,
W574(A) (red residue). The pyrimidine ring in the
sulfonylurea herbicide CE forms a strong π−π stacking with
the indole ring of W574, which is crucial for the tight binding of
sulfonylureas with AtAHAS. For compound I-a, although it
makes hydrophobic contact with W574(A), there does not exist
a π−π stacking. This is in agreement with the W574L inhibition
data, indicating that the mode of action from molecular docking
is reasonable.
In summary, 50 asymmetric aryl disulfides containing [1,2,4]-
triazole groups were synthesized and evaluated as novel plant
AHAS inhibitors via both in vitro enzyme inhibition and in vivo
rape root growth inhibition. Besides the wild type AtAHAS, the
compounds also displayed potent inhibition against mutant
type AtAHAS W574L. 3D-CoMFA contour maps were gene-
rated for design inhibitors with enhanced activity. The binding
mode predicted from molecular docking reasonably explains
the low resistance of the disulfides against W574L. This study
indicates that asymmetric aryl disulfides should be paid more
attention to discover novel herbicides to fight the resistance of
current AHAS inhibitors.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis procedure of substituted [1,2,4]triazole-3-thiol (A)
and arenesulfenyl chloride (B), analytical data for compounds
I-b−I0v, II-a−II-n, and III-a−III-n; selected bond lengths and
torsion angles for the crystal structure of I-a; alignment for the
training set of the CoMFA model; plot of predictive biological
activity versus the experimental biological activity; and overlay
of docked compounds from FlexX. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +86-(0)22-23499414. Fax: +86-(0)22-23503627.
E-mail: nkwjg@nankai.edu.cn.
Funding
We acknowledge financial support from the Natural Science
Foundation of China (No. 21272128), Key Natural Science
Foundation from Tianjin S&T(12JCZDJC25700), National
Basic Research Project of China (No. 2010CB126103), and
National Key Technology Research and Development Program
(No. 2011BAE06B05-3).
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We appreciate associate professor Cong-Wei Niu for her kind
assistance in the measurement of W754L inhibition constants
of the typical disulfides.
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