Anti-Inflammatory Effect of Novel 7-Substituted Coumarin Derivatives through Inhibition of NF-κB Signaling Pathway

Article abstract of DOI:10.1002/cbdv.201800559

A series of novel 7-substituted coumarin derivatives were synthesized and evaluated. Biological screening results obtained by the evaluation of the compounds’ inhibition against LPS-induced IL-6 and TNF-α release in RAW 264.7 cells indicated that most compounds exhibited potent anti-inflammatory activity. Among them, N-(3-methoxybenzyl)-2-[(2-oxo-2H-chromen-7-yl)oxy]acetamide (2d) showed the best activity. The potential targets of title compound 2d were reversely screened with the molecular modeling software, Discovery Studio 2017 R2. Screening and molecule docking results showed that 2d could bind to the active site (NLS Polypeptide) of NF-κB p65, and this binding affinity was confirmed by surface plasmon resonance (SPR) analysis. Furthermore, Western blot assay showed that 2d remarkably blocked the NF-κB signaling pathway in vitro. Collectively, all these findings suggested that compound 2d might be a promising lead compound worthy of further pursuit.

Full text of DOI:10.1002/cbdv.201800559

Title: Anti-inflammatory effect of Novel 7-substituted coumarin
derivatives through inhibition of NF-κB signaling pathway
Authors: Chaoyu Mu, Mingfei Wu, and Zeng Li
This manuscript has been accepted after peer review and appears as an
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To be cited as: Chem. Biodiversity 10.1002/cbdv.201800559
Link to VoR: http://dx.doi.org/10.1002/cbdv.201800559

10.1002/cbdv.201800559
Chemistry & Biodiversity
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Anti-inflammatory effect of novel 7-substituted coumarin derivatives through
inhibition of NF-B signaling pathway
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Chaoyu Mu a, b, Mingfei Wu a and Zeng Li a, *
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a School of Pharmacy, Anhui Medical University, Hefei 230032 China.
b Department of Clinical Laboratory, Huaibei Miner’s General Hospital, Huaibei 235000 China.
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* Corresponding author.
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E-mail address: lizeng@ahmu.edu.cn
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Abstract: A series of novel 7-substituted coumarin derivatives was synthesized and
evaluated. Biological screening results obtained by the evaluation of the compounds’
inhibition against LPS-induced IL-6 and TNF- release in RAW 264.7 cells indicated
that most compounds exhibited potent anti-inflammatory activity. Among them,
compound 2d showed the best activity. The potential targets of title compound 2d
were reverse screened with the molecular modeling software-Discovery Studio 2017
R2. Screening and molecule docking results showed that 2d may bind the active site
(NLS Polypeptide) of NF-B p65, and this binding affinity was confirmed by surface
plasmon resonance (SPR)analysis. Furthermore, western blot assay showed that 2d
remarkably blocked the NF-B signaling pathway in vitro. Collectively, all these
findings suggested that compound 2d might be a promising lead compound worthy of
further pursuit.
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Keywords: coumarin derivative, anti-inflammatory, 2d, reverse screen, NF-B
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Introduction
Inflammation is the response of living tissues to cellular injury and involves innate
and adaptive immunity [1]. The purpose of inflammation is to localize and eliminate
causative agents, limit tissue injury, and restore tissue to normality. Thus,
inflammation is one of the most common and important basic pathological processes
involved in infectious diseases [2], tissue damage [3], initiation and promotion of
cancer [4], and development of atherosclerosis [5]. Classic anti-inflammatory drugs
can be divided into two types: steroidal anti-inflammatory drugs (SAIDs) and
non-SAIDs (NSAIDs). The former is a type of hormone and have many physiological
effects. When administered for inflammatory diseases, they may cause many side
effects [6]. The latter inhibit COX-1 and COX-2 simultaneously; however, they may
cause adverse gastric effects that lead to patient intolerance [7]. Therefore, to aim at
the mechanisms of inflammation, exploring and developing new anti-inflammatory
drugs is important.
Coumarins, a class of natural organic compounds that contains a benzopyrone core,
have multiple biological activities, such as antimicrobial, anticancer,
anti-inflammation, antitubercular, and antioxidant activity; inhibition of platelet
aggregation; and cardiovascular protection [8]. The anti-inflammatory properties of
coumarins are attributed to their inhibition of the production of TNF-α and other
pro-inflammatory cytokines by activated macrophages [9]. Moreover, various
coumarin-related derivatives serve as inhibitors of the lipoxygenase and
cyclooxygenase pathways of arachidonate metabolism [10-11]. Given its favorable
anti-inflammatory activity, a coumarin framework has been used for chemical
modification for identifying novel derivatives with improved pharmacological or
pharmacokinetic profiles.
Substituted aniline, benzylamine, and phenethylamine moieties belong to an
important class of organic compounds that is widely used in medicinal chemistry.
These substituents are often present in anti-inflammatory drug constructs. Thus, we
synthesized some coumarin derivatives hybridized with substituted aniline or benzene
methylamine moieties as possible anti-inflammatory agents (Scheme 1). In this study,
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we selected LPS-stimulated RAW264.7 cells as a cellular model to evaluate the
anti-inflammatory activity of target compounds.
Results and discussion
Chemistry
Coumarin derivatives (2a–2k) were synthesized from two steps, as described in
Scheme 1. Intermediate 1 was obtained under relatively mild conditions by
condensing 7-hydroxycoumarin with ethyl bromoacetate in the presence of K2CO3 as
base in DMF at 60 °C. Reacting the intermediate 1 with the substituted aniline or
benzene methylamine in ethanol at 78 °C afforded the target compounds 2a–2k.
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Scheme 1. Synthesis route of coumarin derivatives 2a-2k. (i) Ethyl Bromoacetate,
K2CO3, acetone, 12 h, rt; 5% sodium hydroxide, 2 h, rt; (ii) Aniline or benzene
methylamine, CH2Cl2, 2 h, rt.
MTT assay
MTT was first used to screen the influences of coumarin derivatives on the
proliferation of LPS-activated RAW264.7 cells. The results (Table s1, Figure 1)
indicated that stimulator LPS (0.5–1 g/mL) and all coumarin derivatives (1–10 M)
in the presence of LPS (1 g/mL) showed little toxic effects on RAW 264.7 cells.
Even at high concentrations, LPS (2 g/mL) and several coumarin derivatives (2c, 2e,
2f, 2i, and 2k at 100 M) presented certain toxic effects. Thus, we further chose the
concentrations of 1 g/mL LPS and 10 M coumarin derivatives in the subsequent
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analyses.
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Figure 1. Effects of indicated concentrations of coumarin derivatives on viability of
LPS-activated RAW264.7 cells. All measurements were obtained in triplicate. Values
are presented as mean ± SD.
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The detection of key pro-inflammatory cytokine
The detection of key pro-inflammatory cytokine induced by LPS-activated RAW
264.7 cells is an essential step for an anti-inflammatory agent [12]. The
anti-inflammatory activity of coumarin derivatives on the pro-inflammatory mediators
IL-6 and TNF- release of LPS-activated RAW 264.7 cells is illustrated in Figure 2.
Compared with the LPS group, the levels of TNF-α and IL-6 were statistically
significantly lower in LPS-stimulated macrophages of most coumarin derivatives
(such as 2c, 2d, and 2e). Among them, compound 2d was the most potent coumarin
derivative (inhibition rate up to -50% compared with LPS). Preliminary SARs showed
that substituent R substantially affected the anti-inflammatory activity and the
methoxy group was better than that of halogen substitution. The methoxy group at
3-position was also important. These results indicated that coumarin derivatives could
markedly inhibit the LPS-induced release of various proinflammatory cytokines with
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potential anti-inflammatory activity.
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Figure 2. Effects of coumarin derivatives (10 M) on IL-6 and TNF- production in
LPS-activated RAW264.7 cells. All measurements were obtained in triplicate. Values
are presented as mean ± SD. In=Indomethacin (10 M). Statistical significance
compared with the LPS group was indicated, *p <0.05, **p <0.01.
Reverse virtual screening and Molecule docking
Computer-aided simulation on the construction of a pharmacophore model based
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on the function of the active ligand-building pharmacophore model and
receptor-ligand crystal complex can shorten experiments remarkably [13]. For this
research, we used reverse virtual screening to validate that coumarin derivative 2d
had a good binding relationship with NF-B p65 (PDB 1MY5) [14] (Figure 3). Thus,
NF-B was considered a possible target of 2d. Further analysis of the results was
conducted by molecule docking using C-DOCKER in Discovery Studio 2017 R2. The
protein crystal structure of NF-B p65 (PDB 1MY5) was used. As shown in Figure 4,
a typical docking pose demonstrated that compound 2d could bind to the active site
(NLS Polypeptide) of p65. Two hydrogen bonds interacted between 2d and p65 NLS
Polypeptide: one was between the -O- group of the lactone and residue of ASP293,
and the other was between the -NH- group with ASP293. The coumarin core of 2d
was inserted into the active site deeply. 2d also exhibited the - stacking interaction
between the aromatic ring and the residue of LEU207 and PRO260. Additionally,
certain weak interactions, including pi-alkyl, carbon-hydrogen bonds, and van der
Waals, contributed to the binding affinity of 2d with NF-B p65.
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Figure 3. Profiling of the predicted protein targets of 2d via Discovery Studio 2017
R2. The Y-axis represents the compound 2d, and the X-axis indicates the predicted
pharmacophore models (pharmacological targets). The color from blue to red
represents a higher fit value and a better fit.
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Figure 4. (A) 3D model of the interaction between compound 2d with the active site
of NF-B p65. (B) 2D model of the interaction between compound 2d with the active
site of NF-B p65.
Binding affinity of the compound 2d to NF-B p65
SPR is a useful technique to monitor molecular reactions in real time, which has
been applied to investigate the interactions between small molecule and proteins. To
confirm the binding affinity of coumarin derivative 2d with NF-B p65, SPR
experiments was performed. Figure 5 shows the SPR sensorgrams of 2d binding to
the immobilized NF-B p65 at the concentration of 12.5, 25, 50, 100, 200 and 400
nM. The association of 2d with NF-B p65 was evaluated using the equilibrium
dissociation constant (KD) by fitting the sensogram with a 1:1 (Langmuir) binding fit
model. The results showed that 2d had a high binding affinity toward NF-B p65
with a KD value of 2.83ⅹ10-7 M.
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Figure 5. SPR sensograms for coumarin derivative 2d binding to the immobilized
NF-B p65. Concentration range: 400, 200, 100, 50, 25, and 12.5 nM for the curves
from the bottom curve upward. Binding plots used to determine the KD value for 2d
with NF-B p65 in the inner panel.
Western blot assay
The NF-B signaling pathway is important in the critical juncture of inflammation
[15]. NF-B is often located in the cytoplasm in non-activated cells as a polymer that
consists of p50, p65, and IkB proteins. In response to an activation signal, NF-B
becomes phosphorylated and degraded [16]. Furthermore, various pro-inflammatory
agents, including interleukins, cytokines, chemokines, iNOS, and COX-2, are
promoted in transcription [17]. To understand the effect of compound 2d on LPS-
stimulated NF-B signaling pathway, the expression of the relative protein of p65,
phospho p65, COX-2, and INOS were checked by western blot assay. As shown in
Figure 6, the title compound 2d remarkably decreased the phosphorylation of p65,
and certain downstream signaling molecules, such as iNOS and COX-2, were also
inhibited. Combined with the reverse virtual screening results, these findings
indicated that 2d is a potent inhibitor of NF-B activity that exhibits
anti-inflammatory activity.
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Figure 6. Coumarin derivative 2d inhibited the activation of NF-B signaling
pathway in LPS-activated RAW264.7 cells. Western blot analysis (A) and quantitative
data of protein (B) BAY11-7082 was used as the positive control. #p < 0.05, ##p < 0.01
compared with the negative control group. *p < 0.05, ** p < 0.01 compared with the
LPS group. Values are presented as mean ± SD.
Conclusion
In summary, to identify original, effective lead compounds that can serve as an
anti-inflammatory agent, a series of 7-substituted coumarin derivatives were
synthesized and evaluated. Most compounds exhibited potent anti-inflammatory
activity, especially compound 2d. The preliminary mechanism study found that 2d
may bind the active site (NLS Polypeptide) of NF-B p65 to block the NF-B
signaling pathway. The results were confirmed by SPR and Western blot assay. The
data presented in this work indicated that coumarin derivative 2d’s anti-inflammatory
role may be partly due to its inhibitory effect on the NF-B signaling pathway.
Additional studies and tests on its mechanism of anti-inflammatory activity are
underway.
Experimental Section
Chemistry
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All required chemicals and solvents were purchased from Sigma-Aldrich (Munich,
Germany). Melting points were determined on a XT4MP apparatus (Taike Corp.,
Beijing, China), and are uncorrected. 1H-NMR spectra are recorded using TMS as the
internal standard in CDCl3 solutions on a Bruker 400 MHz instrument (Bruker,
Karlsruhe, Germany). High-resolution electron impact mass spectra (HRMS) were
recorded under electron impact (70 eV) condition using a Micro Mass GCT CA 055
instrument.
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Synthesis of intermediate 1
A mixture of 7- hydroxycoumarin (6.17 mmol), ethyl bromoacetate (9.15 mmol)
and K2CO3 (4.69 g, 33.91 mmol) in acetone (30 mL) was heated under reflux for 12h.
After cooling to room temperature, the mixture was evaporated to dryness. The
residue was dissolved in ethanol (50 mL) was refluxed with 5% sodium hydroxide (5
mL) for 2 h. The mixture was evaporated to dryness, then dissolved into water and
acidified with HCl (6 M). A white precipitate was formed, filtered, and washed with
cool water, dried and recrystallized from ethanol to afford 1, yield 67%. m.p.
1
210-211℃; H NMR (400 MHz, DMSO) δ 7.99 (d, J = 9.5 Hz, 1H), 7.70 – 7.56 (m,
1H), 7.02 – 6.90 (m, 2H), 6.29 (d, J = 9.5 Hz, 1H), 4.77 (s, 2H). HRMS (ESI) m/z:
calcd for C11H8O5 [M+H]+: 221.0444, found 221.0448.
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General procedures for the preparation of target compounds 2a-2k
A suspension compound 1 (0.945mmol) in dichloromethane (20 mL) was stirred at
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room temperature with oxalyl chloride (1.42mmol), DMF (1mL) for 2 h. After
cooling, the mixture was evaporated to dryness and the residue dissolved in
dichloromethane (20 mL). The solution was refluxed with substituted aniline,
benzylamine or phenethylamine (1.134 mmol) for 5 h. Then the solvent was poured
into water and extracted with dichloromethane (50 mL × 3). The solution was dried
over anhydrous Na2SO4 and concentrated. The residues were purified by flash
chromatography with mineral ether/ethyl acetate (2:1, v/v) elution.
2-((2-oxo-2H-chromen-7-yl)oxy)-N-(m-tolyl)acetamide (2a)
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White solid (63 % yield); m.p. 187-188 ℃; H NMR (400 MHz, CDCl3) δ 8.13 (s,
1H), 7.66 (d, J = 9.5 Hz, 1H), 7.45 (dd, J = 11.4, 8.4 Hz, 2H), 7.38 (d, J = 8.1 Hz, 1H),
7.24 (d, J = 7.9 Hz, 1H), 7.03 – 6.91 (m, 3H), 6.32 (d, J = 9.5 Hz, 1H), 4.67 (s, 2H),
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2.36 (s, 3H). HRMS (ESI) m/z: calcd for C18H15NO4 [M+H]+: 310.1074, found
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310.1078.
N-(2-methoxyphenyl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide (2b)
1
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White solid (72 % yield); m.p. 191-193 ℃; H NMR (400 MHz, CDCl3) δ 8.87 (s,
1H), 8.38 (dd, J = 8.0, 1.5 Hz, 1H), 7.66 (d, J = 9.5 Hz, 1H), 7.46 (d, J = 9.3 Hz, 1H),
7.10 (td, J = 7.9, 1.6 Hz, 1H), 6.99 (td, J = 7.8, 1.2 Hz, 1H), 6.97 – 6.93 (m, 2H), 6.91
(dd, J = 8.1, 1.2 Hz, 1H), 6.32 (d, J = 9.5 Hz, 1H), 4.69 (s, 2H), 3.90 (s, 3H). HRMS
(ESI) m/z: calcd for C18H15NO5 [M+H]+: 326.1023, found 326.1026.
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N-(2-methoxybenzyl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide (2c)
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White solid (54 % yield); m.p. 182-183 ℃; H NMR (400 MHz, CDCl3) 1H NMR
(400 MHz, CDCl3) δ 7.64 (d, J = 9.5 Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.25 – 7.20
(m, 2H), 6.90 – 6.82 (m, 4H), 6.75 (s, 1H), 6.30 (d, J = 9.5 Hz, 1H), 4.59 (s, 2H), 4.49
(d, J = 5.8 Hz, 2H), 3.80 (s, 3H). HRMS (ESI) m/z: calcd for C19H17NO5 [M+H]+:
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340.1179, found 340.1182.
N-(3-methoxybenzyl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide (2d)
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White solid (66 % yield); m.p. 179-180 ℃; 1H NMR (400 MHz, CDCl3) δ 7.64 (d, J
= 9.5 Hz, 1H), 7.42 – 7.37 (m, 1H), 7.31 – 7.23 (m, 1H), 6.89 – 6.81 (m, 6H), 6.30 (d,
J = 9.5 Hz, 1H), 4.59 (s, 2H), 4.54 (d, J = 6.1 Hz, 2H), 3.83 (s, 3H). HRMS (ESI) m/z:
calcd for C19H17NO5 [M+H]+: 340.1179, found 340.1175.
N-(4-methoxybenzyl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide (2e)
White solid (47 % yield); m.p. 191-193 ℃; 1H NMR (400 MHz, CDCl3) δ 7.64 (d, J
= 9.5 Hz, 1H), 7.41 (d, J = 8.7 Hz, 1H), 7.25 – 7.18 (m, 2H), 6.94 – 6.82 (m, 4H),
6.75 (s, 1H), 6.29 (d, J = 9.5 Hz, 1H), 4.59 (s, 2H), 4.49 (d, J = 5.9 Hz, 2H), 3.80 (s,
3H). HRMS (ESI) m/z: calcd for C19H17NO5 [M+H]+: 340.1179, found 340.1175.
N-(2-chlorobenzyl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide (2f)
White solid (51 % yield); m.p. 186-187 ℃; 1H NMR (400 MHz, CDCl3) δ 7.64 (d, J
= 9.5 Hz, 1H), 7.47 – 7.32 (m, 3H), 7.25 (dd, J = 5.8, 3.5 Hz, 2H), 7.00 (s, 1H), 6.87
(dd, J = 8.6, 2.5 Hz, 1H), 6.83 (d, J = 2.4 Hz, 1H), 6.30 (d, J = 9.5 Hz, 1H), 4.64 (d, J
= 6.2 Hz, 2H), 4.59 (s, 2H). HRMS (ESI) m/z: calcd for C18H14ClNO4 [M+H]+:
344.0684, found 344.0687.
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N-(4-fluorobenzyl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide (2g)
White solid (67 % yield); m.p. 179-180 ℃; 1H NMR (400 MHz, CDCl3) δ 7.64 (d, J
= 9.5 Hz, 1H), 7.42 (d, J = 9.1 Hz, 1H), 7.30 – 7.27 (m, 2H), 7.03 (dd, J = 11.9, 5.4
Hz, 2H), 6.91 – 6.79 (m, 3H), 6.30 (d, J = 9.5 Hz, 1H), 4.60 (s, 2H), 4.53 (d, J = 6.0
Hz, 2H). HRMS (ESI) m/z: calcd for C18H15FNO4 [M+H]+: 328.0980, found
328.0977.
N-(4-bromobenzyl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide (2h)
White solid (71 % yield); m.p. 193-194 ℃; 1H NMR (400 MHz, CDCl3) δ 7.64 (d, J
= 9.5 Hz, 1H), 7.51 – 7.44 (m, 2H), 7.42 (d, J = 9.3 Hz, 1H), 7.18 (d, J = 8.5 Hz, 2H),
6.86 (q, J = 2.5 Hz, 3H), 6.30 (d, J = 9.5 Hz, 1H), 4.60 (s, 2H), 4.51 (d, J = 6.1 Hz,
2H). HRMS (ESI) m/z: calcd for C18H14BrNO4 [M+H]+: 388.0179, found 388.0179.
N-(2-fluorophenethyl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide (2i)
White solid (68 % yield); m.p. 195-196 ℃; 1H NMR (400 MHz, CDCl3) δ 7.65 (d, J
= 9.5 Hz, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.19 (dt, J = 17.0, 3.6 Hz, 2H), 7.10 – 6.97 (m,
2H), 6.82 (dt, J = 4.1, 2.4 Hz, 2H), 6.60 (s, 1H), 6.31 (d, J = 9.5 Hz, 1H), 4.52 (s, 2H),
3.63 (q, J = 6.7 Hz, 2H), 2.92 (t, J = 6.8 Hz, 2H). HRMS (ESI) m/z: calcd for
C19H16FNO4 [M+H]+: 342.1136, found 342.1138.
N-(4-fluorophenethyl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide (2j)
White solid (77 % yield); m.p. 192-193 ℃; 1H NMR (400 MHz, CDCl3) δ 7.65 (d, J
= 9.5 Hz, 1H), 7.41 (d, J = 8.3 Hz, 1H), 7.25 (d, J = 7.5 Hz, 2H), 7.09 (d, J = 8.4 Hz,
2H), 6.79 (dd, J = 11.1, 2.6 Hz, 2H), 6.49 (s, 1H), 6.31 (d, J = 9.5 Hz, 1H), 4.53 (s,
2H), 3.61 (dd, J = 13.2, 6.9 Hz, 2H), 2.84 (t, J = 7.0 Hz, 2H). HRMS (ESI) m/z: calcd
for C19H16FNO4 [M+H]+: 342.1136, found 342.1140.
N-(4-bromophenethyl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide (2k)
White solid (79 % yield); m.p. 196-197 ℃; 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J
= 9.5 Hz, 1H), 7.40 (dd, J = 10.9, 8.4 Hz, 3H), 7.04 (d, J = 8.2 Hz, 2H), 6.79 (dd, J =
13.3, 2.2 Hz, 2H), 6.48 (s, 1H), 6.32 (d, J = 9.5 Hz, 1H), 4.53 (s, 2H), 3.60 (q, J = 6.8
Hz, 2H), 2.82 (t, J = 7.0 Hz, 2H). HRMS (ESI) m/z: calcd for C19H16BrNO4 [M+H]+:
402.0335, found 402.0331.
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Cell culture
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Mouse macrophage cell line RAW 264.7 were cultured in cell culture bottles in
DMEM medium (Hyclone, USA) supplemented with 10% fetal bovine serum（FBS,
sijiqing, Hangzhou, Zhejiang, China. All cell cultures were maintained at 37℃ in a
humidified incubator at an atmosphere of 5% CO2. All of the operations provided by
approved guidelines and regulations.
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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
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Proliferation of cells was measured using MTT assay. The cells were seeded on
96-well culture plates, until the cells reached 70%-80% confluency, and then treated
with LPS (0, 0.5, 1, 2 g/mL) for 24 h. Repeat these steps, cells were pretreated with
coumarin derivatives (1 M, 10 M and 100 M) for 24 h after stimulation with 1
g/ml LPS for 24 h. MTT solution (5 mg/ml, Invitrogen) was added to each well and
the resulting mixture was incubated for 4 h at 37 °C. Finally, removing the
supernatant, 150μl DMSO was added to each well, and the plates were read at 490 nm
wavelength. All measurement were obtained in triplicate.
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The detection of pro-inflammatory cytokine IL-6 and TNF-
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The RAW 264.7 cells were stimulated with LPS (1 g/mL) for 24 h and then
treated with indomethacin, coumarin derivatives (10 M) for 24 h. IL-6 and
TNF-levels in the culture media were measured by ELISA and were normalized by
the total protein. The IL-6 and TNF- ELISA Kitwere purchased from
Sigma-Aldrich Co (St. Louis, MO, USA). All measurement were obtained in triplicate.
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Reverse virtual screening and Molecular docking
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To understand the potential interactions between the tested drug and selected
protein, reverse direction finding were performed using Discovery Studio 2017 R2
(DS) in this study. The ligand structure was drawn by using the ChemDraw2010
program. Ligand structures were optimized by using DS. Protein and ligand were
prepared for the reverse target and docking simulation by adding partial charges and
hydrogen with the aid of DS. The proteins can be acquired from the database of
Protein Data Bank (PharmaDB pharmacophores, New York,, NY, USA). Ligand and
pharmacophores were matched in DS.
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The X-ray crystal structure of NF-B p65 (PDB 1MY5) complexed with the
inhibitor was obtained from the RCSB Protein Data Bank. Vina docking encoded in
DS 2017 R2 software was employed to identify the potential binding of 2d to
NF-B p65. Docking parameters were set to default values. All docked poses of 2d
were clustered using a tolerance of 2 Å for RMSD and were ranked on the basis of
the binding docking energies. The lowest energy conformation in the most populated
cluster was selected for subsequent study.
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SPR assay
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SPR experiments were performed with a Biacore T200 apparatus on CM5 sensor
chips (GE Healthcare, Fairfield, CT, USA), following the published protocol [18].
Ligand solutions (at 12.5, 25, 50, 100, 200 and 400 nM.) were prepared with running
buffer by serial dilutions from stock solutions. The sensorgrams were analyzed with
the Biacore T200 evaluation software (version 2.0, Fairfield, CT, USA). The kinetic
parameters, including association rate constants (ka), dissociation rate constants (kd),
and kinetic dissociation constant (KD), were calculated by Biacore T200 evaluation
software 2.1 (Fairfield, CT, USA).
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Western Blot assay
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The RAW 264.7 cells were stimulated with LPS (1 g/mL) for 24 h then treated
with various concentrations of the compound 2d (10 and 20 M) and Bay 11-7082
(0.3 g/ml) for 24 h. The samples of total cellular protein extracts were loaded and
separated by SDS-PAGE and were transferred on PVDF membranes (Beyotime
Biotechnology, Haimen, China). The membranes were blocked with 5% dehydrated
skim milk in TBST for 2 hr at room temperature. The blots were washed thrice in
TBST buffer and were incubated overnight at 4 °C with primary antibodies against
-actin (1:1000 dilution), NF-B p65 (1:1000 dilution), iNOS (1:1000 dilution),
COX-2 (1:1000 dilution) and phospho NF-B p65 (1:1000 dilution) (all from Cell
Signaling Technology, Inc., Danvers, MA, USA). The blots were washed thrice in
TBST buffer, followed by the addition of secondary antibodies. The unbound
antibodies in each step were washed with TBST three times. The specific proteins in
the blots were visualized using an enhanced chemiluminescence reagent (Thermo
Scientific, Waltham, MA, USA). Immunoreactivity of the membranes were detected
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using the Bio-Rad-Image Lab with an electrochemiluminescence system (Thermo
Fisher Scientific, Waltham, MA, USA). The densitometry of the protein bands was
measured using the ImageJ (NIH image software) and was normalized to their
relevant controls.
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Statistical Analysis
Results are expressed as mean ± standard deviation. Statistical comparisons were
performed using one-way ANOVA followed by the least significant difference test.
The minimum level of significance was p < 0.05.
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Acknowledgments
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This work was supported by the General Financial Grants from the China
Postdoctoral Science Foundation (2015M571916), the National Nature Science
Foundation of China (81302701).
Author Contribution Statement
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Z. L. designed the work, C. M. contributed to the synthesis and of the compound.
C. M. and M. W. evaluation of the biological activity.
Appendix A. Supplementary data
The supplementary data associated with this article can be found in the online
version at…………..
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