S.T. Wua et. al.

/ International Journal of Engineering Science and Technology

Vol. 2(9), 2010, 4263-4277

Docking Prediction for Tumor Necrosis

Factor-α and Five Herbal Inhibitors
S. T. Wuaa, Jui-Chiang Suna＊, Kuei-Jen Leeb & Yu-Ming Sunc

a
Department and Graduate Program of BioIndustry Technology, DAYEH UNIVERSITY, Dacun, Changhua, Taiwan
51591, R.O.C.
b
Department of Bioinformatics, Asia University, Taichung, Taiwan 41354, R.O.C.
c
Department of Computer Science, National Tsing Hua University, Hsing Chu, Taiwan 30013, R.O.C
*Corresponding author.E-mail address: sun22860635@yahoo.com.tw (J.C. Sun).

Abstract

Background & objectives: TNF-α (tumor necrosis factor-α), formerly known as cachectin, is a key cytokine that
influences intermediary metabolism. The 5 herbal medicines curcumin, EGCG (epigallocatechin-3-gallate), sinapyl
alcohol, SYRINGIN, and triptolide were experimentally found to be competitive inhibitors of the activities of
TNF-α. In this study, we predicted and analyzed the ability of these 5 herbal medicines to inhibit TNF-α and
identified binding sites for them.

Methods: A homology model of TNF-α was constructed using SWISS-MODEL Workspace, its energy was
minimized, and its quality was verified using PROCHECK, VERIFY3D, and ERRAT. The protein–ligand
interactions were analyzed by simulating the docking of the 5 herbal medicines using Autodock 4.0. The binding
sites were analyzed using Autodock 4.0 and LIGPLOT.

Results: The results indicate that the 5 medicines are potent inhibitors of TNF-α. The 3 main binding regions and
the common binding sites for the 5 medicines in the 3 binding regions were also identified. We also observed that
curcumin and SYRINGIN docked at the receptor-binding sites of TNF-α. Covalent π–π aromatic interactions or
π–cation interactions were found between sinapyl alcohol and curcumin and the TNF-α cytokine.

Interpretation & conclusion: We found that the descending order of the degree of stability of TNF-α inhibition is
curcumin > sinapyl alcohol > triptolide > SYRINGIN > EGCG. We predicted that sinapyl alcohol and curcumin
are the strongest inhibitors of TNF-α because of the covalent bonds they form with TNF-α. The 5 herbal medicines
tested in this study have a common binding site–Cys129–in TNF-α. TNF-α activation can be inhibited more
efficiently when all 5 medicines are simultaneously used as a herbal medicine complex rather than when each is
used alone.

Key words residue ; cytokine ; ligand ; docking ; π–π aromatic interaction ; π–cation interaction

1. Introduction

The cytokine TNF (tumor necrosis factor)-α is produced by several types of cells, especially macrophages, and
has a jelly roll-like structure. At low levels, this cytokine maintains homeostasis by regulating circadian rhythm [1].
However, if TNF-α is present at high levels in the human body, the risk of mortality may be increased [2].

This characteristic, i.e., the toxicity, of TNF-α is important for the development of atherosclerotic lesions promoted
by the expression of adhesion molecules on endothelial cells, which is induced by the cytokine [3] [4]. TNF-α
interferes directly with the metabolic pathways of TGs (triglycerides) and cholesterol [5] [6] [7], thereby increasing
the concentration of phospholipase A2 [8]. Phospholipase A2 hydrolyzes the phospholipids in LDL (low-density
lipoprotein), generates FAs (fatty acids) that oxidize LDL [9], and increases the amount of oxidized LDL among the
sphingolipids [10]. Oxidation renders LDL more atherogenic.

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If TNF-α over-expressed in human skeletal muscle cells during inflammation, it may induce a generalized
insulin-resistant state [11]. In the adipose tissue, TNF-α stimulates lipolysis to increase the amount of FFAs (free
fatty acids) in the plasma [12] and inhibits insulin signaling by stimulating the p55TNF receptor and activating
sphingomyelinase [13]. These may be the reasons for the development of insulin resistance. In the glucose
metabolic pathways, TNF-α inhibits the actions of adiponectin and stimulates those of leptin. The production of
leptin is increased in these circumstances, and the insulin-resistant state may be indirectly induced [14]. Further,
TNF-α stimulates monocyte chemotactic protein-1 expression and production [15], whereby the recruitment of
macrophages into the adipose tissue is increased and the inflammatory state is triggered, once again resulting in
insulin resistance.

In adipocytes, adiponectin and TNF-α each inhibit the synthesis of the other [16]. The action of insulin is
influenced by adiponectin: insulin resistance and obesity are inversely correlated with the plasma adiponectin
concentration [17] [18]. In humans, low plasma adiponectin concentrations may induce the development of type 2
diabetes [19]. The JNK (c-Jun N-terminal kinase) pathway is active in obesity and diabetes, and TNF-α activates
JNK to inhibit adiponectin production [20].

Considering all these points, we can infer that the use of anti-TNF-α therapy to treat RA (rheumatoid arthritis),
cardiovascular morbidity, obesity, insulin resistance, endothelial dysfunction, and other inflammatory disorders may
yield good results [21] [22] [23] [24].

Curcumin is the principal curcuminoid found in the popular Indian spice turmeric, which is a member of the ginger
family Zingiberaceae. Curcumin may downregulate the steady-state levels of TNF-α [25], block LPS
(lipopolysaccharide), and reduce the production of TNF-α [26]. In in vitro conditions, curcumin also inhibits
TNF-α gene expression in human OA synoviocytes and chondrocytes [5].

EGCG (epigallocatechin-3-gallate) is a polyphenol found in tea [16]. It can block the interaction between tumor
promoters and cells, thereby inhibiting the expression of TNF-α mRNA and the release of TNF-α [18].

SYRINGIN is isolated by activity-guided fractionation of the EtOAc (ethyl acetate) extracts of the stem bark of
Magnolia sieboldii. SYRINGIN was found to significantly inhibit both TNF-α production by LPS-stimulated
RAW264.7 cells and CTLL-2 (CD8+ T cell) proliferation in a dose-dependent manner [21] [27].

Sinapyl alcohol is the hydrolysate of SYRINGIN. It inhibits LPS-induced TNF-α production by macrophages even
more potently than SYRINGIN [28].

Triptolide extracted from TWHF (Tripterygium wilfordii Hook F) may suppress TNF-α [29] [9]. Triptolide has
been found to be a safe and efficient drug for the treatment of patients with RA [30]. It exhibits inhibitory effect in
corneal fibroblasts stimulated with the proinflammatory TNF-α [31], and in bovine chondrocytes, triptolide
suppresses the TNF-α-induced expression of ADAMTS-4 [32].

In this paper, we used SWISS-MODEL Workspace [33] [34] [35] [36] [37] to perform homology modeling of
TNF-α, and the quality of this homology model was verified. The docking of 5 herbal medicines—curcumin,
EGCG, sinapyl alcohol, SYRINGIN, and triptolide (which were previously described to competitively inhibit the
activities of TNF-α)—with the homology model of TNF-α was assessed using Autodock 4.0 [22] [38] and
LIGPLOT [31].The abilities of inhibitions, degree of stability to inhibit TNF-α, binding sites, and relationships of
interactions of the five herbal medicines with TNF-α cytokine were discussed.

The rest of this paper is organized as follows. In Section 2, the details of the materials and methods used are
presented. In Section 3, the results of each docking step are given and discussed. Finally, the conclusion is
presented in Section 4.

2. Materials and Methods

Homology model creation. SWISS-MODEL Workspace [33] [34] [35] [36] [37] was used to create a complex
macromolecular TNF-α model. Homology modeling for TNF-α was performed with Chain B, Tumor Necrosis

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Factor Alpha, R31d Mutant (PDB ID: 1A8MB) as the template. Energy minimization was performed using
SwissPdb Viewer [34] (http://www.expasy.org/spdbv). The generated model was entered into the UCLA
bioinformatics server Structural Analysis and Verification Server (SAVES; http://nihserver.mbi.ucla.edu/SAVES/)
for evaluating its quality using PROCHECK [39][40], VERIFY3D [41], and ERRAT [42].

Crystallographic analysis of ligands. ChemSpider (http://www.chemspider.com/) is a free access server

structure-centric community for chemists and is integrated with a multitude of other online services. ChemSpider
was used for crystallographic analysis of the ligands—SYRINGIN, sinapyl alcohol, curcumin, EGCG, and
triptolide.

Docking operations. Autodock 4 with ADT [22] [38] was the main software employed to conduct the docking
calculations on the TNF-α homology model with the 5 ligand crystals. We executed the docking calculations in 2
phases described as follows.

Phase 1. In the homology modeling, polar hydrogen atoms were added to TNF-α macromolecules, and
non-polar hydrogen atoms were merged, with Gasteiger charges assigned for the ligands. The rotatable bonds of
all ligands were set to be rotatable. The center of the grid box was set to the center of the macromolecule, and the
scope of the grid box included the entire macromolecule (dimensions of the grid box: 64 × 64 × 64; grid spacing:
0.831 Å). A genetic algorithm was used with the local search method. A population size of 150 and 10 millions
energy evaluations were used for 100 times searches. The 3 best conformations were selected from the lowest
docking energy identified among the different pockets of the macromolecule for each ligand.

Phase 2. We executed the second run of the docking operations on the 3 best conformations for each docked ligand
identified in Phase 1. The docking steps and parameters are identical to those in Phase 1, except that each grid box
was set to the center of a ligand, and the scope of the grid box included only the nearest pocket of the
macromolecule (dimensions of the grid box: 52 × 52 × 52; grid spacing: 0.375 Å). We selected the best
conformation with the lowest energy and the highest population of molecules in a particular cluster, with not more
than 1.5 Å (rmsd (root-mean-square deviation)). The H-bond, van der Waals (VDW), and other binding
interactions were then analyzed by ADT. The hydrophobic interactions were analyzed by LIGPLOT [31].

3. Results and Discussion

Homology model of TNF-α. The overall sequence identity between the 1a8mB template and TNF-α was 100%.
The SwissPdb Viewer reduced the energy of the homology model from –349.758 kJ/mol to –8634.983 kJ/mol. In
addition, PROCHECK (there was just 1 non-glycine residue in the disallowed region), VERIFY3D (89.54% of the
residues had an average 3D-1D score > 0.2), and ERRAT (overall quality factor: 83.333) revealed good quality
results for the designed model.

Docking modes and estimated energy. The 5 inhibitors shown in Figure 1 were used as ligands to study their
binding interaction with the active site of TNF-α. We designated each docked ligand as l_i, wherein l is the name
of each inhibitor and i is the index of the docked conformation for ligand l selected in Phase 2 of the docking
operations. The energy results obtained by using Autodock 4.0 are shown in Table I ranked in descending order of
estimated free energy. According to the estimated free energy in Table I, we observed that (1) curcumin, EGCG,
sinapyl alcohol, SYRINGIN, and triptolide strongly inhibit TNF-α. This result corresponds to those of previous
studies [5] [9] [16] [18] [25] [26] [30] [31] [32] [43] [44]. (2) We also found that EGCG_3 is the strongest
inhibitor of TNF-α. (3) The standard deviations of the estimated free energy of binding for curcumin, EGCG,
sinapyl alcohol, SYRINGIN, and triptolide are 0.05, 0.65, 0.21, 0.43, and 0.22, respectively. Therefore, the order
of these 5 inhibitors in terms of the stability of TNF-α inhibition is curcumin > sinapyl alcohol > triptolide >
SYRINGIN > EGCG.

All the ligands showed low internal energy, indicating that the docked conformers were in their most favorable
conformation. The spatial arrangement of the ligands bound to the active site of TNF-α is shown in Figures 2 and
3. The order of the ligands in decreasing order of torsional free energy (Table I) is SYRINGIN (3.29) > EGCG
(3.02) > curcumin (2.74) > sinapyl alcohol (1.65) > triptolide (0.55). This indicates that SYRINGIN has the

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maximum rotatable torsion points. Curcumin_1, EGCG_2, sinapyl alcohol_1, sinapyl alcohol_2, triptolide _1, and
triptolide_3 are in the same pocket of TNF-α, as shown in Figure 3(a). Further, curcumin_2, curcumin _3,
SYRINGIN_1, sinapyl alcohol_3, and SYRINGIN_3 are docked in the same pocket of TNF-α, as shown in Figure
3(b). EGCG_3, SYRINGIN_2, and triptolide_2 are docked in the same pocket of TNF-α (Figure 3(c)).
According to the results shown in Figure 3, we predicted that TNF-α activation is inhibited more efficiently when
curcumin, EGCG, sinapyl alcohol, SYRINGIN, and triptolide are used simultaneously.

Interactions between inhibitors and TNF-α residues. VDW and H-bond interactions were analyzed by Autodock
4.0. The spatial relations of these interactions for each ligand and the interacting residue are shown in Figures 5
and 6. The summaries for the VDW and H-bond interactions are presented in Tables II and Table III, respectively.
The hydrophobic interactions were assessed by LIGPLOT, and the results for each pocket described in Figure 3 are
shown in Figure 4. From Figures 3 and 4 and Tables II and III, we can recognize the binding areas for Pockets A,
B, and C as follows: For Pocket A, the residue sets in the binding area are (Lys125, Gly126, Gln127, Cys129,
Pro130), (Thr165, Pro166, Glu167, Ala169, Glu170, Ala171), and (Arg198, Asp200, Tyr201, Leu202, Asp203,
Phe204, Ala205). For Pocket B, the residue sets in the binding area are (Arg66, Pro68, Ser69), (Arg91, Arg92,
Ala95, Leu96, Leu97, Ala98, Asn99, Gly100, Val101, Glu102 Leu103, Arg104, Asp105, Asn106, Gln107), and
(Lys150, Ser193, Ile196, Pro199). For Pocket C, the residue sets in the binding area are (Gly128, Cys129),
(Ser159, Pro160, Cys161, Gln162, Arg163), and (Lys172, Pro173, Trp174, Tyr175, Glu176). We also found that
curcumin and SYRINGIN docked at the receptor-binding sites of TNF-α. Syringin_1docked at the Asn90 and
Ala95 receptor-binding sites, while curcumin_3 docked at Leu89 and Asn90. Thus, curcumin and SYRINGIN
may influence or even interrupt the signal transduction between TNF-α and its receptor, thereby suppressing the
inflammation induced by TNF-α.

From Figures 3 and 4 and Tables II and III, it can be seen that Cys129 is the common binding site for all 5 inhibitors.
The results of analysis of the common binding sites for Pockets A, B, and C are described as follows: (1) The
common binding sites for curcumin, EGCG, sinapyl alcohol, and triptolide in Pocket A are Gln127, Cys129, Pro130,
Pro166, Ala171, and Tyr201. (2) That for curcumin and SYRINGIN in Pocket B is Leu97. For curcumin and
sinapyl alcohol, the common binding sites in Pocket B are Asp105 Asn106. (3) The common binding sites for
EGCG, SYRINGIN, and triptolide in Pocket C are Cys129, Pro160, Trp174, and Tyr175.

Analysis of the covalent π–π aromatic and π–cation interactions is shown in Figure 7. Tyr201 has a π–π aromatic
interaction and Lys126 has a π–cation interaction with curcumin_1 (Figure 7(a)). Ser193 is involved in a π–cation
interaction with curcumin_2 (Figure 7(b)). With sinapyl alcohol_2, Tyr201 shares a π–π aromatic interaction while
Lys125 shares a π–cation interaction (Figure 7(c)). These findings indicate that curcumin and sinapyl alcohol are
good inhibitors of inflammation.

4. Conclusions

The 5 herbal medicines curcumin, EGCG, sinapyl alcohol, SYRINGIN, and triptolide that competitively bind
to TNF-α were examined using a TNF-α model. We inferred the following with regard to the docking results.

Remark 1. We predict that the 5 herbal medicines are good inhibitors of TNF-α and the degree of stability of
TNF-α inhibition decreases in the order curcumin > sinapyl alcohol > triptolide > SYRINGIN > EGCG. We also
predict that if all 5 inhibitors are simultaneously used as a herbal medicine complex, inflammation will be inhibited
more effectively than if they are used individually.

Remark 2. Several interactions such as hydrophobic, VDW, and H-bond interactions were observed between
the herbal medicines and the active sites of TNF-α. The herbal medicines share common binding sites within all 3
pockets of TNF-α. The Cys129 residue is the common binding site for all 5 inhibitors. Further, curcumin and
SYRINGIN docked at the receptor-binding sites of TNF-α.

Remark 3. π–π aromatic interactions or π–cation interactions were detected for between the ligands sinapyl
alcohol and curcumin and TNF-α. We predicted that these 2 herbal medicines are strong inhibitors of
TNF-α-induced inflammation.

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(a) (b) (c)

(d) (e)
Figure 1. Structures of 5 inhibitors. (a). curcumin (b). EGCG (c) . sinapyl alcohol. (d). SYRINGIN (e). triptolide

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(a) (b)

(c)
Figure 2. Docking mode of all inhibitors. (a), (b), and (c) represented 3 sides of the complex. The molecular surface was mapped as a
solvent-excluded surface.

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(a).Pocket A (b).Pocket B

(c). Pocket C

Figure 3. Cluster docked ligands in the same pocket of TNF-α. The docked ligands are in stick mode. TNF-α is displayed as secondary structure.

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(a).Pocket A (b).Pocket B

(c). Pocket C

Figure 4. Cluster docked ligands with hydrophobic interactions in the same pocket of TNF-α. The docked ligands are in stick mode. Residues that
involved in hydrophobic interaction are shown as solvent-excluded surface.

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(a).curcumin_1 (b).curcumin_2 (c).curcumin_3

(d).EGCG_1 (e).EGCG_2 (f).EGCG_3

(g). sinapyl alcohol_1 (h). sinapyl alcohol_2 (i). sinapyl alcohol_3

Figure 5. Van Der Waals (VDW) and hydrogen bonding interaction for ligand: curcumin, EGCG, and sinapyl alcohol. Interacted residues are
shown in stick model. Docked ligands are shown as line and solvent-excluded surface. VDW radius are mapped as wireframe and colored by
atom type. Lines formed by small green balls are represented as H bonding interactions. Docked ligands and interacted residues are colored by
atom types: Carbons that are aliphatic colored by white, Nitrogen colored by blue, Oxygen colored by red, Sulfur colored by yellow, Hydrogen
colored by cyan.

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(a). SYRINGIN_1 (b). SYRINGIN_2 (c). SYRINGIN_3

(d).triptolide_1 (e).triptolide_2 (f).triptolide_3

Figure 6. Van Der Waals (VDW) and hydrogen bonding interaction for ligand: SYRINGIN and triptolide. The representations of are as described
in Figure 5.

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(a). curcumin_1 (b). curcumin_2

(c). sinapyl alcohol_2

Figure 7. π –π type aromatic interaction and π –cation interaction. Residues are represented as stick mode. Ligands with the interactions are in
line mode. Ligands and Residues are colored by atom types described as in Figure 5.

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Table I. Energies (in kcal/mol) and Ki value calculated using AUTODOCK 4.0
Energy Type (1) Estimated (2) Estimated (3) Final (4) Final Internal (5) (6) vdW (7)Electr (8) Unbound
&
Free Energy of Inhibition Intermolecula Energy Torsional + Hbond ostatic System's
Ligand Binding Constant, r Energy Free + desolv Energy Energy
um (Ki) Energy Energy

sinapyl alcohol_3 -4.16 885.52 -5.7 -0.51 1.65 -5.42 -0.28 -0.4
sinapyl alcohol_1 -4.4 596.45 -6.04 -0.41 1.65 -5.91 -0.13 -0.4
sinapyl alcohol_2 -4.67 375 -6.25 -0.47 1.65 -5.74 -0.52 -0.4
SYRINGIN_3 -5.09 184.27 -6.78 -2.75 3.29 -6.49 -0.3 -1.15
SYRINGIN_2 -5.47 98.28 -7.45 -2.47 3.29 -6.96 -0.48 -1.16
EGCG_2 -5.62 76.03 -7.9 -1.22 3.02 -7.54 -0.36 -0.48
SYRINGIN_1 -6.13 32 -7.6 -2.77 3.29 -7.49 -0.11 -0.95
curcumin_1 -6.19 28.78 -8.62 -0.9 2.74 -8.28 -0.33 -0.58
curcumin_2 -6.2 28.64 -8.61 -0.92 2.74 -8.24 -0.37 -0.58
curcumin_3 -6.31 23.66 -8.7 -0.94 2.74 -8.47 -0.22 -0.58
triptolide_3 -6.31 23.7 -6.49 -0.54 0.55 -6.39 -0.1 -0.18
EGCG_1 -6.44 19.04 -8.79 -1.2 3.02 -8.42 -0.37 -0.54
triptolide_2 -6.66 13.19 -6.79 -0.6 0.55 -6.65 -0.14 -0.18
triptolide_1 -6.84 9.76 -7.01 -0.56 0.55 -6.79 -0.22 -0.18
EGCG_3 -7.2 5.26 -9.65 -1.1 3.02 -8.94 -0.71 -0.53 *
(1)=(3)+(4)+(5)-(8)
*Temperature is 298.15K

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Table II. Hydrogen bonding interaction and energy for each bond.
Ligands Residues Invoved in Hydrogen Bonding & Energy of Each Bond
crcumin_1 LYS125(-6.263) GLN127(-2.407)
GLN85(-1.402
crcumin_2 ARG104(-5.267) ASN106(-2.385) LYS150(-3.42)
/-4.349)
crcumin_3 GLU102(-0.676) ASN106(-3.893)
SER155( -7.671
/-1.747 TYR179 (-0.485 VAL183( -4.768
EGCG_1
/-1.239 /-3.729) /-2.403)
/-0.823 )
EGCG_2 GLU170(-1.376) ALA171 (-4.983)
EGCG_3 SER159 (-2.856) PRO160 (-2.159 ) ARG163 (-4.989) LSY172(-1.67 )TYR175(-4.983) GLU176(0.039)
ALA171(-0.167
sinapyl alcohol_1 THR165( -6.125)
/-7.215)
sinapyl alcohol_2 LYS125(-6.225) LEU202(-1.559) ALA205 (-3.95)
sinapyl alcohol_3 LEU86(-4.881) ASP105(-3.801) LYS150(-3.555)
ASN99 (2.844
SYRINGIN_1 ASN90 (-0.845) ALA95 (-1.567)
/-5.347)
TYR175(-5.521
SYRINGIN_2 GLU176 (-3.402)
/-0.811)
SYRINGIN_3 SER69 (-3.542) GLY100 (-3.034) VAL101(-1.272)
LYS125(-2.162
triptolide_1 ALA205(-4.652)
/-0.887)
triptolide_2 TYR175(-6.749)
triptolide_3 GLN127 (-4.638) ALA171 (-5.786)
*If one residue has more than one Hydrogen bonding interactions with one inhibitor, the energy of bonds were split
by slash.

Table III. VDW interactions

Ligands Residues Invoved in Van Der Waals Interaction

curcumin_1 LYS125 GLN127 GLY126 CYS129 PRO130 PRO166 ALA171 TYR201
curcumin_2 GLN85 GLN87 ASN106 GLN107 ARG104 ASP105 LYS150 SER193 GLU195
curcumin_3 GLN87 TRP88 LEU89 ASN90 ARG91 LEU97 GLU102 LEU103 ASP105 ASN106
EGCG_1 LEU154 SER155 ALA156 TYR179 LEU180 GLY181 GLY182 VAL183 PHE184
EGCG_2 GLN127 CYS129 PRO130 THR165 PRO166 GLU167 ALA169 GLU170 ALA171
EGCG_3 GLY128 CYS129 SER159 PRO160 CYS161 GLN162 ARG163 LYS172 TRP174 GLU176
sinapyl GLN127 CYS129 PRO130 THR165 PRO166 GLU170 ALA171
sinapyl LYS125 ASP200 TYR201 LEU202 ASP203 PHE204 ALA205
sinapyl GLN85 LEU86 ASP105 ASN106 LYS150 SER193 ILE196 PRO199
SYRINGIN_1 PRO68 ASN90 ARG91 ARG92 ALA95 LEU96 LEU97 ALA98 ASN99
SYRINGIN_2 CYS129 PRO160 GLN162 ARG163 PRO173 TRP174 TYR175 GLU176
SYRINGIN_3 ARG66 PRO68 SER69 LEU97 ALA98 ASN99 GLY100 VAL101 GLU102
triptolide_1 LYS125 GLY126 GLN127 ASP200 TYR201 LEU202 PHE204 ALA205
triptolide_2 GLY128 CYS129 PRO160 LYS172 TRP174 TYR175
triptolide_3 GLN127 CYS129 PRO130 PRO166 ALA171 ARG198 ASP200 TYR201
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