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Introduction
The prostaglandin endoperoxide H synthase (PGHS),
cyclooxygenase (COX), is in charge of prostaglandin
synthesis[1]. Arachidonic acid (AA) is catalyzed into
prostaglandin H2 (PGH2) through the dual functions of
PGHS[2]. Several prostanoids, which maintain normal physiologies,
are metabolized from the precursor
PGH2[3,4]. Two isozymes have been identified: COX-1 is generally expressed in most
tissues and cells, and the production of COX-1 is maintain
several physiological functions[5,6]; COX-2 is inducible by
growth factors, cytokines, and
endotoxins[7], and the prostanoids act as the potent mediators of fever and
inflammation[8]. At present, many clinical reports have found that
tumorigenesis of some certain tissues and cells is correlated
with COX-2[9_11]. COX-2 has been found to induce
angiogenic factors regulated
angiogenesis[10,11], specifically in colorectal cancer cells. Some carcinogenesis could be
reduced by inhibiting the overexpression of COX-2. For
centuries, non-steroidal anti-inflammatory drugs
(NSAIDs)[12] have been taken as inhibitors of COX-2 to ease
pain[13]. These drugs even showed the efficiency to decrease incidence of
esophageal, gastric, and colorectal
cancers[14,15]. The two isozymes, COX-1 and COX-2 have high structure similarity
(root mean square difference, RMSD <1.0 Å) and
approximately 60% identity for shared
residues[16]. Furthermore, with an emphasis on the similarity of active sites, there are
only 2 different amino acids between the isoforms, His513
and Ile523 of human COX-1, and Arg499 and Val509 of
human COX-2[17]. According to the above reasons, most
NSAIDs are not selective for both isozymes. A consequence
is several side-effects, which is as a result of the
biosynthesis being inhibited[18]. Several studies have reported that
long-term aspirin usage might induce stomach
ulcers[19_21]. A strategy of developing novel drugs with higher selectivity
is important, and the efficacy and side-effects should be
considered.
The commercial compounds, resveratrol and NS-398, are
known specific inhibitors of COX-1 and COX-2,
respectively[22,23]. Resveratrol and NS-398 were recruited as the control
compounds for this study. The xanthone derivatives were
extracted from Garcinia mangostana
(mangosteen) and have several biological activities, including the effect of
anti-inflammatory, anti-bacterial, and skin-infection
free[24]. These derivatives were used reasonably against the inflammatory
function of COX-2. Furthermore, one of the derivatives
shows a potent cytotoxic effect against
hepatoma[25]. The derivatives, α-mangostin and
γ-mangostin, have the ability of preventing AA from binding to the active sites of both
isozymes[26_28]. In this study, hydroxyxanthones were
investigated. A further structure-based research
methodology of virtual screening was employed to analyze the
selectivity of the 2 isozymes. The program LigandFit (Accelrys,
San Diego, CA, USA) was used for the docking
procedures[29]. Several scoring functions, including dock score, piecewise
linear potential (PLP)[30], and potential of mean force
(PMF)[31], were taken to evaluate the docking results. The dock score
is a minus value of the summation of ligand/receptor
interaction energy and ligand internal energy. By considering 4
PLP atom-type predefinitions, the PLP score indicates the
sums of all atomic pairwise interactions of the
ligand/receptor complex. The PMF calculates all the interatomic pairs of
the ligand/receptor complex. Because the values of PLP and
PMF are minus, it would be added a minus prefix before each
the indicator, ie -PLP and -PMF. A higher score may suggest
a higher affinity, as the values of energy would be presented
with the sign reversed. The interaction energy between ligand
and receptor was calculated under the force field at the
Department of Chemistry at Harvard Macromolecular
Mechanics (CHARMm) according to the following
equation[32]:
Einteraction=Ecomplex
_(Eligand+Ereceptor
),
E: energy.
These calculations of scoring functions and interaction
energy represented the purpose of evaluating the ligand and
protein interactions. It might suggest the prediction of
selec-tivity to both isozymes and the directions of further rational
drug design.
Materials and methods
Homology modeling Molecular simulation was performed
under Discovery Studio modeling 1.7 (Accelrys, San Diego,
CA, USA). The protein templates were obtained from
Protein Data Bank (PDB). Ovine prostaglandin
H2 synthase-1 in complex with alpha-methyl-4-biphenylacetic acid (Ovis aries;
PDB ID: 1Q4G)[33], and AA bound to the COX-active site of
COX-2 (Mus musculus; PDB ID: 1CVU)[34]. The sequences
of human COX-1 (Swiss Prot accession No P23219) and
COX-2 (Swiss Prot accession No P35354) were acquired from
the Swiss Prot website, and the signal residues were excluded
from 1 to 23 for human COX-1 and 1 to 17 for human COX-2
before sequence alignment. The sequence identity between
the template and the target protein for COX-1 and COX-2
was 93.9% and 87.6%, respectively.
Docking prediction and interaction energy calculation
The simulated structure was defined as "SBD_RECEPTOR"
in the program, and the cavities as the binding sites within
the receptor were discovered by computational prediction
corresponding to the known active site within the key
residues, Arg120, Leu352, Tyr355, Tyr348, Trp387, Ser530,
and Leu 531 for COX-1[35]. For the same residues of human
species, actually the residue numbers should decrease one
residue number based on ovine species and distributed over
the same space in each structure, such as Arg120 to Arg119.
Xanthone derivatives were prepared by ChemOffice 2005
software (Cambridge Scientic Computmg, Cambridge, Massachusetts, USA), including sketch and energy
minimization (MM2 force field)[36]. These derivatives, compounds
A_H, were employed as the ligands while performing the
docking process. NS-398 and resveratrol were recruited as
the controls. By using LigandFit, compounds A_H were
docked into a cavity, which was located in the active site.
All the ligands were flexible in the docking procedure, while
the receptor was fixative. The poses of all the compounds
were arranged by the LigandFit module in the active site by
Monte Carlo trials, and the dock score was calculated
relying on the generated pose by the Dreiding energy grid force
field. Scoring functions, PLP, and PMF, scored the docking
results for each compound within the receptor. After
docking, the energies of the ligand/receptor complexes were
minimized. Then the interaction energy of each complex was
calculated according to the premised equation.
Results
Structure modeling and analysis of xanthone derivatives
Both human isozymes were built under comparative modeling.
The sequence identity between the template and the target
protein for COX-1 and COX-2 was 93.9% and 87.6%,
respec-tively. With the high similarity and identity of the protein
templates, the built protein structures were well folded.
According to the Ramachandron plot and Verify3D of each
crystal and simulated structures, the amino acids of the outer
regions were located at the peripheral residues and anchor
motif (data not shown). The anchor motif was far from the
active site, so it was reasonable to suppose that there was
no influence on the mechanism of AA catalyzing. All the
derivatives employed in this study are represented in Figure
1. There were 2 tetrahydroxyxanthones, 2 trihydroxyxan-thones,
and 4 dihydroxyxanthones. The compound A,
1,3,6,7-tetrahydroxyxanthone, revealed some similarities with
garcinone E. Compared with another tetrahydroxyxanthone,
compounds A and B indicate a wider structure with the
meta-hydroxyl group than the ortho-hydroxyl group. Compound
C present as a narrower structure than compound A.
Compound D is shorter in the length than compound C. Half of
the hydroxyl groups are on compound E than on compound B.
Binding-site definition and scoring function evaluation
The predictive binding sites of COX-1 and COX-2 were
matched with the actual active sites, respectively. A further
interaction analysis was conducted following the study of
the structure-based design. The scoring functions showed
the ranking of each compound in the fixed receptor. By
considering the scores and poses of all the compounds in
the lobby of the protein, better ligands were obtained. The
interactivon energy between ligand and receptor was
considered after compound screening, while lower interaction
energy indicated a more stable binding status.
Docking results and interaction energy calculations of
conformations of the marked ligands Compound A showed
a higher interaction force with both COX isozymes (Table 1).
The scoring functions in Table 1 suggest that the scaffold of
1,3,6,7-hydroxyxanthone interacted well with the active site.
Notably, compound A bound to Ser529, as COX-1 was
similar to aspirin which could acetylate Ser529 to prevent the
binding of AA[32,37]. For lower interaction energy, compound
A might be more stable than resveratrol and NS-398 in the
active sites of COX-1 and COX-2, respectively. The docking
pose of NS-398 was similar to SC-558 in another protein
template (PDB ID: 6COX). In addition, the simulation results of
these 2 control compounds, resveratrol and NS-398,
corresponded well with biological data. We suggest that the
simulation protein structures (human COX-1 and COX-2) and all
the simulation docking results were reliable. The docking
results of compounds A, B, and F, and the receptors were
displayed through molecular simulation as shown in Figure
2. It could be deduced that compound A might have better
inhibitory ability to both isoforms. From our observations,
we found that compound B had more inhibitory potency to
COX-1 than COX-2 through the calculation of interaction
energy. The active site of COX-1 showed a tunnel-like
structure, while the site of COX-2 was roomier than COX-1.
With the scoring functions showing the same tendency, it
was indicated that the agents with slimmer structures were
more suitable in COX-1 than in COX-2. Compound B
revealed an interesting result; the docking poses were
different between the complexes of compound B in COX-1 and
COX-2. The docking pose of compound B was like the pose
of the docked compound A and it gained a higher dock score
and a more stable energy status (Figure 2C). Although the
docking conformation of compound B had no hydrogen bond
predicted, it might be steadier in the space around the
residues than the pose in Figure 2D. Compounds C and F
showed potent selectivity to COX-2 with obvious differences
in the interaction energy (188.57 and 215.56 kJ/mol),
respec-tively. Through the structure-based study, compound F was
shown to have a hydrogen bond with Ser529 in COX-1, as
indicated in Figure 2E, while the horizontal orientation of
compound F in the active site of COX-2 showed that the
pose in Figure 2F resembled the conformation of compound
B in both COX-1 and COX-2. One strong hydrogen bond
with 1.79 Å was observed in the compound F/COX-2 complex.
Depending on this conformation, compound F could achieve
lower interaction energy and the higher dock score within
the receptor. It could be suggested that compound F might
be a potent novel lead compound for COX-2. Additionally,
compounds D, E, G, and H obtained stable statuses while
they docked with COX-2 than with COX-1. The
conformations of D, E, and G were similar, with compound F in the
active site of both protein structures (Figure 2E, 2F),
respectively.
Discussion
By considering the scoring functions, a better
conformation of compounds could be ranked. These conformations
influenced the integral stability of the ligand and receptor
complex, but the stability of energy within the
ligand/receptor complex would be the major emphasis of real time. Lower
interaction energy would imply more stable ligand/receptor
complexes.
According to structure-based researching methodology,
the feasible prediction of binding sites was calculated, and
every molecular docking result was analyzed through the
scoring functions, interaction energy, and structure
con-formation. Compounds A, C, and D showed lower
interaction energy compared to NS-398 in COX-2. Compound B
revealed selectivity with COX-1 and lower interaction
energy than resveratrol in COX-1. Notably, compound A, D,
and H also had better binding forces than resveratrol. It is
interesting to investigate the docking poses of compounds
B and E because of the difference in 2 of the hydroxyl groups.
Compound B could bind to the gate of active site, Arg119
and Tyr354, of COX-1 counting on the two more hydroxyl
groups and thus gaining more stable than compound E in
the lobby. Furthermore, the different hydroxyl groups
between compounds F and G changed the results slightly. The
binding poses were added in the supplementary part. It was
noticed that compound G in the active site of COX-1 gained
1 hydrogen bond with Tyr354 by its 5'-hydroxyl group. For
the adaption of this pose, the 2'-hydroxyl group of
compound G bound to Tyr384 instead of Ser529 because the
scaffold of this ligand docked with the upper position than
compound F in COX-1.
Compound A bound well to both isozymes by LigandFit.
Compound B showed selective potency of COX-1, while
compound F showed selectivity to COX-2 through
structure-based research. Because the cavity of the COX-2
catalytic site was larger than COX-1, these non-shape-specific
compounds were docked into the site non-specifically. Due
to this reason, the interaction energy varied with different
docking poses. Further pharmacophores should be
generated for the purpose of studying a quantitative
structure_activity relationship between the complexes, and the design
of more efficient inhibitors should be the next step.
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