Key words
Murraya paniculata
- Rutaceae - coumarin - kimcuongin - murracarpin - AutoDock/Vina - soluble epoxide
hydrolase (sEH)
Abbreviations
AUDA:
12-(3-adamantan-1-yl-ureido) dodecanoic acid
DHETs:
dihydroxyeicosatrienoicdiols
EETs:
epoxyeicosatrienoic acids
Ki:
inhibitor constant
Km:
Michaelis-Menten constant
PHOME:
3-phenyl-cyano(6-methoxy-2-naphthalenyl)methyl ester-2-oxiraneacetic acid
PLIP:
protein-ligand interaction profiler
sEH:
soluble epoxide hydrolase
The sEH is the main enzyme that catalyzes the metabolism of EETs into the more polar
and usually less potent metabolites DHETs [1]. Early studies have indicated that chemical compounds that can inhibit the sEHsʼ
activity and stabilize the endogenous EET levels may represent potential therapeutic
agents for cardiovascular disease [2], [3] and the onset of several other diseases [4], [5].
Murraya paniculata (L.) Jack (Rutaceae), local name “Nguyet que”, is mostly grown as ornaments for its
glossy green foliage and white fragrant flowers. It is used as a medicinal plant in
Vietnamese traditional medicine. There were several publications of phytochemical
studies of Murraya (see Supporting Information). In this study, we describe the inhibitory activity
against the sEH of coumarins isolated from this plant and explain this effect by molecular
modelling.
The methanol extract of the dry leaves of M. paniculata was suspended with water and subsequently fractioned with n-hexane, chloroform, and ethyl acetate. From the n-hexane fraction, two sterols, β-sitosterol (6) and β-stigmasterol (7), were isolated. From the chloroform fraction of the leaves of M. paniculata, five coumarins (1–5) were subsequently isolated through column chromatography including mexoticin (1) [6], omphalocarpin (2) [7], murrangatin (3) [8], kimcuongin (4), and (−)-murracarpin (5; [Fig. 1]). The latter two coumarins are reported to possess vasorelaxing activity [9].
Fig. 1 Structures of the isolated compounds (1–5).
The in vitro the inhibitory activity of the isolated coumarins (1–5) from M. paniculata leaves was investigated based on the hydrolysis of the sEH on an artificial fluorescent
substrate, PHOME, with AUDA as a positive control (IC50 15.7 ± 2.7 nM; [Fig. 2]). With the exception of compounds 1 and 5, coumarins 2, 3, and 4 exhibited potential sEH inhibitory activity (assessed at the sEH concentration of
25 µM; [Table 1]). Their IC50 values were found to be 3.2 ± 4.5, 13.9 ± 6.5, and 2.2 ± 4.7 µM, respectively ([Fig. 2] and [Table 1]).
Fig. 2 Inhibitory effect of the isolated coumarins (1–5) on sEH activity determined using the fluorometric method. The sEH activity is expressed
as the percentage of control activity (n = 3). AUDA (IC50 15.7 ± 2.7 nM) was used as a positive control.
Table 1 A In vitro sEH inhibitory activity with IC50 values, kinetic study of coumarins (1–5) and ν
max, Km, and Ki,exp values [Substrate concentration of 0.75 µM. AUDA was used as a positive control 15.7 ± 2.7
(nM)]. B Predicted and experimental binding parameters of coumarins 2–4 to sEH enzyme.
|
No.
|
Compounds
|
A. Kinetic parameters
|
B. Predicted and experimental binding parameters
|
|
IC50 (µM)
|
Binding mode
|
ν
max
|
Km
|
Ki,exp = 1/Km
|
ΔG (kcal/mol)
|
Ki,pred
|
pKi, pred
|
IC50, pred (µM)
|
pKi,exp
|
|
ND = not determined
|
|
1
|
Mexoticin
|
> 25
|
ND
|
|
|
|
|
|
|
|
|
|
2
|
Omphalocarpin
|
2.2 ± 4.7
|
Mixture (competitive/noncompetitive)
|
520.6
|
4.922
|
0.203 169
|
− 6.4
|
2.02E-05 M
|
4.69
|
2.5
|
5.66
|
|
3
|
Murrangatin
|
13.9 ± 6.5
|
noncompetitive
|
628.8
|
4.903
|
0.203 956
|
− 6.8
|
1.03E-05 M
|
4.99
|
(ND)
|
4.87
|
|
4
|
Kimcuongin
|
3.2 ± 4.5
|
noncompetitive
|
651.8
|
3.573
|
0.279 876
|
− 7.8
|
1.89E-06 M
|
5.72
|
(ND)
|
5.49
|
|
5
|
Murracarpin
|
> 25
|
ND
|
|
|
|
|
|
|
|
|
Kinetic parameters including maximum velocity (ν
max), Km, Ki, and the mode of inhibition of the potent sEH inhibitors 2, 3, and 4 were obtained using the sEH enzyme as shown in [Table 1]. According to the Ki,exp value, omphalocarpin (2) showed a higher possibility to interact with the sEH enzyme. In the presence of
the two inhibitors 3 and 4, the Km values of the sEH enzyme were found to be similar, while the ν
max and Km values were gradually decreased and even lower than those of the reaction without
the presence of the inhibitor (0.0 µM; [Fig. 3]). This suggested that the inhibitory activity of 3 and 4 on the sEH followed the noncompetitive binding mode and the coumarins might affect
the enzyme-substrate complex. While in the case of the reaction catalyzed by coumarin
2, the ν
max value decreased, but the Km value increased compared to that of the reaction without inhibitor. These facts suggest
that the inhibitory activity of coumarin 2 on sEH was different from those in the presence of coumarins 3 and 4. It might follow a mixture (competitive/noncompetitive) binding mode.
Fig. 3 Effects of coumarins 2, 3, and 4 on the activity of enzyme sEH for hydrolysis of the PHOME substrate.
In order to investigate the binding mechanisms of the active coumarins to the sEH
enzyme, molecular docking using AutoDock Vina software was carried out, where each
coumarin was manually docked at the active site of the human sEH (complexed with ligands;
[Fig. 4] and Tables 1 S and 2 S, Supporting Information). All three coumarins have hydrophobic interactions to acid
amines Asp335A, Leu408A, Leu499A, His524A, and Trp525A ([Fig. 5]). The smallest hydrophobic bond belonged to omphalocarpin (2) binding to Leu499A with 2.66 Å. Kimcuongin (4) prefered to binding to phenylalanine [Phe267A (3.89 Å) and Phe381A (3.55 and 3.97 Å].
The estimated hydrogen bond formed between the carbonyl groups of the coumarins and
the residues showed that the best docked conformation was determined for kimcuongin
(4) with Tyr383A and Tyr466A of the sEH active site from 2.13–2.77 Å (Table 2 S, Supporting Information).
Fig. 4 A Docked complexes of omphalocarpin (2, blue), murrangatin (3, magenta), and kimcuongin (4, green) into human sEH shown in cartoon representation. B PLIP 3D-interaction map of the ligands with sEH residues playing an essential role
for binding.
Fig. 5 ABS-Scan energy plot. ΔΔG values recorded after alanine scanning mutation of the single residues involved
in the binding of omphalocarpin (blue), murrangatin (magenta), and kimcuongin (green).
During the docking process, the protein was considered to be rigid while the ligands
and those amino acids inside the pocket were flexible. The AutoDock output results
represented the docking scores as Gibbs free energy of binding (ΔG) values, further converted to the predicted inhibition constants (pKi,pred). The designed compounds (2, 3, and 4) were found to have excellent binding affinity to the enzyme, showing binding energies
as ΔG values of − 6.4, − 6.6 and − 7.8 kcal/mol, respectively. The negative values of
ΔG indicated that the coumarins bind to sEH spontanously and these values also proved
that the compounds possess potential sEH enzyme inhibitory binding activities. Three
compounds occupy the same cavity (Fig. 1 S, Supporting Information) with only some differences in amino acid residue involvement
because of their dihedral rotation and conformational mismatch. The small variance
in ΔG values and binding posed inside the pocket may be attributed to the differences
in the position of the functional groups in the selected compounds. The ΔG values were further converted to pKi,pred, where Ki was calculated by the formula Ki = IC50/1 + [S]/Km = exp(ΔG/R × T), so IC50 = exp(ΔG/R × T) × 1 + [S]/Km ([Table 1]).
The docking results of the three compounds were analyzed by a bioinformatic tool,
giving an interaction diagram and a table of interaction data for each binding site.
For all compounds, the binding was dominated by hydrophobic interactions and hydrogen
bonds. The three diagrams, in agreement with the ΔG calculation results, showed that compounds 2 and 3 had a similar set of binding profiles (seven hydrogen interactions and one hydrogen
bond), while 4 had the best binding outline (nine hydrogen bonds and two hydrogen bonds; [Fig. 4]).
The amino acids presented at the binding site are computationally mutated to alanine
in an ABS-Scan tool to perform an in silico alanine scanning mutagenesis for binding site residues in the protein-ligand complex
([Fig. 5]). The binding energy is computed for each mutant and the corresponding energy differencies
(ΔΔG) values between the wild-type protein and the mutated one are also calculated. The
ΔΔG profile shared by murrangatine (3) and kimcuongin (4) was not totally repeated by omphalocarpin (2), which showed the smallest binding energy. The docked poses of coumarin derivatives
clearly demonstrated the binding positions of the ligand with the enzyme. The main
binding force is due to the interactions of Tyr466 and Tyr383 with the coumarin derivatives,
while other important interactions were found with amino acid residues Trp 525, Trp
336, and Phe 267. The coumarin kimcuongin (4), furthermore, showed good affinity towards Tyr 383, Gln 384, and Phe 387, which
were responsible for better sEH binding energy and its potential inhibitory activity.
In summary, among five coumarins isolated from the chloroform fraction of the leaves
and stems of M. paniculata, three coumarins, omphalocarpin (2), murrangatin (3), and kimcuongin (4), showed sEH inhibitory activity with IC50 values 2.2 ± 4.7, 13.9 ± 6.5, and 3.2 ± 4.5 µM, respectively, in noncompetitive (3, 4) and mixture-kinetic mode (2). Using a computational approach, kimcuongin (4) showed the best binding outline, characterized by the smallest binding energy (ΔG = − 7.8 kcal/mol), a good affinity towards Tyr 383, Gln 384, and Phe 387, and its
potential sEH inhibitory activity. These three coumarins might be worthy to further
investigate in order to develop a new scaffold of therapeutic agents for cardiovascular
diseases.
Materials and Methods
The leaves and twigs of M. paniculata were collected in Cuc Phuong National Park, Hoa Binh Province, North Vietnam. The
plant was identified by botanist Dr. Tran The Bach, Institute of Ecology and Biological
Resources (VAST). A voucher specimen (C-425) is deposited in the herbarium of the
Institute of Natural Products Chemistry (VAST), Hanoi, Vietnam.
Dried powdered leaves and twigs of M. paniculata (3.2 kg) were extracted with MeOH to yield a black crude MeOH extract (120 g). The
crude MeOH extract was suspended in hot MeOH-water (1 : 1, v/v) and successively partitioned
with n-hexane, dichloromethane (DCM), ethyl acetate (EtOAc), and water to give the corresponding
solvent-soluble fractions n-hexane (6.7 g), chloroform (7.2 g), EtOAc (16.7 g), and water (60 mL). Repeated chromatography
of n-hexane and chloroform fractions on silica gel columns with different eluting solvents
resulted in the separation of compounds 1–5 and 6, 7, respectively.
Supporting information
General experimental procedures, extraction and isolation, sEH bioassays, and molecular
modelling are described in detail in Supporting Information.
Acknowledgements
This research was supported by the Ministry of Science and Technology (MOST) for the
Vietnam-Korea Project (Code 52/2011/NĐT) and the National Foundation for Science and
Technology Development of Vietnam (NAFOSTED), grant No. 104.01–2010.25.
