Pradeep Paudel1, Su Hui Seong1, Srijan Shrestha1,2, Hyun Ah Jung3, Jae Sue Choi1. 1. Department of Food and Life Science, Pukyong National University, Busan 48513, Republic of Korea. 2. Discipline of Pharmacology, School of Medicine, Faculty of Health Science, The University of Adelaide, Adelaide, South Australia 5005, Australia. 3. Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju 54896, Republic of Korea.
Abstract
In recent years, Cassia seed extract has been reported as a neuroprotective agent in various models of neurodegeneration, mainly via an antioxidant mechanism. However, no one has previously reported the effects of Cassia seed extract and its phytochemicals on human monoamine oxidase (hMAO) enzyme activity. The seed methanol extract, the solvent-soluble fractions, and almost all isolated compounds displayed selective inhibition of hMAO-A isozyme activity. Interestingly, compounds obtusin (3), alaternin (8), aloe-emodin (9), questin (12), rubrofusarin (13), cassiaside (15), toralactone 9-O-β-gentiobioside (26), and (3S)-9,10-dihydroxy-7-methoxy-3-methyl-1-oxo-3,4-dihydro-1H-benzo[g]isochromene-3-carboxylic acid 9-O-β-d-glucopyranoside (38) showed the most promising inhibition of the hMAO-A isozyme with IC50 values of 0.17-11 μM. The kinetic study characterized their mode of inhibition and molecular docking simulation predicted interactions with Ile-335 and Tyr-326 in support of the substrate/inhibitor selectivity in respective isozymes. These results demonstrate that Cassia seed extract and its constituents inhibit hMAO-A enzyme activity with high selectivity and suggest that they could play a preventive role in neurodegenerative diseases, especially anxiety and depression.
In recent years, Cassia seed extract has been reported as a neuroprotective agent in various models of neurodegeneration, mainly via an antioxidant mechanism. However, no one has previously reported the effects of Cassia seed extract and its phytochemicals on humanmonoamine oxidase (hMAO) enzyme activity. The seed methanol extract, the solvent-soluble fractions, and almost all isolated compounds displayed selective inhibition of hMAO-A isozyme activity. Interestingly, compounds obtusin (3), alaternin (8), aloe-emodin (9), questin (12), rubrofusarin (13), cassiaside (15), toralactone 9-O-β-gentiobioside (26), and (3S)-9,10-dihydroxy-7-methoxy-3-methyl-1-oxo-3,4-dihydro-1H-benzo[g]isochromene-3-carboxylic acid 9-O-β-d-glucopyranoside (38) showed the most promising inhibition of the hMAO-A isozyme with IC50 values of 0.17-11 μM. The kinetic study characterized their mode of inhibition and molecular docking simulation predicted interactions with Ile-335 and Tyr-326 in support of the substrate/inhibitor selectivity in respective isozymes. These results demonstrate that Cassia seed extract and its constituents inhibit hMAO-A enzyme activity with high selectivity and suggest that they could play a preventive role in neurodegenerative diseases, especially anxiety and depression.
The typical structural and physiological
properties of brain neurons
decline with aging, accompanied by variable degrees of cognitive decline
(Alzheimer’s disease; AD), movement disorder (Parkinson’s
disease; PD), and excitotoxicity (Huntington disease; HD), which are
often grouped together as neurodegenerative diseases (NDs).[1] The etiopathology and affected brain regions
differ among the NDs; however, they all share progressive dysfunction,
collapse the neuronal network, neuronal cell death, neuroinflammation,
and oxidative damage to the brain.[2]Among the greatest health threats of the 21st century, cognitive
frailty demands a great deal of attention.[3] The incidence of NDs increases with age, and the population in developed
countries is rapidly aging. The number of people with PD is expected
to reach 8.7 million by 2040,[4] so it will
claim an increasing portion of world healthcare budgets, with an estimated
current cost in excess of US$600 billion worldwide.[2] PD is a chronic and progressive disorder of the central
nervous system that affects the motor system. It is the second most
frequent neurodegenerative disorder and is characterized by resting
tremor, rigidity, and bradykinesia due to the loss of dopaminergic
(DA) neurons of the midbrain.[5] Though PD
is a subject of great concern, its exact etiology remains unclear.
Researchers have postulated that oxidative stress, mitochondrial dysfunction,
neuroinflammation, and apoptosis lead to DA neuronal damage.[6−10] In PD, progressive loss of DA neurons occurs in the substantia nigra
pars compacta. Humanmonoamine oxidase (hMAO) is a flavoenzyme that
catalyzes the oxidative deamination of various bioamines; its inhibitors
were the first developed antidepressants. Because the major depressive
disorder is estimated to be the second leading cause of disease worldwide,[11] the need for hMAO inhibitors is urgent. hMAO
has two isozymes (hMAO-A and -B) that differ in their sensitivity
to inhibitors and substrate specificity.[12]MAO is an enzyme of crucial interest because it catalyzes
the major
inactivation pathway for the catecholamine neurotransmitters: adrenaline,
noradrenaline, dopamine, and even 5-hydroxytryptamine.[13] MAO is responsible for alterations in the level
of neurotransmitters in the central nervous system, and imbalanced
neurotransmitter levels are linked to the biochemical pathology of
many neurogenic disorders, including depression, AD, and PD.[14] Studies of the therapeutic effect of MAO enzyme
inhibitors in depression have characterized two isomeric forms, hMAO-A
and hMAO-B. The selectivity of a molecule to these isoforms determines
its therapeutic activity. hMAO-A is responsible for the metabolism
of norepinephrine, serotonin, and tyramine. Therefore, hMAO-A selective
inhibitors have antidepressant activity. On the other hand, hMAO-B
selectively metabolizes dopamine, so selective hMAO-B inhibitors have
been used to treat PD.Because MAO involves the oxidative deamination
of primary, secondary,
and tertiary amines to their corresponding aldehydes and free amines,
which generates hydrogen peroxide (H2O2), oxidative
stress is believed to be involved in a variety of NDs, including AD,
PD, and amyotrophic lateral sclerosis, because neurons are sensitive
to H2O2.[15−18] Similarly, amyloid β protein toxicity in AD
is caused by increased levels of H2O2 and the
accumulation of lipid peroxides.[19] Therefore,
the development of new hMAO inhibitors from natural sources has attracted
significant attention.Cassia obtusifolia Linn seed is
well-known in traditional Chinese medicine (TCM) for its pronounced
effects, including as a vision improver, aperient, diuretic, antiasthenic,
cholesterol-lowering agent, and blood-pressure reducer. The seed extract
has also been reported as a therapy for NDs.[20] In a current report, C. obtusifolia seed extract protected the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced
degeneration of DA neurons in the substantia nigra and striatum of
PDmice.[21] Furthermore, Cassia seed extract
exhibited neuroprotective effects via its anti-inflammatory effects,
upregulation of BDNF expression, and CREB phosphorylation.[22] Cassia seed also has a long history of use against
inflammation,[23] type II diabetes,[24] hepatotoxicity,[25,26] bacterial
infections,[27] AD,[28] oxidative stress,[29] tumor progression,[30] and mutagenesis.[31] Recently, we have reported the possible role of rubrofusarin in
comorbid diabetes and depression via PTP1B and hMAO inhibition.[32] However, despite many reports about the different
pharmacological actions of the seed extract, reports on the specific
compounds that produce particular activities are insufficient. Therefore,
to be more specific, we evaluated the hMAO inhibition potential of
isolated compounds (Figure ) and performed enzyme kinetics and molecular docking simulations.
Figure 1
Chemical
structures of compounds isolated from C.
obtusifolia Linn seeds.
Chemical
structures of compounds isolated from C.
obtusifolia Linn seeds.
Results
In Vitro
Human MAO Inhibition by a MeOH Extract and Solvent-Soluble
Fractions of C. obtusifolia Linn Seeds
The methanol extract of C. obtusifolia Linn seeds and its different solvent-soluble fractions were evaluated
for their potential to inhibit hMAO. As presented in Table , the methanol extract of Cassia
seeds demonstrated good inhibition against both isozymes of the hMAO
enzyme, hMAO-A (IC50: 86.89 ± 3.80 μg/mL), and
hMAO-B (IC50: 196.00 ± 6.82 μg/mL). Among the
solvent-soluble fractions, the EtOAc fraction showed the most potent
inhibition of hMAO-Aactivity (IC50: 20.82 ± 5.04
μg/mL), followed by the CH2Cl2 fraction
(IC50: 40.06 ± 3.58 μg/mL) and n-BuOH fraction (IC50: 93.49 ± 0.27 μg/mL).
Similarly, the EtOAc fraction showed the most potent hMAO-B inhibition
(IC50: 56.28 ± 3.13 μg/mL), followed by the
CH2Cl2 fraction (IC50: 98.51 ±
12.53 μg/mL) and n-BuOH fraction (IC50: 246.50 ± 10.31 μg/mL). All of the solvent-soluble fractions
inhibited hMAO-A better than hMAO-B, with a selectivity index (SI)
of approximately 2.5. From each fraction, we isolated compounds with
different skeletons (anthraquinone, naphthopyrone, naphthalene, and
naphthalenic lactone) and evaluated their ability to inhibit hMAO.
The inhibition result is presented in Table .
Table 1
Human MAO-A and MAO-B
Inhibition by
the Methanol Extract and Solvent-Soluble Fractions of C. obtusifolia Seeds
IC50 value for MAO isozymes (μg/mL ± SD)a
samples
hMAO-A (n = 3)
hMAO-B (n = 3)
SIb
methanol extract
86.89 ± 3.80
196.00 ± 6.82
2.25
CH2Cl2 fraction
40.06 ± 3.58
98.51 ± 12.53
2.46
EtOAc fraction
20.82 ± 5.04
56.28 ± 3.13
2.70
n-BuOH fraction
93.49 ± 0.27
246.50 ± 10.31
2.64
deprenyl HClc
9.19 ± 0.54
0.12 ± 0.03
0.013
clorgylined
0.00397 ± 0.75
63.41 ± 1.20
15 972.29
The 50% inhibition concentrations
(IC50, μg/mL) are expressed as the mean ± SD
of triplicate experiments.
The selectivity index (SI) was determined
by the hMAO-B to hMAO-A ratio of IC50 values.
Selective hMAO-B reference inhibitor
(IC50, μM).
Selective hMAO-A reference inhibitor,
and values (IC50, μM) were extracted from the literature.[54]
Table 2
Human MAO-A and MAO-B Inhibition by
Compounds from Cassia obtusifolia Seeds
The 50% inhibitory concentration
(IC50) values (μM) were calculated from a log dose
inhibition curve and expressed as the mean ± SD of triplicate
experiments.
The selective
index (SI) was determined
by the ratio of hMAO-B IC50/hMAO-A IC50.
Selective hMAO-B reference inhibitor.
Selective hMAO-A reference
inhibitor,
and values were extracted from the literature.[54]
The 50% inhibition concentrations
(IC50, μg/mL) are expressed as the mean ± SD
of triplicate experiments.The selectivity index (SI) was determined
by the hMAO-B to hMAO-Aratio of IC50 values.Selective hMAO-B reference inhibitor
(IC50, μM).Selective hMAO-A reference inhibitor,
and values (IC50, μM) were extracted from the literature.[54]The 50% inhibitory concentration
(IC50) values (μM) were calculated from a log dose
inhibition curve and expressed as the mean ± SD of triplicate
experiments.The selective
index (SI) was determined
by the ratio of hMAO-B IC50/hMAO-A IC50.Selective hMAO-B reference inhibitor.Selective hMAO-A reference
inhibitor,
and values were extracted from the literature.[54]
In Vitro Human MAO Inhibition
by Anthraquinones
A total
of twenty-three anthraquinones were isolated in the form of either
aglycones or glycosides. As tabulated in Table , all anthraquinones except for 1, 2, and 7 showed moderate to potent inhibition
of hMAO-A isozyme activity. Potency varied depending on the nature,
number, and position of the functional groups in the anthracene-9,10-dione
(anthraquinone) skeleton. The most promising inhibitor of hMAO-A was 12, which had an IC50 value of 0.17 ± 0.01
μM, followed by 9 (IC50: 2.47 ±
0.14 μM), 8 (IC50: 5.35 ± 0.09
μM), and 4 (IC50: 11.12 ± 0.60
μM). In addition to their hMAO-A isozyme inhibition, 8 and 12 exhibited the best inhibition of hMAO-B among
these compounds. Only a few compounds were active against hMAO-B,
indicating that the anthraquinones have preferential/selective inhibition
of hMAO-A. Glycosylation of the active aglycones retarded activity;
the pattern of activity was aglycones > glucopyranoside > gentiobioside
> triglucoside > tetraglucoside. Interestingly, for inactive
aglycones 1 and 2, glycosylation induced
inhibition of
isozyme-A but not isozyme-B.
In Vitro Human MAO Inhibition by Naphthopyrones
In
the list of isolated naphthopyrones (Table ), 13, 15, and 20 were the most active against the hMAO-A isozyme, with IC50 values of 5.90, 11.26, and 16.32 μM, respectively.
Inhibition of hMAO-Bactivity was moderate only for 20 and was mild for the other glycosides. As presented in Table , glycosylation of 13 retarded its inhibition of the MAO enzyme. Unlike the activity
pattern for the anthraquinone glycosides, the inhibition pattern for
the naphthopyrone glycosides was tetraglucoside (35)
> triglucoside (33) > gentiobioside (28)
> glucopyranoside (22).
In Vitro Human MAO Inhibition
by Naphthalenes and Naphthalenic
Lactones
Six naphthalenes and naphthalenic lactones were
evaluated for their hMAO inhibitory potential (Table ). These compounds were obtained as glycosides.
Compounds 26 and 38 had IC50 values
< 10 μM for hMAO-A. Interestingly, 26 did not
show any observable inhibitory effect on hMAO-B up to 400 μM,
whereas 38 moderately inhibited isozyme-B, with an IC50 value of 96.15 ± 3.35 μM. Compounds 16, 19, 27, and 37 moderately
inhibited isozyme-A, but their activity toward isozyme-B was negligible.Overall, the enzyme inhibition assay showed that compounds 3, 8, 9, 12, 13, 15, 26, and 38 were
the most potent (IC50: 0.17 to 11.26 μM) and highly
selective for hMAO-A inhibition.
Enzyme Kinetics of hMAO
Inhibition
Because compounds 3, 8, 9, 12, 15, 26, and 38 most potently inhibited
the hMAO isozymes, we selected them for the enzyme kinetics study.
Enzyme kinetics of 13 has been reported in our recent
report.[32] The results of the kinetic study
are presented using Michaelis–Menten plots, Lineweaver–Burk
plots, and secondary plots (Figures –4) and summarized in Table . As tabulated in the table, 26 and 38 showed selective and noncompetitive inhibition of hMAO-A, with Ki values of 4.30 and 9.57 μM, respectively,
and 9 showed a competitive mode of hMAO-A inhibition,
with a Kic value of 0.5 μM. Similarly,
the mode of selective hMAO-A isozyme inhibition by 15 was mixed-type, with a Kic value of
6.26 μM and a Kiu value of 12.32
μM. The mode of inhibition of the equipotent compound 8 over both isozymes was also a mixed-type. For hMAO-A inhibition,
it showed a Kic value of 3.97 μM
and a Kiu value of 9.33 μM, and
for hMAO-B inhibition, it had a Kic value
of 2.41 μM and a Kiu value of 4.46
μM. The mode of hMAO-A inhibition by 3 and 12 was competitive. The anthraquinone 12 exhibited
the most potent inhibition of hMAO-A, with a Kic value of 0.13 μM. In addition, it showed competitive
inhibition of hMAO-B, with a Kic value
of 4.14 μM.
Figure 2
Michaelis–Menten plots, Lineweaver–Burk
plots, and
secondary plots of compound 3 (A, B, C, and D), 8 (E, F, G, and H), and 9 (I, J, K, and L) for
hMAO-A inhibition.
Figure 4
Michaelis–Menten plots, Lineweaver–Burk plots, and
secondary plots of compound 8 (A, B, C, and D) and 12 (E, F, G, and H) for hMAO-B inhibition.
Table 3
Enzyme Kinetics of hMAO Inhibition
by Compounds from C. obtusifolia Seeds
compounds
inhibition
typea
Ki valueb
hMAO-A
3
competitive
6.15
8
mixed-type
3.97c, 9.33d
9
competitive
0.50
12
competitive
0.13
13e
mixed-type
4.38c, 4.22d
15
mixed-type
6.26c, 12.32d
26
noncompetitive
4.30
38
noncompetitive
9.57
hMAO-B
8
mixed-type
2.41c, 4.46d
12
competitive
4.14
Inhibition type
was determined by
the Lineweaver–Burk plot.
Inhibition constant was determined
by the Secondary plot.
Binding
constant of an inhibitor
with a free enzyme (Kic).
Binding constant of an inhibitor
with an enzyme–substrate complex (Kiu).
Data reproduced from
our recent
publication.[32]
Michaelis–Menten plots, Lineweaver–Burk
plots, and
secondary plots of compound 3 (A, B, C, and D), 8 (E, F, G, and H), and 9 (I, J, K, and L) for
hMAO-A inhibition.Michaelis–Menten
plots, Lineweaver–Burk plots, and
secondary plots of compound 12 (A, B, C, and D), 15 (E, F, G, and H), 26 (I, J, K, and L), and 38 (M, N, O, and P) for hMAO-A inhibition.Michaelis–Menten plots, Lineweaver–Burk plots, and
secondary plots of compound 8 (A, B, C, and D) and 12 (E, F, G, and H) for hMAO-B inhibition.Inhibition type
was determined by
the Lineweaver–Burk plot.Inhibition constant was determined
by the Secondary plot.Binding
constant of an inhibitor
with a free enzyme (Kic).Binding constant of an inhibitor
with an enzyme–substrate complex (Kiu).Data reproduced from
our recent
publication.[32]
In Silico Molecular Docking Simulation of hMAO Inhibition
Small changes in a parent structure affected the potency and selectivity
of hMAO isozyme inhibition in the in vitro assay. Therefore, to explain
the variations in potency and selectivity over the two isozymes, we
used AutoDock 4.2 to study the docking of the active compounds in
the site cavities of hMAO-A (2z5x)[33] and
hMAO-B (2v5z)[34] (Figure ). The most active compounds (3, 8, 9, 12, 13, 15, 26, and 38) were selected
to predict their binding energies and the residues that interact with
the enzyme. The reference inhibitors harmine and safinamide were used
to validate the docking results for the A- and B-isozymes, respectively.
The molecular docking simulation results are tabulated in Tables and 5 and depicted in Figures –8.
Figure 5
Molecular docking of
hMAO-A (A) and hMAO-B (B) binding with active
compounds from C. obtusifolia, along
with positive controls. The chemical structures of 3, 8, 9, 12, 15, 26, 38, harmine, safinamide, and FAD are shown
in pink, green, orange, yellow, purple, olive, cyan, blue, red, and
black, respectively.
Table 4
Molecular
Interaction of the hMAO-A
(2z5x) Active Site with Active Compounds and the Reported Inhibitor
Harmine
Binding energy, which indicates
binding affinity and capacity for the active site of hMAO-B.
All amino acid residues from the
enzyme–inhibitor complexes were determined using AutoDock 4.2
and Discovery studio.
Catalytic
inhibition mode.
Allosteric
inhibition mode.
Reported
hMAO-B inhibitor (coligand
of 2v5z).
Figure 8
Molecular docking of hMAO-B binding with compounds 8 and 12, along with positive controls. The chemical
structures of 8, 12, and FAD are shown in
green, yellow, and black, respectively. Close-up of 8 (A and D: catalytic inhibition, B and E: allosteric inhibition)
and 12 (C and F: catalytic inhibition) binding sites
showing the interaction between the ligand and hMAO-B.
Molecular docking of
hMAO-A (A) and hMAO-B (B) binding with active
compounds from C. obtusifolia, along
with positive controls. The chemical structures of 3, 8, 9, 12, 15, 26, 38, harmine, safinamide, and FAD are shown
in pink, green, orange, yellow, purple, olive, cyan, blue, red, and
black, respectively.Binding
energy, which indicates
binding affinity and capacity for the active site of hMAO-A.All amino acid residues from the
enzyme–inhibitor complexes were determined using AutoDock 4.2
and Discovery studio.Catalytic
inhibition mode.Allosteric
inhibition mode.Reported
hMAO-A inhibitor (coligand
of 2z5x).Binding energy, which indicates
binding affinity and capacity for the active site of hMAO-B.All amino acid residues from the
enzyme–inhibitor complexes were determined using AutoDock 4.2
and Discovery studio.Catalytic
inhibition mode.Allosteric
inhibition mode.Reported
hMAO-B inhibitor (coligand
of 2v5z).
Molecular Docking Simulation
of hMAO Inhibition by Anthraquinones
As tabulated in Table , ligands 3, 8, 9,
and 12 bound competitively to the catalytic site with
the best pose, as indicated by their low binding energies. The lowest
binding energy was predicted for the most potent compound 12 (−9.37 kcal/mol), followed by 8 and 9 (approx. −8.80 kcal/mol) and 3 (−8.51
kcal/mol). In addition to the catalytic site, 8 bound
to the allosteric site with a binding energy of −8.30 kcal/mol,
demonstrating that it is a mixed-type inhibitor.Ligands 3, 8, 9, and 12 have
a 9,10-dioxoanthracene scaffold in common, with different substituents
in the peripheral aromatic rings that played a key role in the ligand–enzyme
interaction, thereby affecting the potency and selectivity of their
hMAO inhibition. The 2-OH group of 3 was involved in
the formation of H-bonds that interacted with Cys323 and Thr336 at
the active catalytic site of hMAO-A (Figure A,B). Similarly, other predicted interacting
residues were Ile-335, Tyr-444, FAD, Cys323, Tyr-407, Phe208, Ile325,
phe208, Ile180, and Leu337, which possibly stabilized the enzyme–ligand
complex. As depicted in the enzyme kinetics, 8 bound
to both the catalytic (−8.82 kcal/mol) and allosteric (−8.30
kcal/mol) sites of the hMAO-A enzyme. At the catalytic site, OH groups
at the 1-, 3-, and 8-positions formed H-bonds with Phe208, Thr336,
and Asn181, respectively (represented by the green dotted lines in Figure D). Similarly, the
6-methyl group was involved in the interaction with FAD, Tyr-407,
and Tyr-444. In contrast, at the allosteric site, the 7-OH group interacted
with Ala111 and Phe112 via H-bonding (Figure B). In addition, OH groups at the 1- and
3-positions displayed H-bond interactions with His488 and Asn125.
However, no interaction was observed with the 8-OH group at the allosteric
site. Compound 9 bound to the catalytic site of the hMAO-A
enzyme with −8.80 kcal/mol binding energy by forming two H-bond
interactions. Specifically, the 1-OH group and the 3-hydroxymethyl
group showed H-bond interactions with FAD and Tyr197, respectively
(Figure E,F). In addition,
three aromatic rings interacted with Tyr-407 and Ile-335, and the
8-OH group interacted with Ile180. The most active compound (12, which had a submicromolar IC50 value) displayed
the lowest binding energy (−9.37 kcal/mol) among the test and
reference compounds, forming H-bond interactions with Tyr-444, FAD,
and Phe208. In addition, Tyr-444 and FAD were involved in π–sigma
and π–alkyl interactions (Figure G,H).
Figure 6
Close-up of the compounds 3 (A and B), 8 (C and D), 9 (E and F), 12 (G and H),
and 15 (I and J) binding sites showing the interaction
between the inhibitors and catalytic site residues of hMAO-A. The
chemical structures of 3, 8, 9, 12, 15, harmine, and FAD are shown in
pink, green, orange, yellow, purple, blue, and black, respectively.
Figure 7
Close-up of the compounds 8 (A and B), 15 (C and D), 26 (E and F), and 38 (G and
H) binding sites showing the interaction between the inhibitors and
allosteric site residues of hMAO-A. The chemical structures of 8, 15, 26, and 38 are
shown in green, purple, olive, and cyan, respectively.
Close-up of the compounds 3 (A and B), 8 (C and D), 9 (E and F), 12 (G and H),
and 15 (I and J) binding sites showing the interaction
between the inhibitors and catalytic site residues of hMAO-A. The
chemical structures of 3, 8, 9, 12, 15, harmine, and FAD are shown in
pink, green, orange, yellow, purple, blue, and black, respectively.Close-up of the compounds 8 (A and B), 15 (C and D), 26 (E and F), and 38 (G and
H) binding sites showing the interaction between the inhibitors and
allosteric site residues of hMAO-A. The chemical structures of 8, 15, 26, and 38 are
shown in green, purple, olive, and cyan, respectively.Similarly, two compounds (8 and 12) exhibited
potent inhibition of hMAO-B enzyme activity. Therefore, these two
compounds were docked in the crystal structure of the hMAO-B enzyme
(2v5z) (Figure ), and the binding energies and predicted
interactions with amino acid residues are presented in Table . Safinamide, a coligand of
2v5z, was used as a reference ligand to validate the docking results.
As shown in Table , compound 8 bound to the catalytic site with the minimum
binding energy (−8.85 kcal/mol), and its key interactions involved
H-bonds (Tyr-435, Cys172, Tyr-326), π–sigma (Leu171,
Tyr-398, FAD), π–sulfur (Cys172), π–π
stacked (Tyr-398, Tyr-435, Tyr-326), and π–alkyl (Leu171,
Cys172, and Ile199) interactions (Figure A,D). Similarly, at the allosteric site, 8 formed five H-bonds (Gln206, Pro102 × 2, Ile199, and
Leu171) and two π–sigma (Leu171 and Ile199), alkyl (Pro104
and Leu164), and π–alkyl (Trp199 and Ile199) interactions
with a binding energy of −8.42 kcal/mol (Figure B,E). Compound 12, on the other
hand, bound to the catalytic site by forming major H-bond interactions
with FAD, Cys172, and Tyr-435 (Figure C,F). Other interacting residues that possibly stabilized
the hMAO-B–12 complex were Tyr-435, FAD, Tyr-398,
Tyr-326, Leu171, Ile199, Leu171, Cys172, and Ile198. Compounds 8 and 12 shared common interacting residues (Tyr-435,
FAD, Ile199, Tyr-398, Cys172, and Leu171) with the reference ligand
(safinamide).Molecular docking of hMAO-B binding with compounds 8 and 12, along with positive controls. The chemical
structures of 8, 12, and FAD are shown in
green, yellow, and black, respectively. Close-up of 8 (A and D: catalytic inhibition, B and E: allosteric inhibition)
and 12 (C and F: catalytic inhibition) binding sites
showing the interaction between the ligand and hMAO-B.
Molecular Docking Simulation of hMAO Inhibition by Naphthopyrones
The active glycoside 15 bound to the allosteric cavity
of hMAO-A with a binding energy of −7.59 kcal/mol by forming
H-bond interactions with Asp328, Arg172, Glu327, and Glu329 and having
other interactions with His187, Leu176, Pro186, and Tyr175. Interestingly,
multiple H-bond interactions were observed between Glu327, Glu329,
and the −OH groups of the sugar moiety of this glycoside. Furthermore, 15 bound to the catalytic site with higher affinity by forming
four H-bond interactions (Gln215 and Cys323 with the glycosyl −OH
group and Ile180 and Asn181 with the 10-OH group), as shown in Figure I,J.
Molecular Docking
Simulation of hMAO Inhibition by Naphthalenes
and Naphthalenic Lactones
Compounds 26 and 38, two active glycosides, bound to the allosteric cavity
of hMAO-A with binding energies of −6.06 and −7.83 kcal/mol,
respectively. With higher binding energy, compound 38 bound to the allosteric site by forming H-bond interactions with
Asp328, Glu327, Glu329, and Lys357 and other interactions with His187
and Pro186. Interestingly, multiple H-bond interactions were observed
between Glu327, Glu329, and the −OH groups of the sugar moiety
of these two glycosides. Similarly, for compound 26,
different −OH groups from the sugar moieties at the C-9 position,
the −OH group at the C-10 position, and the ketone group at
the C-1 position displayed multiple H-bond interactions with four
residues (Thr276, Met300, Tyr410, and Glu188) with a binding energy
of −6.06 kcal/mol. In addition, an aromatic ring was involved
in a π–alkyl interaction with Ala302 and Cys398. Though
both of these inhibitors have a common parent structure and bound
to the allosteric pocket, their interacting residues were completely
different. Both inhibitors showed an equal number of H-bond interactions.
Discussion
With aging, the incidence of NDs increases, making
the neurodegenerative
disease a common diagnosis in the elderly. Therefore, developing disease-
or gene-modifying drugs to counteract the progression of NDs is a
hot research topic and one of the biggest challenges of modern pharmacology.[35] Despite much primary research on the causes
and pathogenic features of NDs, progress toward effective treatments
has been frustratingly slow.[36] The most
probable barriers hindering the development of neuronal drugs are
(a) timely relapse of symptomatic relief from treatments using the
enzyme inhibition approach already approved by the FDA, (b) potential
side effects and food and drug interactions, (c) high R&D expenditures,
and (d) a small success rate in clinical trials (Phase II and Phase
III).[36,37] Therefore, research on TCM is trending in
the hope of discovering novel, safe, and better-tolerated therapeutics
that take advantage of different treatment strategies. In this study,
we have evaluated the role of C. obtusifolia Linn seeds, a well-known treatment in TCM, in neurodegenerative
diseases by screening the seed extract against hMAO activity following
bioassay-guided isolation.C. obtusifolia seeds contain mainly
anthraquinones, naphthopyrones, and their glycosides, along with other
fatty acids and amino acids. The MeOH-seed extract and three solvent-soluble
fractions exhibited good inhibition of the hMAO enzyme, and the selectivity
of inhibition was toward the hMAO-A isozyme. Upon bioassay-guided
isolation, eleven anthraquinone aglycones (1–6, and 8–12), twelve anthraquinoneglycosides (7, 14, 17, 18, 21, 23–25, 30, 31, 34, and 36), one naphthopyrone aglycone (13) and its glycosides
(22, 28, 32, 34, and 36), and other glycosides (15, 16, 19, 20, 26, 27, 29, 37, and 38)
were isolated in pure form and evaluated for their inhibitory potential
against hMAO enzyme activity. Most of the isolated compounds displayed
prominent inhibition of the hMAO-A isozyme.After the bioassay-guided
isolation, we sought to draw some insights
about the structure–activity relationship of the isolated compounds.
Numerous 9,10-anthraquinone, naphthopyrone, and naphthalene analogues
(aglycones and their glycosides) were evaluated for their inhibitory
effect on hMAO enzymes, and the structure–activity relationship
was investigated. From the list of anthraquinone analogues available
(Table ), it was suggestive
that simple dihydroxyanthraquinone (1 and 2) did not show anti-hMAO activity. However, O-methylation
of the hydroxyl groups favorably influenced inhibitory activity (1 and 2 vs 3–6). Interestingly, trihydroxyanthraquinone (5 and 10) and tetrahydroxyanthraquinone (8) exhibited
potent activity. Compound 10 is a 1,6,8-trihydroxy-3-methylanthracene-9,10-dione
that is present in relatively high quantity in Cassia seeds. In the
enzyme inhibition assay, 10 displayed moderate inhibition
of hMAO-A (IC50: 23.27 ± 1.16 μM) and mild inhibition
of hMAO-B (IC50: 54.67 ± 0.74 μM). An additional
7-hydroxy group in 10 enhanced the potency of hMAO-A
inhibition by four times and the potency of hMAO-B inhibition by 14
times (as depicted by the activity of 8). Similarly,
the substitution of an 8-OH group in 10 by an −OCH3, as in 12, further elevated the potency toward
both isozymes. In particular, 8-OCH3 greatly enhanced hMAO-A
inhibition (10 vs 12). Interestingly, masking
the 6-OH group of 10 and replacing the 3-methyl group
with a hydroxymethyl group produced compound 9, which
was 10-fold more potent in hMAO-A inhibition. However, these changes
completely masked the hMAO-B inhibition potential. Furthermore, replacing
the −OH groups at the 1-, 6-, and 7-positions of 8 with an −OCH3 group and adding an −OH group
at the 2-position (as in 3), reduced the hMAO-A inhibition
activity by half and completely abolished the hMAO-B inhibition effect
(8 vs 3). Compound 17, the
fully O-methylated analogue of 14, had
different positions for the O-glucoside and had approximately
five times more inhibitory activity against hMAO-A than 14.To further understand the stereochemistry between the active
anthraquinones
and the hMAO enzyme, we performed structure-based molecular modeling.
The free binding energy of 8 and 9 at the
catalytic site of the hMAO-A enzyme was similar (−8.8 kcal/mol);
however, the potency of 9 was twice that of 8. A greater number of interactions was predicted for 8 when these two compounds were docked at the active site cavity of
hMAO-A. On the contrary, 9 had two unique H-bond interactions
with FAD and Tyr197 that were not observed in the binding of 8. Because MAO is a flavin-containing enzyme, FAD is vital
for enzyme activity, and tyrosine residues are critical for FAD binding,
enzyme folding, and enzyme activity.[38] Therefore,
the H-bond interaction of 9 with FAD and Tyr197 concomitantly
explains its higher potency. Similarly, 12 was the most
potent hMAO-A inhibitor tested here. Compared with the reference inhibitor
and other test inhibitors, it had the lowest binding energy (−9.37
kcal/mol). All of the inhibitors interacted with at least one of the
tyrosine residues. However, 12 had H-bond and π–sigma
interactions with the most functional tyrosine residue (Tyr-444) and
two additional H-bond interactions with FAD and Phe208. Because the
hydroxyl groups of Tyr-444 and Tyr-435 are more important for substrate
binding than those of Tyr-407 and Tyr-398,[39]12 displayed potent inhibition by preventing the substrate
from binding to the active site cavity of the enzyme. In hMAO-B inhibition, 8 and 12 exhibited potent inhibition, and the
potency of 8 was twice that of 12. Compound 8 bound to catalytic and noncatalytic sites of hMAO-B. Though
the predicted binding energy for 12 was lower than that
of 8, multiple bond interactions with Tyr-435, cys172,
and Leu171 might have enhanced the stability of the compound 8–enzyme complex. Most of the hMAO inhibitors interacted
with Ile-335 at the binding site, indicating why the Cassia compounds
selectively inhibit the hMAO-A enzyme: Ile-335 in hMAO-A and Tyr-326
in hMAO-B play a crucial role in substrate/inhibitor selectivity.[33] Other than the dual inhibitors (8 and 12), the active compounds in this study selectively
inhibited the hMAO-A isozyme. Overall, the structural insights and
molecular docking prediction concluded that (a) the most potent inhibition
of hMAO-A (by 9 and 12) was because they
had the most favorable interactions (H-bond interactions with functional
tyrosine residues and cofactor FAD) in the reactive site of the enzyme,
(b) compounds with low activity and binding affinity lacked a H-bond
interaction with FAD, and (c) the bulky group sugar in the glycosides
hampered their interaction with the enzyme, producing weak inhibition.In a previous report,[40] Fujimoto and
colleagues evaluated four anthraquinones (emodin, chrysophanol, questin,
and physcion) from the fungi Anixiella micropertusa against the mouse liver MAO enzyme and found that emodin was the
only moderate inhibitor (IC50: 37 μM). However, in
another study,[41] six anthraquinones (emodin,
rhein, chrysophanol, aloe-emodin, physcion, and 1,8-dihydroxyanthraquinone)
were inactive against ratMAO enzyme. These two studies demonstrated
the biased effect of emodin on mouseMAO inhibition. Interestingly,
here, we found potent inhibition of hMAO-Aactivity by 9 and 12 and moderate inhibition by 10.
The type of MAO enzyme, i.e., human vs mouse, must be the reason for
these discrepant findings. Though hMAO-A and mouseMAO-A have 92%
sequence identity, differential sensitivity to phentermine inhibition
suggests that structural and functional differences exist between
them.[42]Oxidative deamination by
hMAO liberates H2O2, a powerful oxidizer, and
induces oxidative stress, a root cause
of several NDs. As a neuroprotective agent, Cassia seed extract protects
neuronal cells from scopolamine- or bilateral common carotid artery
occlusion-induced cell damage via anti-inflammatory and antioxidant
responses (by attenuating iNOS and COX-2 levels and increasing the
expression of pCREB and BDNF)[22] and acetylcholinesterase
inhibition.[43] Similarly, an ethanol extract
protected hippocampal cells against mitochondrial toxin (3-nitropropionic
acid; 3-NP) and reduced N-methyl-D-aspartate-induced cell death by
attenuating dysregulated Ca2+.[20] Excitotoxicity and oxidative stress are two pathways that lead to
neurodegeneration due to glutamic acid, and hMAO-A isozyme inhibitors
(but not hMAO-B inhibitors) prevent this glutamate toxicity.[44] Our results in this study reveal selective hMAO-A
inhibition by Cassia seed constituents. Whether these inhibitors protect
neuronal cells from toxic 3-NP and glutamic acid or are responsible
for the previously reported neuroprotective activity of the seed extract
remains to be explored. In addition, how these inhibitors act on G-protein
coupled receptors for the management of NDs requires urgent study.In conclusion, C. obtusifolia Linn
seed is a well-known TCM treatment whose neuroprotective activity
has recently been described. In this study, we have explored the role
of Cassia seed and its metabolites against neurodegenerative diseases,
particularly focusing on the hMAO enzyme. The seed extract and its
metabolites selectively inhibited the hMAO-A isozyme. In particular,
aglycons 3, 8, 9, 12, and 13 and glycosides 15, 26, and 38 showed promising inhibition of hMAO-A. Compounds 8 and 12 were active against hMAO-B, too. The
enzyme kinetic study revealed the mode of enzyme inhibition, and an
in silico molecular docking simulation predicted Ile-335 as the determinant
interacting residue for hMAO-A selectivity, and Tyr-407, Phe208, and
Ile180 enhanced the binding affinity. Overall, our findings in this
study are relevant to the future therapeutic study of C. obtusifolia-derived secondary metabolites for
the management of neurodegenerative disorders, particularly anxiety
and depression.
Materials and Methods
Chemicals and Reagents
We purchased hMAO isozymes,
deprenyl HCl, and dimethyl sulfoxide from Sigma-Aldrich Co. (St Louis,
MO). A MAO-Glo assay kit was purchased from Promega (Promega Corporation,
Madison, WI). All other chemicals and solvents used were purchased
from Merck and Fluka, unless otherwise stated.
Plant Material
Raw C. obtusifolia Linn seeds were
purchased from Omni Herb Co. (Daegu, Korea) and
authenticated by Prof. J.-H. Lee (Dongguk University, Gyeongju, Korea).
A voucher specimen (no. 20130302) has been deposited in the laboratory
of Prof. J. S. Choi.
Extraction and Fractionation
The
dried C. obtusifolia Linn seeds (3.0
kg) were refluxed
in methanol (MeOH) for 3 h (6 L × 3 times). After filtration,
the total filtrate was concentrated to dryness in vacuo at 40 °C
to acquire the MeOH extract (430 g). Then, the MeOH extract was suspended
in distilled H2O: MeOH (9:1) and successively partitioned
with dichloromethane (CH2Cl2), ethyl acetate
(EtOAc), and n-butanol (n-BuOH)
to yield the CH2Cl2 (107 g), EtOAc (147 g),
and n-BuOH (76.8 g) fractions, respectively, as well
as the H2O residue (89 g).
Isolation of Compounds
from the CH2Cl2 Fraction
The CH2Cl2 fraction (107
g) was chromatographed over a silica gel column (15 × 100 cm,
63–200 μm particle size, Merck) eluted with CH2Cl2–MeOH (100:0 → 1:1, gradient), which
yielded 10 subfractions (CF1–CF10). Fraction CF1(5.4 g) was
chromatographed on a silica gel column (5 × 80 cm) and eluted
with n-hexane–acetone (500:1, gradient system)
to yield chrysophanol (1, 168 mg) and physcion (2, 250 mg). Fraction CF2 (8.75 g) was chromatographed over
a silica gel column (3 × 60 cm) and eluted with n-hexane–EtOAc (50:1) to afford obtusin (3, 230
mg) and obtusifolin (4, 190 mg). Fraction CF8 (9.5 g)
was chromatographed over a silica gel column (3 × 60 cm) and
eluted with n-hexane–EtOAc (10:1) to obtain
three subfractions (CF8.1–CF8.3). Fraction CF8.1 (1.08 g) was
chromatographed over a silica gel column (2 × 80 cm) using n-hexane–EtOAc (10:1), which produced aurantio-obtusin
(5, 410 mg). Fraction CF8.3 (800 mg) was chromatographed
over a silica gel column (1 × 60 cm) and eluted with CH2Cl2–MeOH (100:1) to afford chryso-obtusin (6, 250 mg). Fraction CF10 (12 g) was chromatographed over
a silica gel column (5 × 80 cm) using CH2Cl2–MeOH (10:1) to yield gluco-obtusifolin (7, 50
mg).
Isolation of Compounds from the EtOAc Fraction
The
EtOAcsoluble fraction (147 g) was subjected to a silica gel column
(15 × 100 cm) and eluted with CH2Cl2–MeOH
(30:1 → 1:1, gradient), which produced 20 subfractions (EFr.1–EFr.20).
Fraction EFr.2 (6.4 g) was subjected to a silica gel column (5 ×
80 cm) using a gradient solvent system of n-hexane–EtOAc
(10:1) to yield alaternin (8, 50 mg), aloe-emodin (9, 60 mg), and emodin (10, 170 mg). Similarly,
EFr.4 (2.2 g) was chromatographed on a silica gel column (5 ×
80 cm) and eluted with n-hexane–EtOAc (5:1)
to yield 2-hydroxyemodin 1-methyl ether (11, 68 mg),
questin (12, 40 mg), and rubrofusarin (13, 54 mg). Subfraction EFr.7 (2.6 g) was chromatographed on a silica
gel column (3 × 80 cm) and eluted with CH2Cl2–MeOH–H2O (15:1:0.1) to yield chryso-obtusin
2-O-glucoside (14, 50 mg), cassiaside
(15, 275 mg), and cassiatoroside (16, 30
mg). Subfraction EFr.16 (200 mg) was chromatographed on a silica gel
column (2 × 80 cm) and eluted with EtOAc–MeOH–H2O (24:3:2) to yield glucoaurantio-obtusin (17, 65 mg).
Isolation of Compounds from the n-BuOH Fraction
The n-BuOHsoluble fraction
(76.8 g) was chromatographed
on a Diaion HP-20 using an H2O-MeOH gradient solvent system
to give H2O (44.6 g), 40% MeOH (3.5 g), 60% MeOH (25.7
g), and 100% MeOH (2.5 g) fractions.The 60% MeOH fraction (25.7
g) was chromatographed on a silica gel column and eluted with CH2Cl2–MeOH–H2O = 10:1:0.1
to yield 11 subfractions (B60M1–B60M11). Fraction B60M3 (570
mg) was chromatographed over a silica gel column (3 × 80 cm)
and eluted with EtOAc–MeOH (20:1) to yield emodin 1-O-β-d-glucopyranoside (18, 18
mg). Fraction B60M3 (920 mg) was subjected to a silica gel column
(3 × 80 cm) with EtOAc–MeOH–H2O (24:3:2)
and yielded 1-hydroxyl-2-acetyl-3,8-dimethoxy-naphthalene 6-O-β-d-apiofuranosyl-(1 → 2)-β-d-glucopyranoside (19, 16 mg), isorubrofusarin
10-O-β-d-glucopyranoside (20, 9.5 mg), and physcion 8-O-β-d-glucopyranoside
(21, 24 mg). Similarly, fraction B60M6 (1.06 g) was chromatographed
over a silica gel column (4 × 80 cm) with EtOAc–MeOH–H2O (24:3:2) and yielded rubrofusarin 6-O-β-d-glucopyranoside (22, 11 mg).Fraction B60M3
(9.0 g) was chromatographed on a silica gel column
with EtOAc–MeOH–H2O (24:3:2) as the eluent
to yield alaternin 1-O-β-d-glucopyranoside
(23, 7.5 mg) and 1-desmethylaurantio-obtusin 2-O-β-d-glucopyranoside (24, 23
mg), along with a large amount of precipitate and mother liquor in
some subfractions. The subfractions with similar thin layer chromatography
patterns were combined, and the precipitate and mother liquor were
separated by filtration. The precipitate was dissolved in MeOH–H2O (2:1) and chromatographed on a silica gel column (4 ×
80 cm) eluted with EtOAc–MeOH–H2O (24:3:2)
to obtain physcion 8-O-β-gentiobioside (25, 13 mg), toralactone 9-O-β-gentiobioside
(26, 85 mg), cassialactone 9-O-β-gentiobioside
(27, 23 mg), and rubrofusarin gentiobioside (28, 76 mg). Similarly, repeated chromatography of the mother liquor
in the silica gel column with EtOAc–MeOH–H2O (24:3:2) yielded isorubrofusarin gentiobioside (29, 15 mg), chrysophanol 1-O-β-gentiobioside
(30, 85 mg), emodin-1-O-β-gentiobioside
(31, 15 mg), and rubrofusarin 6-O-β-d-apiofuranosyl (1 → 6)-O-β-d-glucopyranoside (32, 65 mg). The last subfraction
B60M11 (2.7 g) was chromatographed on a Si gel column eluted with
EtOAc–MeOH–H2O (21:5:3); repeated chromatographic
steps yielded rubrofusarin triglucoside (33, 65 mg),
chrysophanol triglucoside (34, 65 mg), cassiaside B2
(35, 14 mg), and chrysophanol tetraglucoside (36, 17 mg).Similarly, the 40% MeOH fraction (3.5 g) was chromatographed
over
a Si gel (EtOAc–MeOH–H2O, 600:99:81) and
yielded 20 subfractions. Subfraction 7 (174 mg) was subjected to a
Si gel column with EtOAc–MeOH–H2O (30:2:1)
and yielded 12 subfractions (M7-1–M7-12). M7-8 (78 mg) was
further subjected to a reversed phase (RP)-column using 40% MeOH and
yielded (R)-3,4-dihydro-10-hydroxy-3-hydroxymethyl-7-methoxy-3-methyl-1H-naphtho[2,3-c]pyran-1-one 9-O-β-d-glycopyranoside or (3R)-cassialactone
9-O-β-d-glucopyranoside (37, 15 mg). Similarly, subfraction 16 (200 mg) was subjected to repeated
Si gel chromatography eluted with EtOAc–MeOH–H2O, 21:4:3, and purified by an RP-18 (40% MeOH), which produced (3S)-9,10-dihydroxy-7-methoxy-3-methyl-1-oxo-3,4-dihydro-1H-benzo[g]isochromene-3-carboxylic acid
9-O-β-d-glucopyranoside (38, 20 mg).All of the compounds isolated from the different
solvent-soluble
fractions (the CH2Cl2, EtOAc, and n-BuOH fractions of the MeOH extract of C. obtusifolia seeds) were identified by comparing their spectral data with the
published spectral data.[26,29,31,45−49] The purity of the isolated compounds was estimated
to be >98% based on NMR spectra, and the structures of all isolated
compounds are shown in Figure .
In Vitro Human MAO Inhibitory Assay
The hMAO inhibitory
potential of the compounds was evaluated via a chemiluminescent assay
in a white, opaque, 96-well plate using the MAO-Glo kit (Promega,
Madison, WI). All of the experimental conditions and procedures we
used in this study were similar to those reported in our previous
paper.[50] The percent of inhibition (%)
was obtained using the following equation: % inhibition = (Ac – As)/Ac × 100, where Ac is the absorbance of the control, and As is the absorbance of the sample.
Kinetic Parameters in hMAO-A
Inhibition: Michaelis–Menten
Plots, Lineweaver–Burk Plots, and Secondary Plots
Michaelis–Menten plots, Lineweaver–Burk plots, and
secondary plots were used to determine the kinetic mechanisms.[51,52] The reaction mixtures contained three different concentrations of
the MAO substrate (320, 160, and 80 μM for hMAO-A and 32, 16,
and 8 μM for hMAO-B) in the presence or absence of the test
compounds. The Michaelis–Menten constant (Km) and maximum velocity (Vmax) for each enzyme inhibition were measured by Lineweaver–Burk
plots, and the inhibition constant (Ki) was calculated from secondary plots using SigmaPlot 12.0 software
(SPCC, Inc., Chicago, IL).
Molecular Docking
The docking of
the target enzyme
and active compounds was successfully simulated using the AutoDock
4.2 program.[53] X-ray crystallography of
the hMAO A-harmine complex (PDB ID:2Z5X) and hMAO-B-safinamide (PDB ID:2V5Z) was obtained from
the RCSB Protein Data Bank (PDB) website (http://www.rcsb.org/), with respective
resolutions of 2.2 and 1.6 Å.[33,34] The three-dimensional
(3D) structures of 3, 8, 9, 12, 15, and 26 were obtained from
the PubChem Compound database (NCBI), with compound CIDs of 155380,
12548, 10207, 160717, 164146, and 14189968, respectively. Similarly,
the 3D structure of 38 was constructed using Chem3D Pro
v12.0 and adjusted to pH 7.0 using MarvinSketch (ChemAxon, Budapest,
Hungary). To assess the appropriate binding orientations and conformations
of the ligand molecules with different protein inhibitors, an automated
docking simulation was performed using AutoDockTools (ADT). For the
docking calculations, Gasteiger charges were added by default, the
rotatable bonds were set by the ADT, and all torsions were allowed
to rotate. The grid maps were generated using AutoGrid. The docking
protocol for rigid and flexible ligand docking consisted of 10 independent
genetic algorithms; the other parameters used were the ADT defaults.
The results were visualized and analyzed using Discovery Studio (v17.2,
Accelrys, San Diego, CA).
Authors: Luigi De Colibus; Min Li; Claudia Binda; Ariel Lustig; Dale E Edmondson; Andrea Mattevi Journal: Proc Natl Acad Sci U S A Date: 2005-08-29 Impact factor: 11.205
Figure 1
Figure 2
Figure 4
Figure 5
Figure 8
Figure 6
Figure 7