Accepted Manuscript Anti-Alzheimer's disease potential of coumarins from Angelica decursiva and Artemisia capillaris and structure-activity analysis Md. Yousof Ali, Susoma Jannat, Hyun Ah Jung, Ran Joo Choi, Anupom Roy, Jae Sue Choi PII:
S1995-7645(16)00015-8
DOI:
10.1016/j.apjtm.2016.01.014
Reference:
APJTM 180
To appear in:
Asian Pacific Journal of Tropical Medicine
Received Date: 15 November 2015 Revised Date:
20 December 2015
Please cite this article as: Ali MY, Jannat S, Jung HA, Choi RJ, Roy A, Choi JS, Anti-Alzheimer's disease potential of coumarins from Angelica decursiva and Artemisia capillaris and structure-activity analysis, Asian Pacific Journal of Tropical Medicine (2016), doi: 10.1016/j.apjtm.2016.01.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title: Anti-Alzheimer's disease potential of coumarins from Angelica decursiva and Artemisia capillaris and structure-activity analysis
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Authors Md. Yousof Ali1, Susoma Jannat1, Hyun Ah Jung2*, Ran Joo Choi3, Anupom Roy1, Jae Sue Choi1*
1
SC
Affiliations:
Department of Food and Life Science, Pukyong National University, Busan 608-737,
2
Department of Food Science and Human Nutrition, Chonbuk National University,
Jeonju 561-756, Republic of Korea 3
M AN U
Republic of Korea
Angiogenesis& Chinese Medicine Laboratory, Department of Pharmacology,
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University of Cambridge, Cambridge, UK
*
Corresponding author: Jae Sue Choi, Department of Food and Life Science, Pukyong
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National University, Busan 608-737, Republic of Korea. Tel.: +82-51-629-5845
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Fax: +82 51 629 5842
E-mail: [email protected] Hyun Ah Jung, Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju 561-756, Republic of Korea. Tel.: 82-63-270-4882 Fax: 82-63-270-3854 E-mail: [email protected] 1
ACCEPTED MANUSCRIPT Foundation project: This research was supported by the Basic Science Research Program through the National Research Foundation of Korea and funded by the Ministry of Science, ICT & Future Planning (grant No. 2014R1A1A3051684 and
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2012R1A6A1028677).
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This paper has 2 tables and 5 figures
Keywords:
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Umbelliferone 6-carboxylic acid Esculetin Daphnetin Coumarins
BACE1
EP
ABSTRACT
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Cholinesterase
Objective: To use structure–activity analysis to study the anti-Alzheimer's disease
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(anti-AD) activity of natural coumarins isolated from Angelica decursiva and Artemisia capillaries, along with one purchased coumarin (daphnetin). Methods: Umbelliferone, umbelliferone 6-carboxylic acid, scopoletin, isoscopoletin, 7-methoxy coumarin, scoparone, scopolin, and esculetin have been previously isolated; however 2'-isopropyl psoralene was isolated from Angelica decursiva for the first time to evaluate their inhibitory effects against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) enzyme activity. We 2
ACCEPTED MANUSCRIPT scrutinized the potentials of coumarins as cholinesterase and BACE1 inhibitors via enzyme kinetics and molecular docking simulation. Results: Among the test compounds, umbelliferone 6-carboxylic acid, esculetin and daphnetin exhibited potent
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inhibitory activity against AChE, BChE and BACE1. Both esculetin and daphnetin have a catechol group and exhibit significant anti-AD activity against AChE and BChE. In contrast, presence of a sugar moiety and methoxylation markedly reduced the anti-
SC
AD activity of the coumarins investigated in this study. With respect to BACE1 inhibition, umbelliferone 6-carboxylic acid, esculetin and daphnetin contained carboxyl
M AN U
or catechol groups, which significantly contributed to their anti-AD activities. To further investigate these results, we generated a 3D structure of BACE1 using Autodock 4.2 and simulated binding of umbelliferone 6-carboxylic acid, esculetin and daphnetin. Docking simulations showed that different residues of BACE1 interacted with hydroxyl
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and carboxylic groups, and the binding energies of umbelliferone 6-carboxylic acid, esculetin and daphnetin were negative (-4.58, -6.25 and -6.37 kcal/mol respectively). Conclusions: Taken together, our results suggest that umbelliferone 6-carboxylic acid,
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esculetin and daphnetin have anti-AD effects by inhibiting AChE, BChE and BACE1,
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which might be useful against AD.
1. Introduction
Alzheimer’s disease (AD) is the major form of dementia and is a deadly neurodegenerative disease that is progressive in nature and develops through a 3
ACCEPTED MANUSCRIPT multifactorial process. AD is the fourth leading cause of death in developed countries, and predominates in Europe and USA following cardiovascular disease, cancer, and stroke[1]. The two most common hypotheses used to describe the pathology of AD are
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known as the ‘cholinergic hypothesis’ and ‘amyloid hypothesis’. The cholinergic hypothesis suggests that AD is caused by a deficiency in the brain levels of the cerebral neurotransmitter acetylcholine (ACh), which is hydrolyzed by acetylcholinesterase (AChE)[2]. Similarly, butyrylcholinesterase (BChE) activity is increased by 40%–90%
SC
during the progression of AD[3], and BChE inhibition is considered a potentially
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important aspect of treating AD. In addition to the AChE and BChE hypotheses, accumulation of amyloid-β peptide (Aβ) in the brain is widely considered to be critically involved in the pathogenesis of AD[4]. Aβ plaques emerge roughly 15 years before the symptoms of AD appear[5], and once AD develops the cognitive decline caused by neuronal damage cannot be reversed, even after Aβ levels in the brain are
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lowered by immunotherapy[6]. Thus, prevention of Aβ accumulation is considered an important part of preventing AD. Aβ is excised from amyloid-β precursor protein
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through sequential cleavage by aspartic protease β-secretase 1 (BACE1)[4]. Thus, because BACE1 initiates Aβ processing, inhibition of BACE1 activity may be an
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effective way to prevent Aβ accumulation[7]. Coumarins are an important class of natural compounds and are used as additives in both foods and cosmetics[8]. Coumarin has been reported to have antibacterial[9], antioxidant[10], anti-inflammatory and anticoagulant[11], and anti-AD activities[12]. Coumarins are characterized a fused benzene and α-pyrone ring that serves as the structural nucleus. Importantly, a number of studies have demonstrated the ability of coumarins to inhibit AChE[12-14], and the benzopyrone structural nucleus of coumarins is 4
ACCEPTED MANUSCRIPT an essential aspect of hybrid molecules capable of simultaneously inhibiting AChE and AChE-induced β-amyloid aggregation[15]. Likewise, the possibility of numerous chemical substitutions afforded by the structural nucleus makes coumarins interesting
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molecules for drug discovery. As part of our continuing efforts to identify compounds from natural resources that inhibit AChE, BChE, and BACE1, we recently found that the methanolic extract of
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Angelica decursiva (Umbelliferae) (A. decursiva) fulfills these criteria[16]. This plant is used traditionally as an anti-inflammatory, diuretic, expectorant, and diaphoretic, as well
M AN U
as a remedy for colds, influenza, hepatitis, arthritis, indigestion, coughs, chronic bronchitis, pleurisy, typhoid, headaches, flatulence, fever, colic, travel sickness, rheumatism, bacterial and fungal infections, and diseases of the urinary organs[17,18]. Furthermore, extensive investigations of different species of this genus have been carried out in the last decade, and as a result many classes of compounds have been
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isolated including different types of coumarin derivatives[19]. In addition, Artemisia capillaries (A. capillaries) is commonly distributed in sandy areas along the Korean
EP
coastline. This plant has been frequently used in the treatment of liver disease, including hepatitis, jaundice, fatty liver and bilious disorder. Infusions of the buds, stems and
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whole plant of A. capillaris have been used in traditional Chinese medicine primarily as a choleric, anti-inflammatory, antipyretic, and diuretic agent for treating epidemic hepatitis[20,21]. This study deals with the anti-AD activities and the structure-activity relationship of coumarins isolated from A. decursiva and A. capillaris. Specifically, because there is currently no detailed information regarding the molecular interactions between umbelliferone 6-carboxylic acid, esculetin, daphnetin and BACE1, we performed molecular docking analysis and detailed enzyme kinetic analysis in order to 5
ACCEPTED MANUSCRIPT investigate the possibility of using compounds umbelliferone 6-carboxylic acid, esculetin and daphnetin as potent anti-AD drug candidates.
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2. Materials and methods
The 1H- and
13
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2.1. General experimental procedures
C-NMR spectra were determined using a JEOL JNM ECP-400 13
C in deuterated
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spectrometer (Tokyo, Japan) at 400 MHz for 1H and 100 MHz for
chloroform (CDCl3). Column chromatography was conducted using silica gel 60 (70– 230 mesh, Merck, Darmstadt, Germany). All TLC analyses were conducted using precoated Merck Kieselgel 60 F254 plates (20 cm×20 cm, 0.25 mm) and using 50 % H2SO4
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as the spray reagent.
Electric-eel
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2.2. Chemicals and reagents
acetylcholinesterase (BChE,
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butyrylcholinesterase
EC
(AChE,
3.1.1.8),
acetyl
EC3.1.1.7), thiocholine
horse-serum iodide
(ACh),
butyrylthiocholine chloride (BCh), 5,5′-dithiobis [2-nitrobenzoic acid] (DTNB), quercetin, daphnetin and berberine were purchased from E. Merck, Fluka, or SigmaAldrich unless otherwise stated. The BACE1 FRET assay kit (β-secretase) was purchased from Pan Vera Co. (Madison, WI, USA). All chemicals and solvents used for column chromatography were of reagent grade, purchased from commercial sources, and used as received. 6
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2.3. Isolation of coumarins
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Umbelliferone, umbelliferone 6-carboxylic acid, scopoletin, isoscopoletin, 7methoxy coumarin, scoparone, scopolin and esculetin were isolated from A . decursiva
and
A
.
ctively.
2'-Isopropyl
psoralene
was
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capillaris, according to the method described by Zhao et al[18] and Islam et al[21], respe isolated
from
subfraction-
ce
including
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2 of dichloromethane fraction from A. decursiva, and identified by spectroscopic eviden 1
H
and
13
C–
NMR, as well as by comparison with spectral published data[22].
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2.4. In vitro ChE enzyme assay
The inhibitory activities of the isolated coumarins towards ChE were measured using
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the spectrophotometric method developed by Ellman et al[23]. ACh and BCh were used as substrates to assay the inhibition of AChE and BChE, respectively. Each reaction
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mixture consisted of 140 µL sodium phosphate buffer (pH 8.0), 20 µL of test sample solution at a final concentration of 100 µM for all compounds, and 20 µL of either AChE or BChE solution, which were then combined and incubated for 15 min at room temperature. All test samples and positive control (berberine) were dissolved in 10% DMSO. Reactions were initiated upon addition of 10 µL of DTNB and 10 µL of either ACh or BCh, respectively. The enzymatic hydrolysis mediated by AChE or BChE was monitored according to the formation of the yellow 5-thio-2- nitrobenzoate anion at 412 7
ACCEPTED MANUSCRIPT nm for 15 min, which was due to the reaction of DTNB with thiocholine released from ACh or BCh, respectively. All reactions were performed in 96-well plates in triplicate
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and recorded using a microplate spectrophotometer (Molecular Devices).
2.5. In vitro BACE1 enzyme assay
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The in vitro BACE1 enzyme assay was carried out according to the manufacturers recommended protocol with minor modifications. Briefly, a mixture of 10 µL of assay
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buffer (50 mM sodium acetate, pH 4.5), 10 µL of BACE1 (1.0 U/mL), 10 µL of the substrate (750 nM Rh-EVNLDAEFK-Quencher in 50 mM, ammonium bicarbonate) and 10 µL of the test samples were dissolved in 10% DMSO and incubated for 60 min at 25 °C in the dark. The proteolysis of two fluorophores (Rh- EVNLDAEFK-Quencher)
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by BACE1 was determined by monitoring formation of the fluorescent donor (RhEVNL) at wave lengths of 530–545 nm for excitation and 570–590 nm for emission. Fluorescence was measured with a microplate spectrofluorometer (Gemini EM,
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Molecular Devices, and Sunnyvale, CA, USA). Specifically, each reaction was excited
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at 545 nm and the emission intensity was recorded at 585 nm.
2.6. Kinetic parameters of different coumarins towards AChE, BChE and BACE1 inhibition
In order to determine the mechanism of inhibition of the different coumarins, AChE and BChE inhibition was evaluated by monitoring the effects of different concentrations of substrate (0.6 to 0.1 mM for different samples). Specifically, AChE and BChE 8
ACCEPTED MANUSCRIPT inhibition assays were performed as described above, but the substrate concentration was varied. Reactions were initiated upon addition of 10 µL of DTNB and 10 µL of either Ach or Bch at the indicated concentrations. In addition, to determine the kinetic
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mechanisms of the different coumarins towards BACE1, we employed two complementary kinetic methods, namely Lineweaver–Burk and Dixon plots[24,25]. Specifically, Dixon plots for inhibition of BACE1 by umbelliferone 6-carboxylic acid,
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esculetin and daphnetinwere obtained in the presence of different concentrations of substrate: 375 nM (▼); 250 nM (○) and 150 nM (●). Likewise, the test concentrations
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of umbelliferone 6-carboxylic acid, esculetin and daphnetin for the BACE1 inhibition kinetic analysis were 0.1 µM (▼), 1 µM (○), and 10 µM (●) for umbelliferone 6carboxylic acid; 2.5 µM (▼), 12.5 µM (○), and 62.5 µM (●) for esculetin; and 2.5 µM (▼), 25 µM (○), and 100 µM (●) for daphnetin. Inhibition constants (Ki) were
taken as _Ki[25,26].
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determined by interpretation of Dixon plots, where the value of the x-axis intercept was
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2.7. BACE1 molecular docking simulations
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To estimate the structure of the enzyme-inhibitor complex and to ensure accuracy, repeatability, and reliability of docking results, we employed Autodock 4.2 software. In our study, the test concentrations of coumarins for AChE were 100 µM, 50 µM, and 10 µM for umbelliferone 6-carboxylic acid, 100 µM, 20 µM, and 4 µM for esculetin; and 100 µM, 50 µM, and 25 µM for daphnetin. Likewise, the test concentrations for BChE were 100 µM, 50 µM and 25 µM for umbelliferone 6-carboxylic acid; 100 µM, 20 µM, and 1 µM for esculetin; and 50 µM, 25 µM, and 10 µM for daphnetin. Specifically, we 9
ACCEPTED MANUSCRIPT used Autodock 4.2 to dock different coumarins into the binding site of the BACE1 crystallographic structure, which was defined as all residues 5-6 Å from the inhibitor in the original complex. Autodock 4.2 uses a semi-empirical free energy force field to
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predict binding free energies of protein–ligand complexes of known structures and binding energy for both bound and unbound states. Twelve ligand structures were constructed and minimized using Chemdraw and LigandScout software for 2D and 3D
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conformations, respectively. For docking studies, the crystal structure of the BACE1 protein target was obtained from the protein sequence alignment [Protein Data Bank
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(PDB ID 2wjo)]. The 3D structures of umbelliferone 6-carboxylic acid, esculetin and daphnetin were generated and minimized using Chemdraw and LigandScout, for 2D and 3D conformation, respectively.
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2.8. Statistical analysis
All results are expressed as the mean ± SEM of triplicate samples. Results were
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analyzed using one-way ANOVA and Student’s t-test where appropriate (Systat Inc.,
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Evaston, IL,USA). Values of P100
Isoscopoletin
153.77 ± 1.93
110.30 ± 0.39
0.71
146.43 ± 1.08 172.26 ± 0.96
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Umbelliferone
SC
Test compounds
7-Methoxy coumarin Scoparone Scopolin Berberinef
0.64
259.47 ± 0.97
273.39 ± 1.94
1.05
g
>200
173.89 ± 1.19
179.22 ± 0.91
0.717 ± 0.01
7.01 ± 0.31
-
-
1.03 -
>100 >100 >100 13.98 ± 0.51
Final concentration of test samples were 100 µM, dissolved in 10% DMSO; dSI: selectivity
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b,c,e
119.39 ± 0.99
>200
2'-Isopropyl psoralene
Quercetin
186.47 ± 0.86
index (BChE/AChE). fBerberine and gquercetin were used as positive controls for the ChE and
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BACE1 assays, respectively.
26
Table 2
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ACCEPTED MANUSCRIPT
Mode of inhibition and dissociation constants (Ki) of coumarins for AChE, BChE and BACE1 based on enzyme kinetic plot. BChE
Test compounds
Inhibition type a
Ki (µM) b
Umbelliferone 6-carboxylic acid
Noncompetitive
80.99
Esculetin
Noncompetitive
BACE1
Ki (µM)a
Inhibition type c
Ki (µM)d
Noncompetitive
49.78
Mixed
2.08
25.09
Mixed e
16.11
Mixed
17.99
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Inhibition type b
Mixed
19.98
Mixed
7.18
Mixed
16.18
e
-
-
-
-
Competitive
10.45
f
NA
NA
NA
NA
-
-
Quercetin
Berberine
Determined by Dixon plots
c,d
Determined by Dixon and Lineweaver-Burk plots
e,f
Positive controls.
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NA, not tested
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a, b
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Daphnetin
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AChE
27
ACCEPTED MANUSCRIPT Figure and legends
Figure 1. Dixon plot of the inhibition of AChE and BChE by umbelliferone 6-carboxylic acid, esculetin and daphnetin in the presence of different substrate and inhibitor concentrations.
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(A) umbelliferone 6-carboxylic acid [0.6 mM (●); 0.4 mM (○); and 0.2 mM (▼) of AChE], (B) esculetin [0.6 mM (●); 0.4 mM (○); and 0.2 mM (▼) of AChE], (C) daphnetin [0.6 mM (●); 0.3 mM (○); and 0.1 mM (▼) of AChE], (D) umbelliferone 6-carboxylic acid [0.6 mM (●); 0.3 mM (○); and
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0.1 mM (▼) of BChE], (E) esculetin [0.6 mM (●); 0.3 mM (○); and 0.1 mM (▼) of BChE], and (F)
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daphnetin [0.6 mM (●); 0.4 mM (○); and 0.2 mM (▼) of BChE].
Figure 2. Dixon plots of BACE1 inhibition by coumarins. Umbelliferone 6-carboxylic acid. (A), esculetin (B), and daphnetin (C) were tested in the presence of different concentrations of substrate: 150 nM (●), 250 nM (○), 375 nM (▼). Lineweaver-Burk plots for BACE1 inhibition by coumarins. BACE1 inhibition was analyzed in the presence of different concentrations of sample as
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indicated: 0.1 µM (▼), 1 µM (○), and 10 µM (●) for umbelliferone 6-carboxylic acid (D); 2.5 µM (▼), 12.5 µM (○), and 62.5 µM (●) for esculetin (E); and 2.5 µM (▼), 25 µM (○), and 100 µM (●) for daphnetin (F).
Figure 3. (A) Molecular docking model of BACE1 with umbelliferone 6-carboxylic acid (red color)
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and QUD (blue color). (B) Diagram of the ligand interaction of umbelliferone 6-carboxylic acid in the active site of BACE1.
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Figure 4. (A) Molecular docking model of BACE1 with esculetin (magenta color) and QUD (white color). (B) Diagram of the ligand interaction of esculetin in the active site of BACE1. Figure 5. (A) Molecular docking model of BACE1 with daphnetin (magenta color) and QUD (white color). (B) Diagram of the ligand interaction of daphnetin in the active site of BACE1.
28
ACCEPTED MANUSCRIPT
A
C
B
800
1800
1400
1600 1200
1400
600
400
200
0
0 -50
0
50
100
150
-40
-20
0
20
Concentration (µ µ M)
Concentration (µ µM)
D
E
1200
2000
1000
EP
1400
40
60
80
600 400 200 0
100
120
-50
0
50
100
Concentration (µ µ M)
F
TE D
2500
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200
1000 800
SC
600
1/V
400
1200
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800
1/V
1/V
1000
1500
1/V
1/V
AC C
1/V
1500
800
1000
1000
600 400
500
500
200
0
0 -50
0
50
Concentration (µ µ M)
100
150
-40
-20
0
20
40
60
Concentration (µ µM)
80
100
120
0 -20
0
20
Concentration (µΜ )
40
15
ACCEPTED MANUSCRIPT
8
B
A
7
4
C 6
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6
4
2
0 0
5
10
15
-80
-60
-40
-20
Concentration (µΜ) µΜ)
0
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1
0 -5
3
20
40
60
2 1 0
80
-1 00
-50
0
TE D
600
E
500
50
1 00
C o ncentra tio n (µ µΜ )
Concentration (µ µ M)
D
4
SC
2
5
1/V
1/V
1/V
3
F
400
EP
300
1/V
300
200
300
1/V
AC C
1/V
400
200
200
100
100
100
-100
0 0
0
100
1/[S] (nM)
200
300
400
500
-200
-100
0 0
100
200
-1
1/[S] (nM )
-1
300
400
500
-200
0
200 -1
1/[S] (nM)
400
ACCEPTED MANUSCRIPT
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ig 3 A
AC C
QUD
EP
TE D
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Umbelliferone 6carboxylic acid
SC
B
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
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4
ACCEPTED MANUSCRIPT
AC C
EP
TE D
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SC
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5
S1995-7645(16)00015-8
DOI:
10.1016/j.apjtm.2016.01.014
Reference:
APJTM 180
To appear in:
Asian Pacific Journal of Tropical Medicine
Received Date: 15 November 2015 Revised Date:
20 December 2015
Please cite this article as: Ali MY, Jannat S, Jung HA, Choi RJ, Roy A, Choi JS, Anti-Alzheimer's disease potential of coumarins from Angelica decursiva and Artemisia capillaris and structure-activity analysis, Asian Pacific Journal of Tropical Medicine (2016), doi: 10.1016/j.apjtm.2016.01.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title: Anti-Alzheimer's disease potential of coumarins from Angelica decursiva and Artemisia capillaris and structure-activity analysis
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Authors Md. Yousof Ali1, Susoma Jannat1, Hyun Ah Jung2*, Ran Joo Choi3, Anupom Roy1, Jae Sue Choi1*
1
SC
Affiliations:
Department of Food and Life Science, Pukyong National University, Busan 608-737,
2
Department of Food Science and Human Nutrition, Chonbuk National University,
Jeonju 561-756, Republic of Korea 3
M AN U
Republic of Korea
Angiogenesis& Chinese Medicine Laboratory, Department of Pharmacology,
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University of Cambridge, Cambridge, UK
*
Corresponding author: Jae Sue Choi, Department of Food and Life Science, Pukyong
EP
National University, Busan 608-737, Republic of Korea. Tel.: +82-51-629-5845
AC C
Fax: +82 51 629 5842
E-mail: [email protected] Hyun Ah Jung, Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju 561-756, Republic of Korea. Tel.: 82-63-270-4882 Fax: 82-63-270-3854 E-mail: [email protected] 1
ACCEPTED MANUSCRIPT Foundation project: This research was supported by the Basic Science Research Program through the National Research Foundation of Korea and funded by the Ministry of Science, ICT & Future Planning (grant No. 2014R1A1A3051684 and
RI PT
2012R1A6A1028677).
SC
This paper has 2 tables and 5 figures
Keywords:
M AN U
Umbelliferone 6-carboxylic acid Esculetin Daphnetin Coumarins
BACE1
EP
ABSTRACT
TE D
Cholinesterase
Objective: To use structure–activity analysis to study the anti-Alzheimer's disease
AC C
(anti-AD) activity of natural coumarins isolated from Angelica decursiva and Artemisia capillaries, along with one purchased coumarin (daphnetin). Methods: Umbelliferone, umbelliferone 6-carboxylic acid, scopoletin, isoscopoletin, 7-methoxy coumarin, scoparone, scopolin, and esculetin have been previously isolated; however 2'-isopropyl psoralene was isolated from Angelica decursiva for the first time to evaluate their inhibitory effects against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) enzyme activity. We 2
ACCEPTED MANUSCRIPT scrutinized the potentials of coumarins as cholinesterase and BACE1 inhibitors via enzyme kinetics and molecular docking simulation. Results: Among the test compounds, umbelliferone 6-carboxylic acid, esculetin and daphnetin exhibited potent
RI PT
inhibitory activity against AChE, BChE and BACE1. Both esculetin and daphnetin have a catechol group and exhibit significant anti-AD activity against AChE and BChE. In contrast, presence of a sugar moiety and methoxylation markedly reduced the anti-
SC
AD activity of the coumarins investigated in this study. With respect to BACE1 inhibition, umbelliferone 6-carboxylic acid, esculetin and daphnetin contained carboxyl
M AN U
or catechol groups, which significantly contributed to their anti-AD activities. To further investigate these results, we generated a 3D structure of BACE1 using Autodock 4.2 and simulated binding of umbelliferone 6-carboxylic acid, esculetin and daphnetin. Docking simulations showed that different residues of BACE1 interacted with hydroxyl
TE D
and carboxylic groups, and the binding energies of umbelliferone 6-carboxylic acid, esculetin and daphnetin were negative (-4.58, -6.25 and -6.37 kcal/mol respectively). Conclusions: Taken together, our results suggest that umbelliferone 6-carboxylic acid,
EP
esculetin and daphnetin have anti-AD effects by inhibiting AChE, BChE and BACE1,
AC C
which might be useful against AD.
1. Introduction
Alzheimer’s disease (AD) is the major form of dementia and is a deadly neurodegenerative disease that is progressive in nature and develops through a 3
ACCEPTED MANUSCRIPT multifactorial process. AD is the fourth leading cause of death in developed countries, and predominates in Europe and USA following cardiovascular disease, cancer, and stroke[1]. The two most common hypotheses used to describe the pathology of AD are
RI PT
known as the ‘cholinergic hypothesis’ and ‘amyloid hypothesis’. The cholinergic hypothesis suggests that AD is caused by a deficiency in the brain levels of the cerebral neurotransmitter acetylcholine (ACh), which is hydrolyzed by acetylcholinesterase (AChE)[2]. Similarly, butyrylcholinesterase (BChE) activity is increased by 40%–90%
SC
during the progression of AD[3], and BChE inhibition is considered a potentially
M AN U
important aspect of treating AD. In addition to the AChE and BChE hypotheses, accumulation of amyloid-β peptide (Aβ) in the brain is widely considered to be critically involved in the pathogenesis of AD[4]. Aβ plaques emerge roughly 15 years before the symptoms of AD appear[5], and once AD develops the cognitive decline caused by neuronal damage cannot be reversed, even after Aβ levels in the brain are
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lowered by immunotherapy[6]. Thus, prevention of Aβ accumulation is considered an important part of preventing AD. Aβ is excised from amyloid-β precursor protein
EP
through sequential cleavage by aspartic protease β-secretase 1 (BACE1)[4]. Thus, because BACE1 initiates Aβ processing, inhibition of BACE1 activity may be an
AC C
effective way to prevent Aβ accumulation[7]. Coumarins are an important class of natural compounds and are used as additives in both foods and cosmetics[8]. Coumarin has been reported to have antibacterial[9], antioxidant[10], anti-inflammatory and anticoagulant[11], and anti-AD activities[12]. Coumarins are characterized a fused benzene and α-pyrone ring that serves as the structural nucleus. Importantly, a number of studies have demonstrated the ability of coumarins to inhibit AChE[12-14], and the benzopyrone structural nucleus of coumarins is 4
ACCEPTED MANUSCRIPT an essential aspect of hybrid molecules capable of simultaneously inhibiting AChE and AChE-induced β-amyloid aggregation[15]. Likewise, the possibility of numerous chemical substitutions afforded by the structural nucleus makes coumarins interesting
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molecules for drug discovery. As part of our continuing efforts to identify compounds from natural resources that inhibit AChE, BChE, and BACE1, we recently found that the methanolic extract of
SC
Angelica decursiva (Umbelliferae) (A. decursiva) fulfills these criteria[16]. This plant is used traditionally as an anti-inflammatory, diuretic, expectorant, and diaphoretic, as well
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as a remedy for colds, influenza, hepatitis, arthritis, indigestion, coughs, chronic bronchitis, pleurisy, typhoid, headaches, flatulence, fever, colic, travel sickness, rheumatism, bacterial and fungal infections, and diseases of the urinary organs[17,18]. Furthermore, extensive investigations of different species of this genus have been carried out in the last decade, and as a result many classes of compounds have been
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isolated including different types of coumarin derivatives[19]. In addition, Artemisia capillaries (A. capillaries) is commonly distributed in sandy areas along the Korean
EP
coastline. This plant has been frequently used in the treatment of liver disease, including hepatitis, jaundice, fatty liver and bilious disorder. Infusions of the buds, stems and
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whole plant of A. capillaris have been used in traditional Chinese medicine primarily as a choleric, anti-inflammatory, antipyretic, and diuretic agent for treating epidemic hepatitis[20,21]. This study deals with the anti-AD activities and the structure-activity relationship of coumarins isolated from A. decursiva and A. capillaris. Specifically, because there is currently no detailed information regarding the molecular interactions between umbelliferone 6-carboxylic acid, esculetin, daphnetin and BACE1, we performed molecular docking analysis and detailed enzyme kinetic analysis in order to 5
ACCEPTED MANUSCRIPT investigate the possibility of using compounds umbelliferone 6-carboxylic acid, esculetin and daphnetin as potent anti-AD drug candidates.
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2. Materials and methods
The 1H- and
13
SC
2.1. General experimental procedures
C-NMR spectra were determined using a JEOL JNM ECP-400 13
C in deuterated
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spectrometer (Tokyo, Japan) at 400 MHz for 1H and 100 MHz for
chloroform (CDCl3). Column chromatography was conducted using silica gel 60 (70– 230 mesh, Merck, Darmstadt, Germany). All TLC analyses were conducted using precoated Merck Kieselgel 60 F254 plates (20 cm×20 cm, 0.25 mm) and using 50 % H2SO4
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as the spray reagent.
Electric-eel
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2.2. Chemicals and reagents
acetylcholinesterase (BChE,
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butyrylcholinesterase
EC
(AChE,
3.1.1.8),
acetyl
EC3.1.1.7), thiocholine
horse-serum iodide
(ACh),
butyrylthiocholine chloride (BCh), 5,5′-dithiobis [2-nitrobenzoic acid] (DTNB), quercetin, daphnetin and berberine were purchased from E. Merck, Fluka, or SigmaAldrich unless otherwise stated. The BACE1 FRET assay kit (β-secretase) was purchased from Pan Vera Co. (Madison, WI, USA). All chemicals and solvents used for column chromatography were of reagent grade, purchased from commercial sources, and used as received. 6
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2.3. Isolation of coumarins
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Umbelliferone, umbelliferone 6-carboxylic acid, scopoletin, isoscopoletin, 7methoxy coumarin, scoparone, scopolin and esculetin were isolated from A . decursiva
and
A
.
ctively.
2'-Isopropyl
psoralene
was
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capillaris, according to the method described by Zhao et al[18] and Islam et al[21], respe isolated
from
subfraction-
ce
including
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2 of dichloromethane fraction from A. decursiva, and identified by spectroscopic eviden 1
H
and
13
C–
NMR, as well as by comparison with spectral published data[22].
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2.4. In vitro ChE enzyme assay
The inhibitory activities of the isolated coumarins towards ChE were measured using
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the spectrophotometric method developed by Ellman et al[23]. ACh and BCh were used as substrates to assay the inhibition of AChE and BChE, respectively. Each reaction
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mixture consisted of 140 µL sodium phosphate buffer (pH 8.0), 20 µL of test sample solution at a final concentration of 100 µM for all compounds, and 20 µL of either AChE or BChE solution, which were then combined and incubated for 15 min at room temperature. All test samples and positive control (berberine) were dissolved in 10% DMSO. Reactions were initiated upon addition of 10 µL of DTNB and 10 µL of either ACh or BCh, respectively. The enzymatic hydrolysis mediated by AChE or BChE was monitored according to the formation of the yellow 5-thio-2- nitrobenzoate anion at 412 7
ACCEPTED MANUSCRIPT nm for 15 min, which was due to the reaction of DTNB with thiocholine released from ACh or BCh, respectively. All reactions were performed in 96-well plates in triplicate
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and recorded using a microplate spectrophotometer (Molecular Devices).
2.5. In vitro BACE1 enzyme assay
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The in vitro BACE1 enzyme assay was carried out according to the manufacturers recommended protocol with minor modifications. Briefly, a mixture of 10 µL of assay
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buffer (50 mM sodium acetate, pH 4.5), 10 µL of BACE1 (1.0 U/mL), 10 µL of the substrate (750 nM Rh-EVNLDAEFK-Quencher in 50 mM, ammonium bicarbonate) and 10 µL of the test samples were dissolved in 10% DMSO and incubated for 60 min at 25 °C in the dark. The proteolysis of two fluorophores (Rh- EVNLDAEFK-Quencher)
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by BACE1 was determined by monitoring formation of the fluorescent donor (RhEVNL) at wave lengths of 530–545 nm for excitation and 570–590 nm for emission. Fluorescence was measured with a microplate spectrofluorometer (Gemini EM,
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Molecular Devices, and Sunnyvale, CA, USA). Specifically, each reaction was excited
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at 545 nm and the emission intensity was recorded at 585 nm.
2.6. Kinetic parameters of different coumarins towards AChE, BChE and BACE1 inhibition
In order to determine the mechanism of inhibition of the different coumarins, AChE and BChE inhibition was evaluated by monitoring the effects of different concentrations of substrate (0.6 to 0.1 mM for different samples). Specifically, AChE and BChE 8
ACCEPTED MANUSCRIPT inhibition assays were performed as described above, but the substrate concentration was varied. Reactions were initiated upon addition of 10 µL of DTNB and 10 µL of either Ach or Bch at the indicated concentrations. In addition, to determine the kinetic
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mechanisms of the different coumarins towards BACE1, we employed two complementary kinetic methods, namely Lineweaver–Burk and Dixon plots[24,25]. Specifically, Dixon plots for inhibition of BACE1 by umbelliferone 6-carboxylic acid,
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esculetin and daphnetinwere obtained in the presence of different concentrations of substrate: 375 nM (▼); 250 nM (○) and 150 nM (●). Likewise, the test concentrations
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of umbelliferone 6-carboxylic acid, esculetin and daphnetin for the BACE1 inhibition kinetic analysis were 0.1 µM (▼), 1 µM (○), and 10 µM (●) for umbelliferone 6carboxylic acid; 2.5 µM (▼), 12.5 µM (○), and 62.5 µM (●) for esculetin; and 2.5 µM (▼), 25 µM (○), and 100 µM (●) for daphnetin. Inhibition constants (Ki) were
taken as _Ki[25,26].
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determined by interpretation of Dixon plots, where the value of the x-axis intercept was
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2.7. BACE1 molecular docking simulations
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To estimate the structure of the enzyme-inhibitor complex and to ensure accuracy, repeatability, and reliability of docking results, we employed Autodock 4.2 software. In our study, the test concentrations of coumarins for AChE were 100 µM, 50 µM, and 10 µM for umbelliferone 6-carboxylic acid, 100 µM, 20 µM, and 4 µM for esculetin; and 100 µM, 50 µM, and 25 µM for daphnetin. Likewise, the test concentrations for BChE were 100 µM, 50 µM and 25 µM for umbelliferone 6-carboxylic acid; 100 µM, 20 µM, and 1 µM for esculetin; and 50 µM, 25 µM, and 10 µM for daphnetin. Specifically, we 9
ACCEPTED MANUSCRIPT used Autodock 4.2 to dock different coumarins into the binding site of the BACE1 crystallographic structure, which was defined as all residues 5-6 Å from the inhibitor in the original complex. Autodock 4.2 uses a semi-empirical free energy force field to
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predict binding free energies of protein–ligand complexes of known structures and binding energy for both bound and unbound states. Twelve ligand structures were constructed and minimized using Chemdraw and LigandScout software for 2D and 3D
SC
conformations, respectively. For docking studies, the crystal structure of the BACE1 protein target was obtained from the protein sequence alignment [Protein Data Bank
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(PDB ID 2wjo)]. The 3D structures of umbelliferone 6-carboxylic acid, esculetin and daphnetin were generated and minimized using Chemdraw and LigandScout, for 2D and 3D conformation, respectively.
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2.8. Statistical analysis
All results are expressed as the mean ± SEM of triplicate samples. Results were
EP
analyzed using one-way ANOVA and Student’s t-test where appropriate (Systat Inc.,
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Evaston, IL,USA). Values of P100
Isoscopoletin
153.77 ± 1.93
110.30 ± 0.39
0.71
146.43 ± 1.08 172.26 ± 0.96
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Umbelliferone
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Test compounds
7-Methoxy coumarin Scoparone Scopolin Berberinef
0.64
259.47 ± 0.97
273.39 ± 1.94
1.05
g
>200
173.89 ± 1.19
179.22 ± 0.91
0.717 ± 0.01
7.01 ± 0.31
-
-
1.03 -
>100 >100 >100 13.98 ± 0.51
Final concentration of test samples were 100 µM, dissolved in 10% DMSO; dSI: selectivity
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b,c,e
119.39 ± 0.99
>200
2'-Isopropyl psoralene
Quercetin
186.47 ± 0.86
index (BChE/AChE). fBerberine and gquercetin were used as positive controls for the ChE and
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BACE1 assays, respectively.
26
Table 2
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ACCEPTED MANUSCRIPT
Mode of inhibition and dissociation constants (Ki) of coumarins for AChE, BChE and BACE1 based on enzyme kinetic plot. BChE
Test compounds
Inhibition type a
Ki (µM) b
Umbelliferone 6-carboxylic acid
Noncompetitive
80.99
Esculetin
Noncompetitive
BACE1
Ki (µM)a
Inhibition type c
Ki (µM)d
Noncompetitive
49.78
Mixed
2.08
25.09
Mixed e
16.11
Mixed
17.99
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Inhibition type b
Mixed
19.98
Mixed
7.18
Mixed
16.18
e
-
-
-
-
Competitive
10.45
f
NA
NA
NA
NA
-
-
Quercetin
Berberine
Determined by Dixon plots
c,d
Determined by Dixon and Lineweaver-Burk plots
e,f
Positive controls.
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NA, not tested
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a, b
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Daphnetin
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AChE
27
ACCEPTED MANUSCRIPT Figure and legends
Figure 1. Dixon plot of the inhibition of AChE and BChE by umbelliferone 6-carboxylic acid, esculetin and daphnetin in the presence of different substrate and inhibitor concentrations.
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(A) umbelliferone 6-carboxylic acid [0.6 mM (●); 0.4 mM (○); and 0.2 mM (▼) of AChE], (B) esculetin [0.6 mM (●); 0.4 mM (○); and 0.2 mM (▼) of AChE], (C) daphnetin [0.6 mM (●); 0.3 mM (○); and 0.1 mM (▼) of AChE], (D) umbelliferone 6-carboxylic acid [0.6 mM (●); 0.3 mM (○); and
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0.1 mM (▼) of BChE], (E) esculetin [0.6 mM (●); 0.3 mM (○); and 0.1 mM (▼) of BChE], and (F)
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daphnetin [0.6 mM (●); 0.4 mM (○); and 0.2 mM (▼) of BChE].
Figure 2. Dixon plots of BACE1 inhibition by coumarins. Umbelliferone 6-carboxylic acid. (A), esculetin (B), and daphnetin (C) were tested in the presence of different concentrations of substrate: 150 nM (●), 250 nM (○), 375 nM (▼). Lineweaver-Burk plots for BACE1 inhibition by coumarins. BACE1 inhibition was analyzed in the presence of different concentrations of sample as
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indicated: 0.1 µM (▼), 1 µM (○), and 10 µM (●) for umbelliferone 6-carboxylic acid (D); 2.5 µM (▼), 12.5 µM (○), and 62.5 µM (●) for esculetin (E); and 2.5 µM (▼), 25 µM (○), and 100 µM (●) for daphnetin (F).
Figure 3. (A) Molecular docking model of BACE1 with umbelliferone 6-carboxylic acid (red color)
EP
and QUD (blue color). (B) Diagram of the ligand interaction of umbelliferone 6-carboxylic acid in the active site of BACE1.
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Figure 4. (A) Molecular docking model of BACE1 with esculetin (magenta color) and QUD (white color). (B) Diagram of the ligand interaction of esculetin in the active site of BACE1. Figure 5. (A) Molecular docking model of BACE1 with daphnetin (magenta color) and QUD (white color). (B) Diagram of the ligand interaction of daphnetin in the active site of BACE1.
28
ACCEPTED MANUSCRIPT
A
C
B
800
1800
1400
1600 1200
1400
600
400
200
0
0 -50
0
50
100
150
-40
-20
0
20
Concentration (µ µ M)
Concentration (µ µM)
D
E
1200
2000
1000
EP
1400
40
60
80
600 400 200 0
100
120
-50
0
50
100
Concentration (µ µ M)
F
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2500
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200
1000 800
SC
600
1/V
400
1200
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800
1/V
1/V
1000
1500
1/V
1/V
AC C
1/V
1500
800
1000
1000
600 400
500
500
200
0
0 -50
0
50
Concentration (µ µ M)
100
150
-40
-20
0
20
40
60
Concentration (µ µM)
80
100
120
0 -20
0
20
Concentration (µΜ )
40
15
ACCEPTED MANUSCRIPT
8
B
A
7
4
C 6
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6
4
2
0 0
5
10
15
-80
-60
-40
-20
Concentration (µΜ) µΜ)
0
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1
0 -5
3
20
40
60
2 1 0
80
-1 00
-50
0
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600
E
500
50
1 00
C o ncentra tio n (µ µΜ )
Concentration (µ µ M)
D
4
SC
2
5
1/V
1/V
1/V
3
F
400
EP
300
1/V
300
200
300
1/V
AC C
1/V
400
200
200
100
100
100
-100
0 0
0
100
1/[S] (nM)
200
300
400
500
-200
-100
0 0
100
200
-1
1/[S] (nM )
-1
300
400
500
-200
0
200 -1
1/[S] (nM)
400
ACCEPTED MANUSCRIPT
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ig 3 A
AC C
QUD
EP
TE D
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Umbelliferone 6carboxylic acid
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B
ACCEPTED MANUSCRIPT
AC C
EP
TE D
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SC
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4
ACCEPTED MANUSCRIPT
AC C
EP
TE D
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SC
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5
