Food and Chemical Toxicology 89 (2016) 104e111

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Kinetics and molecular docking studies of fucosterol and fucoxanthin, BACE1 inhibitors from brown algae Undaria pinnatifida and Ecklonia stolonifera Hyun Ah Jung a, 1, Md Yousof Ali b, 1, Ran Joo Choi c, Hyong Oh Jeong d, Hae Young Chung d, Jae Sue Choi b, * a

Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju 561-756, Republic of Korea Department of Food and Life Science, Pukyong National University, Busan 608-737, Republic of Korea Angiogenesis & Chinese Medicine Laboratory, Department of Pharmacology, University of Cambridge, Cambridge, UK d College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 July 2015 Received in revised form 11 December 2015 Accepted 20 January 2016 Available online 26 January 2016

Since the action of b-site amyloid precursor protein cleaving enzyme 1 (BACE1) is strongly correlated with the onset of Alzheimer's disease (AD), the development of BACE1 inhibitors as therapeutic agents is being vigorously pursued. In our ongoing research aimed at identifying anti-AD remedies derived from maritime plants, we evaluated the BACE1 inhibitory activities of fucosterol and fucoxanthin from Ecklonia stolonifera and Undaria pinnatifida. In vitro anti-AD activities were performed via BACE1 inhibition assays, as well as enzyme kinetic and molecular docking predictions. Based on enzyme-based assays, fucosterol and fucoxanthin showed noncompetitive and mixed-type inhibition, respectively, against BACE1. In addition, docking simulation results demonstrated that the Lys224 residue of BACE1 interacted with one hydroxyl group of fucosterol, while two additional BACE1 residues (Gly11 and Ala127) interacted with two hydroxyl groups of fucoxanthin. Moreover, the binding energy of fucosterol and fucoxanthin was negative (
10.1 and
7.0 kcal/mol), indicating that hydrogen bonding may stabilize the open form of the enzyme and potentiate tight binding of the active site of BACE1, resulting in more effective BACE1 inhibition. The results suggest that fucosterol and fucoxanthin may be used beneficially in the treatment of AD and provide potential guidelines for the design of new BACE1 inhibitors. © 2016 Elsevier Ltd. All rights reserved.

Keywords: BACE1 Alzheimer's disease Docking analysis Enzyme kinetic Fucosterol Fucoxanthin

1. Introduction Alzheimer's disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and thinking skills, and eventually even the ability to carry out the simplest of tasks (Kim and Kim, 2008). AD is characterized by the existence of two pathological features, namely, amyloid plaques and neurofibrillary tangles. The observation of these features has led to the development of the amyloid cascade hypothesis (Hardy and Higgins, 1992),

Abbreviation: AD, Alzheimer's disease; APP, amyloid precursor protein; Ab, bamyloid; BACE1, b site amyloid precursor protein cleaving enzyme 1; BBB, bloodebrain barrier; COX-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase; NF-kB, nuclear factor kappa-B; ROS, reactive oxygen species. * Corresponding author. E-mail address: [email protected] (J.S. Choi). 1 Hyun Ah Jung and Md. Yousof Ali have contributed equally to this work. http://dx.doi.org/10.1016/j.fct.2016.01.014 0278-6915/© 2016 Elsevier Ltd. All rights reserved.

which is based on large amount of genetic and histopathological data that together suggest AD is a direct consequence of the presence of b-amyloid (Ab) aggregates in the brain (Schnabel, 2011; Crouch et al., 2008). Ab peptides are derived from sequential proteolytic cleavage of the amyloid precursor protein (APP) by b- and g-secretases (Zheng and Koo, 2011). b-Site amyloid precursor protein cleaving enzyme 1 (BACE1) is a membrane-bound aspartyl protease initially as a b-secretase (Vassar et al., 1999; Lin et al., 2000). Studies show that BACE1 protein levels and activity are elevated in sporadic AD brains (Yang et al., 2003), and increasing evidence reveals that BACE1 levels are upregulated under stress conditions such as cerebral ischemia (Wen et al., 2004), hypoxia (Zhang et al., 2007), and oxidative stress (Tamagno et al., 2002), all of which are associated with increased AD incidence. Since its discovery BACE1 has been heavily pursued as a small-molecule drug target (Ghosh et al., 2008). However, while many potent BACE1 inhibitors have been described, only recently have some

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been reported that are able to cross the bloodebrain barrier (BBB) in sufficient quantities to reduce brain Ab concentrations in humans (Varghese, 2010). Recently, the marine organisms serve as a rich source of healthpromoting components (Barrow and Shahidi, 2008). Among marine organisms, edible seaweeds have been identified as an underexploited plant resource and a source of functional foods with beneficial biological activities (Li and Kim, 2011). In addition, edible seaweeds have long been used in food diets as well as traditional remedies in Asian countries mainly China, Japan, and Korea. Various edible marine algae, sometimes referred to seaweed, have attracted special interest as good sources of nutrients. Indeed, one particular interesting feature of edible seaweeds is their richness in phlorotannins, sulfated polysaccharides, carotenoid pigments, phytosterols, and bioactive peptides (Chandini et al., 2008). Fucoxanthin is an orange carotenoid present in edible brown seaweeds such as Undaria pinnatifida (Wakame), Hijikia fusiformis (Hijiki), and Eisenia bicyclis (Arame) (D'Orazio et al., 2012; Kim et al., 2011). Fucoxanthin belongs to the class of non-pro-vitamin A carotenoids. Recent studies have demonstrated that fucoxanthin has remarkable biological properties, including anti-oxidant (Sachindra et al., 2007), anti-tumor (Wang et al., 2012), apoptotic-promoting (Yu et al., 2011), anti-inflammatory (Heo et al., 2012), radical scavenging (Takashima et al., 2012), and anti-diabetic activities (Jung et al., 2012). In addition, fucosterol was found to be the predominant sterol in brown seaweeds, comprising 83e97% of the nchez-Machado et al., 2004). Fucosterol total sterol content (Sa isolated from Ecklonia stolonifera is the most abundant phytosterol and has various biological activities, such as anti-cancer (Khanavi et al., 2012), cholesterol-reducing (Hoang et al., 2012), antidiabetic (Lee et al., 2004; Jung et al., 2013a), antioxidant (Lee et al., 2003), anti-adipogenic (Jung et al., 2014), anti-fungal (Attaur-Rahman et al., 1997), anti-histaminic, anti-cholinergic (Kumar et al., 2009), anti-inflammatory (Yoo et al., 2012; Jung et al., 2013b), and butyrylcholinesteraseeinhibitory activities (Yoon et al., 2008). In the present study, we investigated the anti-AD activities of fucosterol and fucoxanthin via BACE1 inhibition assays. Since there is no detailed information on the mode of inhibition or on BACE1efucosterol and BACE1efucoxanthin molecular interactions, we propose an approach to develop fucosterol and fucoxanthin as potent anti-Alzheimer drug candidates by scrutinizing their molecular docking predictions and enzyme kinetics.

105

Fig. 1. Chemical structures of fucosterol and fucoxanthin.

2.3. In vitro BACE1 enzyme assay The in vitro BACE1 assay was carried out according to the manufacture's recommended protocol with selected modifications. Briefly, mixtures of 10 mL assay buffer (50 mM sodium acetate, pH 4.5), 10 mL BACE1 (1.0 U/mL), 10 mL substrate (750 nM RhEVNLDAEFK-Quencher in 50 mM, ammonium bicarbonate) and 10 mL test samples [final concentration; 100 mM for compounds] dissolved in 10% DMSO were incubated for 60 min at 25  C in the dark. Proteolysis of the fluorescent donor (Rh-EVNL) resulted in an increase in fluorescence at 530e545 nm (excitation) and 570e590 nm (emission). Fluorescence was measured with a microplate spectrofluorometer (Gemini EM, Molecular Devices, Sunnyvale, CA, USA). Mixtures were irradiated at 545 nm and the emission intensity was recorded at 585 nm. The percent inhibition (%) was obtained by the following equation: % inhibition ¼ [1
(S60
S0)/(C60
C0)]  100, where C60 is the fluorescence of the control (enzyme, buffer, substrate) after 60 min of incubation, C0 is the initial fluorescence of the control, S60 is the fluorescence of the test samples (enzyme, sample solution, or substrate) after incubation for 60 min, and S0 is the initial fluorescence of the test samples. To account for a quenching effect of samples, the sample solution was added to reaction mixture C, and any subsequent reduction in fluorescence by the sample was expressed in terms of the IC50 value (mM required to inhibit proteolysis of the BACE1 substrate, by 50%) as calculated from the logdose inhibition curve.

2. Materials and methods 2.1. Chemicals The BACE1 FREF assay kit (b-secretase) was purchased from Pan Vera Co. (Madison, WI, USA). Quercetin was purchased from SigmaeAldrich Co. (St. Loius, MO, USA). All chemicals and solvents used in assays were of reagent grade and were purchased from commercial sources. 2.2. Plant material Fucoxanthin (30 -acetoxy-5,6-epoxy-3,50 -dihydroxy-60 ,70 -didehyro-5,6,7,8,50 ,60 -hexahydro-b,b-carotene-8-one) was isolated from the brown algae U. pinnatifida and E. bicyclis as previously described (Hosokawa et al., 1999). Fucosterol was isolated from E. stolonifera (Jung et al., 2014). Fucoxanthin and fucosterol were identified by spectroscopic methods, including 1H and 13C NMR, as well as by comparison with published spectral data and TLC analysis (Jung et al., 2012, 2013a). The structures of fucoxanthin and fucosterol are shown in Fig. 1.

2.4. Kinetic parameters of fucosterol and fucoxanthin in BACE1 inhibition LineweavereBurk and Dixon plots were used to determine the kinetic mechanism (Lineweaver and Burk, 1934; Dixon, 1953; Cornish-Bowden, 1974). In order to determine the mechanism of BACE1 inhibition, enzymatic reactions at various concentrations of fucosterol and fucoxanthin were evaluated by monitoring the effects of different concentrations of the substrates via Dixon plots (single reciprocal plot). Specifically, Dixon plots for inhibition of BACE1 by fucosterol and fucoxanthin were generated with substrate at 150 nM (C), 250 nM (B), or 375 nM (;). In order to obtain a LineweavereBurk double reciprocal plot, the test concentrations of fucosterol and fucoxanthin in the BACE1 inhibition kinetic analysis were 0 mM (D), 10 mM (;), 50 mM (B), 100 mM (C) for fucosterol and 0 mM (D), 5 mM (;), 20 mM (B), 100 mM (C) for fucoxanthin. The inhibition constants (Ki) were determined by interpretation of Dixon plots, where the value of the x-axis was taken as -Ki (Cornish-Bowden, 1974; Dixon, 1953).

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To estimate the conformation of the enzymeeinhibitor complex and to increase accuracy, repeatability, and reliability of docking results, we used the docking program Autodock 4.2. Specifically, Autodock 4.2 software was used to dock compounds into the binding sites of the crystallographic structures, defined as all residues 5e6 Å from the inhibitor in the original complex. Autodock 4.2 uses a semi-empirical free energy force field to predict binding free energies of proteineligand complexes of a known structure and binding energies for both the bound and unbound states. Twelve ligand structures were constructed and minimized using Chemsketch 3.5 and Omega 2.0 software (Open Eye Scientific Software, Santa Fe, New Mexico, USA), for 2D and 3D conformations, respectively. For docking studies, the crystal structure of the protein targets (NCBI GI 167887469) was obtained from the protein sequence alignment [Brookhaven protein Data Bank (PDB ID 2wjo)]. The 3D structure of fucosterol and fucoxanthin was constructed and minimized using Chemsketch 3.5 and Omega 2.0 software (OpenEye Scientific Software), for 2D and 3D conformations, respectively. The predicted protein-ligand complexes were optimized and ranked according to the empirical scoring function TM score (Structural alignment tool, sheba3.1), which estimates the binding free energy of the ligandereceptor complex. 2.6. Statistical analysis Statistical significance was analyzed via one-way ANOVA and Student's t-test (Sysat In., Evaston, IL, USA), and p < 0.05 was used as the cutoff for statistical significance. All results are presented as the mean ± S.E.M. of triplicate samples. 3. Results 3.1. BACE1 inhibitory activity of fucosterol and fucoxanthin In order to evaluate the anti-AD activity of the fucosterol and fucoxanthin from E. stolonifera and U. pinnatifida, the inhibitory activities of fucosterol and fucoxanthin against BACE1 were evaluated and the results are shown in Table 1 and Fig. 2. Fucoxanthin exhibited promising BACE1 inhibitory activity with an IC50 value of 5.31 ± 0.9 mM, whereas the positive control quercetin had an IC50 value of 10.19 ± 0.8 mM. On the other hand, fucosterol showed significant BACE1 inhibitory activity with an IC50 value of 64.12 ± 1.0 mM. Our results suggest that the ability of fucosterol and fucoxanthin to inhibit BACE1 might make them strong candidates for the treatment of AD by reducing Ab aggregates in the brain. 3.2. Kinetic parameters of fucosterol and fucoxanthin In an attempt to explain the mode of enzymatic inhibition of fucosterol and fucoxanthin, kinetic analyses were performed using

Table 1 BACE1 inhibitory activities and constants of fucosterol and fucoxanthin. Test compound

IC50 (mM)a

Ki (mM)b

Inhibition type

Fucosterol Fucoxanthin Quercentinc

64.12 ± 1.0 5.31 ± 0.9 10.19 ± 0.8

64.59 7.19 NA

Noncomparative Mixed NA

a Final concentration of test samples were 100 mM, dissolved in 10% DMSO. 50% Inhibition concentrations expressed as the mean ± S.E.M. of triplicates. b Inhibition constants (Ki) determined by interpretation of the Dixon plot. c Quercetin used as a positive control.

Inhibition (%)

2.5. Molecular docking simulation of BACE1 inhibition with Autodock 4.2

90

Fucosterol

80

Fucoxanthin

70

Quercetin

60 50 40 30 20 10 0 0

2

10 Concentration (μM)

50

100

Fig. 2. Concentration-dependent BACE1 inhibitory activity of fucosterol and fucoxanthin. Quercetin was used as a positive control.

different concentrations of substrate (150, 250, and 375 nM) and inhibitor. The Dixon plot is a graphical method [plot of 1/enzyme velocity (1/V) against inhibitor concentration (I)] for determination of the type of enzyme inhibition and was used to determine the dissociation or inhibition constant (Ki) for the enzymeeinhibitor complex (Cornish-Bowden, 1974). In this way, the inhibition constants (Ki) of fucosterol and fucoxanthin were determined by interpretation of Dixon plots, where the value of the x-axis was taken as eKi. The concentrations of compounds tested were as follows: fucosterol (10, 50 and 100 mM) and fucoxanthin (5, 20 and 100 mM). The reaction mixture consisted of the same BACE1 assay method described above. For noncompetitive inhibition, the x-axis represents eKi when 1/V ¼ 0. As shown in Fig. 3A and C and Table 1, fucosterol exhibited noncompetitive inhibition with a respective Ki value of 64.59 mM, while fucoxanthin (Fig. 3B and D) exhibited mixed type inhibition with a Ki value of 7.19 mM. Typically, a lower Ki value indicates tighter enzyme binding and a more effective inhibitor. Thus, our results suggest that fucosterol and fucoxanthin may be good candidates as BACE1 inhibitors. 3.3. Molecular docking study of the inhibitory activity of fucosterol and fucoxanthin against BACE1 The molecular docking models of fucosterol (yellowish green color), fucoxanthin (coral pink color), quercetin (pink color), and compound QUD (light green color) are illustrated in Fig. 4AeD, respectively. The ligandeenzyme complexes with fucosterol, fucoxanthin, quercetin, and compound QUD were stably posed in the same pocket of BACE1 by Autodock 4.2. As illustrated in Fig. 5AeD and Table 2, the corresponding ligand interactions of fucoxanthin in the active site of BACE1 consisted of two hydrogenbonding interactions between the Gly11 and Ala127 residues of the enzyme. Further, two hydroxyl groups of fucoxanthin and eight residues Thr231, Tyr198, Thr232, Tyr71, Ile110, Ile118, Leu30, and Ile126 of the enzyme participated in hydrophobic interactions with the methyl group of fucoxanthin, while the corresponding ligand interactions of fucosterol in the active site of BACE1 consisted of one hydrogen-bonding interaction between Lys224 of BACE1 and one hydroxyl group of fucosterol, while the seven residues Ile118, Tyr71, Ile226, Thr231, Val332, Phe108, and Val69 of the enzyme participated in hydrophobic interactions with the methyl group of fucosterol. The ligand interactions of quercetin in the active site of BACE1 consisted of the six hydrogen-bonding interactions between

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Fig. 3. LineweavereBurk plots for BACE1 inhibition of fucosterol (A) and fucoxanthin (B). BACE1 inhibition was analyzed in the presence of different concentrations of fucosterol and fucoxanthin as follows: 0 mM (D), 10 mM (;), 50 mM (B), 100 mM (C) for fucosterol and 0 mM (D), 5.0 mM (;), 20 mM (B), 100 mM (C) for fucoxanthin. Dixon plots for BACE1 inhibition of fucosterol (C) and fucoxanthin (D). Fucosterol and fucoxanthin were tested in the presence of different concentration of substrate: 150 nM (C), 250 nM (B), 375 nM (;).

the Val69, Trp76, Ser35, Asn37, Ser36, and Ile126 residues of the enzyme and four hydroxyl groups and one oxygenated carbon group of quercetin, while two residues Tyr71 and Phe108 of the enzyme participated in hydrophobic interactions with the methyl group of quercetin. In addition, the binding energies of the compounds were negative:
10.1 kcal/mol for fucosterol,
7.0 kcal/mol for fucoxanthin,
6.8 kcal/mol for quercetin, and
10.8 kcal/mol for compound QUD. These results indicated that additional hydrogen bonding might stabilize the open form of the enzyme and potentiate tighter binding to the active site of BACE1, resulting in more effective BACE1 inhibition.

4. Discussion Recently, a great deal of interest has been placed on isolating novel bioactive compounds from marine resources because of their numerous beneficial health effects. Among marine resources, marine algae are valuable sources of structurally diverse bioactive compounds, and have the potential to be used in place of artificial compounds as functional ingredients in the food industry. In the present study, we focused on the seaweed-derived bioactive compounds fucoxanthin and fucosterol and their potential anti-AD effects. U. pinnatifida, known more commonly as wakame, is one species of brown seaweed containing valuable pharmacological compounds such as fucoxanthin and fucosterol. In addition, U. pinnatifida has several biological activities such as prevention of

hyperglycemia (Maeda et al., 2009), suppression of chemicalinduced mammary tumors (Vishchuk et al., 2011), antihypertension (Sato et al., 2002), and anti-obesity (Woo et al., 2009). In addition, E. bicyclis and E. stolonifera are two species of brown algae that contain fucosterol and fucoxanthin (Hosokawa et al., 1999). Both of these brown algae species are ecologically and economically important, and are commonly consumed as foodstuffs together with U. pinnatifida. For these reasons, production and application of bioactive constituents of U. pinnatifida, E. bicyclis, and E. stolonifera as potential therapeutic foods has become an increasingly important research topic. As a part of our ongoing search for therapeutic agents against AD from natural marine sources, we investigated the BACE1 inhibitory activities of fucoxanthin and fucosterol isolated from U. pinnatifida and E. stolonifera. We found that fucoxanthin and fucosterol inhibited BACE1 activity in vitro. Specifically, as shown in Fig. 2A, both fucoxanthin and fucosterol inhibited BACE1 in a concentration-dependent manner, with respective IC50 values of 5.31 ± 0.9 and 64.12 ± 1.0 mМ, which compared favorably to the positive control quercetin, which had an IC50 of 10.19 ± 0.8 mМ. We were next interested in the kinetics of BACE1 inhibition by fucoxanthin and fucosterol, and thus performed additional assays to generate LineweavereBurk plots and Dixon plots (Lineweaver and Burk, 1934; Cornish-Bowden, 1974; Dixon, 1953) As shown in Table 1, varying substrate and inhibitor concentrations revealed that fucoxanthin exhibits mixed-type inhibition of BACE1 with a Ki

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Fig. 4. Molecular docking models for BACE1 inhibition of fucosterol (A, coral pink color), fucoxanthin (B, yellowish green color), quercetin (C, pink color), and QUD (D, light green color). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Ligand interaction diagram of fucosterol (A), fucoxanthin (B), quercetin (C), and QUD (D) in the active site of BACE1.

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Table 2 Binding sites of fucosterol and fucoxanthin in BACE1 using the Autodock 4.2 molecular docking program. Test compounds

Autodock 4.2 score (Kcal/mol)

No. of H-bond

H-bonding interacting residues

Hydrophobic interacting residues

Fucosterol


10.1

1

Lys224

Fucoxanthin


7.0

2

Ala127, Gly11

Quercetin


6.8

6


10.8

4

Val69, Trp76, Ser35, Asn37, Ser36, Ile126 Asp32, Asp228, Gly230

Ile226, Thr231, Val332, Phe108, Val69, Tyr71, Ile118 Ile118, Leu30, Ile126, Thr231, Tyr198, Thr232, Tyr71, Ile110 Tyr71, Phe108

QUD

value of 7.19 mM, indicating that fucoxanthin binds to both the allosteric site of the free enzyme and to the enzymeesubstrate complex. On the other hand, fucosterol was identified as a noncompetitive-type BACE1 inhibitor with a Ki value of 64.59 mM, and thus exhibits tighter binding to the free enzyme and enzymeesubstrate complex. Taken together, these results suggest that both fucoxanthin and fucosterol are effective inhibitors of BACE1. Based on the inhibitory activities of fucoxanthin and fucosterol as well as complementary analysis of enzyme kinetics using two kinetic methods, we next performed molecular docking simulations of fucoxanthin and fucosterol with BACE1 using Autodock 4.2 software (Morris et al., 2009; Bustanji et al., 2009). The docking results of fucoxanthin and fucosterol showed negative binding energies (
7.0 kcal/mol and
10.1 kcal/mol, respectively) indicative of higher affinity enzyme-inhibitors and tighter binding capacity of the inhibitors to the active site of BACE1 (Fig. 4). Autodock 4.2 can also be used to simulate the inhibitors into the binding sites of enzymes at a distance of 5e6 Å. In this way, molecular docking studies are proving to be powerful methods to predict substructures that fit into the pockets of the corresponding enzyme followed by inhibition/or activation. The molecular docking models of fucosterol (yellowish green color), fucoxanthin (coral pink color), quercetin (pink color) and compound QUD (2-amino-3-[(1R)-1cyclohexyl-2-[(cyclohexylcarbonyl) amino]ethyl]-6phenoxyquinazolin-3-ium) (light green color) are illustrated in Fig. 4AeD. Compound QUD is the most potent nonpeptic BACE1 inhibitor according to results in the protein data bank (PDB). The ligandeenzyme complexes with fucosterol, fucoxanthin, quercetin or compound QUD were stably posed in the same pocket of the BACE1 by Autodock 4.2. As illustrated in Fig. 5, the corresponding ligand interactions of fucosterol in the active site of BACE1 consisted of one hydrogen-bonding interactions between the Lys224 residues of the enzyme and one hydroxyl groups of fucosterol, while seven residues of BACE1 (Ile118, Tyr71, Ile226, Thr231, Val332, Phe108, and Val69) were found to participate in hydrophobic interactions with the methyl group of fucosterol. On the other hand, the corresponding ligand interactions of fucoxanthin in the active site of BACE1 comprised two hydrogen-bonding interactions between the Gly11 and Ala127 residues of the enzyme and the two hydroxyl groups of fucoxanthin, and eight residues Thr231, Tyr198, Thr232, Tyr71, Ile110, Ile118, Leu30, and Ile126 of BACE1 participated in hydrophobic interactions with the methyl group of fucoxanthin. Moreover, the ligand interactions of quercetin in the active site of BACE1 consisted of six hydrogen-bonding interactions between Val69, Trp76, Ser35, Asn37, Ser36 and Ile126 residues of the enzyme and four hydroxyl groups and one oxygenated carbon group of quercetin, while the two residues Tyr71 and Phe108, of the enzyme participated in hydrophobic interactions with the methyl group of quercetin. Taken together, our in vitro analyses and molecular docking data indicated that both fucoxanthin and fucosterol have a strong ability to inhibit BACE1, suggesting their potential to prevent AD by blocking b-amyloids development.

Tyr71, Phe108, Ile118

Both human genetic evidence and clinical histopathological findings indicate that inhibition of amyloidogenic processing of APP remains one of the most promising therapeutic approaches for AD. Recent failures of clinical trial have highlighted the need for more effective therapeutic molecules with which to test the amyloid hypothesis (Cummings, 2010). BACE1 is a key target for therapeutic AD drug development because it is at the top of the amyloidogenic cascade for Ab production. Indeed, elevated protein levels and enzymatic activity of BACE1 in AD brains leads to increased bcleavage of APP and Ab production, suggesting that BACE1 contributes significantly to AD pathogenesis and may be a useful therapeutic target for AD treatment. This notion is supported by findings that deletion or inhibition of BACE1 prevents amyloid pathology and cognitive deficits in APP transgenic mice (Luo et al., 2001; Fukumoto et al., 2010). In recent years, many other hypotheses have been proposed to explain the complexity and multifactorial pathogenesis of AD, including oxidative stress, disruption of homeostasis by metal ions and neuroinflammation (Blennow et al., 2006; da Rocha et al., 2011). Currently, oxidative stress is considered one of the major causative factors of AD, unifying a number of other sequential or individual pathophysiologic events. Oxidative damage in the brain of AD patients is a result of excessive production of free radicals induced by Ab, functional alteration in mitochondria, inadequacy of energy supplementation, production of inflammatory mediators, and alteration of antioxidant defenses (Liu et al., 2007; Legg, 2011). Modulation of cellular oxidative processes is closely related to the redox properties of metals ions such as copper (Cu2þ) and zinc (Zn2þ), which influence the processes of protein aggregation, a critical step in many neurodegenerative diseases. In the case of AD, APP and Ab are able to form high affinity complexes with Cu2þ promoting their aggregation. During the last two decades many potent BACE1 inhibitors have been described; however, few have successfully displayed a balanced in vitro potency. The initial examples of BACE1 inhibitors, like most inhibitors of aspartate proteases, originated from substrate and transition state analog-based design. This development resulted in large polar compounds with a high total polar surface area, exhibiting many rotatable bonds and numerous hydrogen bond donor and hydrogen bond acceptors, making them prohibitive for permeability, especially through the blood brain barrier (BBB). In this way, preliminary development of anti-AD drugs must account for penetration through plasma membranes and the BBB. For this reason, it has been proposed that BACE1 inhibitors should be of relatively low-molecular weight and exhibit high lipophilicity. Ab plaques have recently been implicated in oxidative stressinduced BBB dysfunction, leading to neurotoxicity. Indeed, Ab accumulation might an play essential role in neuroinflammatory responses, whereby activation of nuclear factor kappa-B (NF-kB) as a pivotal transcription factor accelerates release of inflammatory mediators including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and cytokines, as well as reactive oxygen species (ROS) and nitric oxide (Sastre et al., 2008). Previous

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reports have revealed that fucoxanthin and fucosterol have antioxidant, anti-inflammatory, and radical scavenging activities (Lee et al., 2003; Sachindra et al., 2007; Takashima et al., 2012; Heo et al., 2012). As such, fucoxanthin and fucosterol may possess beneficial pivotal anti-AD properties in addition to BACE1 inhibition by virtue of scavenging free radicals and ROS in neuronal cells as well as inhibition of Ab-inducing inflammatory responses and general oxidative stress in the brains of patients with AD. 5. Conclusions

b-Secretase is a major enzyme responsible for Ab production and is thus a therapeutic target for the development of inhibitor drugs. In this study, the marine natural compounds fucosterol and fucoxanthin were shown to significantly inhibit b-secretase activity and thus may be good template for anti-AD drugs. Our results also provide important mechanistic insights into binding mechanism of these compounds to BACE1. Taken together, the enhanced predicted activity, high binding score, and presence of crucial drug-like molecular properties provides substantial evidence for consideration of fucosterol and fucoxanthin as potent inhibitors for prospective treatment of AD. This information may also be of high value for design and development of novel drugs against AD. Therefore, U. pinnatifida and E. stolonifera, and the components fucosterol and fucoxanthin contained therein may have beneficial uses in the development of therapeutics and preventive agents against AD. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2012R1A6A1028677). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.fct.2016.01.014. References Atta-ur-Rahman, Nasreen, A., Akhtar, F., Shekhani, M.S., Clardy, J., Parvez, M., Choudhary, M.I., 1997. Antifungal diterpenoid alkaloids from Delphinium denudatum. J. Nat. Prod. 60, 472e474. Barrow, C., Shahidi, F., 2008. Marine nutraceuticals and Functional Foods. CRC Press, New York, NY, USA, pp. 185e187. Blennow, K., de Leon, M.J., Zetterberg, H., 2006. Alzheimer's disease. Lancet 368, 387e403. Bustanji, Y., Al-Masri, I.M., Qasem, A., Al-Bakri, A.G., Taha, M.O., 2009. In silico screening for non-nucleoside HIV-1 reverse transcriptase inhibitors using physicochemical filters and high-throughput docking followed by in vitro evaluation. Chem. Biol. Drug Des. 74, 258e265. Chandini, S.K., Ganeshan, P., Suresh, P.V., Bhaskar, N., 2008. Seaweeds as a source of nutritionally beneficial compounds-A review. J. Food Sci. Technol. 45, 1e13. Cornish-Bowden, A., 1974. A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors. Biochem. J. 137, 143e144. Crouch, P.J., Harding, S.M.E., White, A.R., Camakaris, J., Bush, A.I., Masters, C.L., 2008. Mechanisms of Ab mediated neurodegeneration in Alzheimer's disease. Int. J. Biochem. Cell Biol. 40, 181e198. Cummings, J., 2010. What can be inferred from the interruption of the semagacestat trial for treatment of Alzheimer's disease. Biol. Psychiatry 68, 876e878. D'Orazio, N., Gemello, E., Gammone, M.A., de Girolamo, M., Ficoneri, C., Riccioni, G., 2012. Fucoxantin: a treasure from the sea. Mar. Drugs 10, 604e616. da Rocha, M.D., Viegas, F.P., Campos, H.C., Nicastro, P.C., Fossaluzza, P.C., Fraga, C.A.M., Barreiro, E.J., Viegas, C., 2011. The role of natural products in the discovery of new drug candidates for the treatment neurodegenerative

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