Biological evaluation and in silico docking study of γ-linolenic acid as a potential BACE1 inhibitor

journal of functional foods 10 (2014) 187–191

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Biological evaluation and in silico docking study of γ-linolenic acid as a potential BACE1 inhibitor Kumju Youn a, Jinhyuk Lee b,c, Eun-Young Yun d, Chi-Tang Ho e, Mukund V. Karwe e, Woo-Sik Jeong f, Mira Jun a,* a

Department of Food Science and Nutrition, Dong-A University, Busan 604-714, Republic of Korea Korean Bioinformation Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea c Department of Bioinformatics, University of Sciences and Technology, Daejeon 305-350, Republic of Korea d Department of Agricultural Biology, National Academy of Agricultural Science RDA, Suwon 441-100, Republic of Korea e Department of Food Science, Rutgers University, New Brunswick, NJ 08901, USA f Department of Food & Life Science, College of Biomedical Science & Engineering, Inje University, Gimhae 621749, Republic of Korea b

A R T I C L E

I N F O

A B S T R A C T

Article history:

Sequential proteolytic cleavage of amyloid precursor protein (APP) by β-secretase (BACE1)

Received 23 April 2014

is a crucial process in β-amyloid peptide (Aβ) generation, which further forms into neuro-

Received in revised form 29 May

toxic amyloid plaques that are considered to be a pivotal hallmark in the development and

2014

progress of Alzheimer’s disease (AD). Hence, the inhibition of BACE1 has emerged as a cred-

Accepted 4 June 2014

ible target for the prevention and/or treatment of AD. In this study, γ-linolenic acid (GLA)

Available online

was discovered as a novel BACE1 specific inhibitor. GLA non-competitively suppressed BACE1 activity with an IC50 value of 7.6 × 10−5 M and Ki value of 3.5 × 10−5 M. In addition, we dem-

Keywords:

onstrated the calculated docking poses of GLA to human BACE1 and revealed the interac-

Alzheimer’s disease

tions of GLA with the allosteric site of the enzyme bound with the OH group of CYS359.

β-secretase (BACE1)

Our findings provide a novel possibility of GLA to be efficacious for the prevention of AD

β-amyloid peptide (Aβ)

and provide scaffolds to explore more potent natural BACE1 inhibitors.

γ-linolenic acid

1.

Introduction

Alzheimer’s disease (AD) is the most common form of agerelated neurodegenerative disorders leading to dementia. Critical pathological features of AD are characterized by generation of extracellular amyloid neuritic plaques and intracellular neurofibrillary tangles (NFT) composed of β-amyloid peptide (Aβ) * Corresponding author. Tel.:+82 51 200 7323; fax: +82 51 200 7535. E-mail address: [email protected] (M. Jun). http://dx.doi.org/10.1016/j.jff.2014.06.005 1756-4646/© 2014 Elsevier Ltd. All rights reserved.

© 2014 Elsevier Ltd. All rights reserved.

and hyperphosphorylated tau proteins, respectively (Tong et al., 2005). Aβ, the central component of amyloid plaque, is generated by the consecutive endoproteolytic cleavage of Aβ precursor protein (APP) by two proteases, β- and γ-secretase. In the amyloidogenic pathway, β-site APP cleaving enzyme (BACE1) leaves APP at the N-terminal end of Aβ and then this cleavage produces a secreted ectodomain of APP (sAPPβ) and the C99 fragment. γ-Secretase gives intramembranous cleavage on

188

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the C99 fragment releasing Aβ of varying length which form aggregates that lead to be the predominant species in amyloid plaque (Hardy & Allsop, 1991). Aβ is neurotoxic in several modes. It induces oxidative stress causing generation of oxidized protein and DNA, lipid peroxidation, inflammatory responses, mitochondrial dysfunction, and disruption of Ca2+ homeostasis (Butterfield, Castegna, Lauderback, & Drake, 2002). Aβ, furthermore, was proved to associate with phosphorylation, cleavage and aggregation of tau protein which is the other distinctive pathological characteristic in AD (De Felice et al., 2008). In the amyloid cascade hypothesis, Aβ production is the rate limiting step and modulation and/or clearance of brain Aβ levels by inhibiting the key enzyme BACE1 is believed to be one of the priority drug targets in AD prevention. It has been demonstrated that the increased activity and overexpression of BACE1 enhanced marked increases in sAPPβ and Aβ peptide species levels, whereas the downregulation of BACE1 diminished Aβ production (Sinha et al., 1999). The Phase I clinical trials showed that BACE1 inhibitors were able to lower cerebrospinal fluid (CSF) Aβ levels (Jeppsson et al., 2012). On the other hand, both function and structure of γ-secretase yet remain unknown and the enzyme, moreover, is involved in notch signaling pathway that controls cell fate decision. γ-Secretase inhibitors are displayed to have neurotoxic side effects in vitro and in vivo causing significant developmental defects (Shimmyo, Kihara, Akaike, Niidome, & Sugimoto, 2008). γ-Linolenic acid (GLA, 18:3) is the first derivative in the conversion of linoleic acid to arachidonic acid (AA) by the action of enzyme Δ-6-desaturase. GLA is mostly found in plant seed oils of borage, evening primrose and black currant (Chang et al., 2010). Various studies suggested that GLA is unique among the n-6 polyunsaturated fatty acid (PUFA) family members in its potential to inhibit tumor growth, metastasis and inflammatory responses, which the end product AA apparently cannot (Janssen & Kiliaan, 2014; Montserrat-de la Paz, García-Giménez, Ángel-Martín, Marín-Aguilar, & Fernández-Arche, 2013). Recent findings indicated that GLA exerted selective tumoricidal effects in various cancers such as breast, pancreas, colon, and brain (Kapoor & Huang, 2006). Moreover, GLA was proven to restore nerve blood flow, normalize nerve conduction velocity, and adjust the impaired nerve function in diabetic animal models (Dines, Cameron, & Cotter, 1995; Horrobin, 1997). It modulated interleukin-1β-induced changes in anxiety-like behavior, monoaminergeic neurotransmitters and brain inflammation in rats (Song, Manku, & Horrobin, 2008). In contrast to such various GLA studies on inflammation, cancers, diabetes and neuropathy, no study has examined any potential impact or association of Alzheimer’s disease with GLA. In our continuous research for discovering natural BACE1 inhibitors, encouraging series of previous works on GLA kept our attention. Furthermore, one of the fatty acids, oleic acid was proven to suppress in vitro human BACE1 activity in our previous study (Youn et al., 2014). Therefore, the possibility of GLA as a BACE1 inhibitor was evaluated and moreover, in silico docking studies were performed to investigate the specific binding sites of the inhibitor in recombinant BACE1.

2.

Materials and methods

2.1.

General

Fluorescence and optical density were measured with a BioTEK ELISA microplate fluorescence reader FLx 800 and BioTEK ELx 808 (Winooski, VT, USA), respectively. A BACE1 assay kit was purchased from Pan Vera (Madison, WI, USA.). α-Secretase (TACE) and substrates were obtained from R&D systems (Minneapolis, MN, USA). Trypsin, chymotrypsin, elastase and their substrates were obtained from Sigma-Aldrich (St. Louis, MO, USA). GLA (>99% purity) was also purchased from Sigma-Aldrich. All other chemicals used were of the highest grade commercially available.

2.2. Enzymatic assay of BACE1, TACE and other serine proteases For the solubility problem of high hydrophobicity of GLA, 5 nM of Tween 20 was added in each enzymatic assay that showed no effect on the enzyme activity. Fluorometric BACE1 and TACE assays were determined using the previously described method (Youn, Jeong, & Jun, 2013). Trypsin, chymotrypsin and elastase were assayed according to the protocol described in the reference (Youn et al., 2014).

2.3.

Evaluation of the inhibition kinetics on BACE1

The mode of BACE1 inhibition was determined by the graphical views of Dixon plot, Lineweaver–Burk plot, and its secondary plot. The BACE1 inhibitory activity of GLA was measured with different concentrations of the substrates. The inhibition constants (Ki) were determined by interpretation of the Dixon plot, where the value of the x-axis implies −Ki. The data were acquired as mean values of 1/V, the inverse of the increment of fluorescence intensity per min (min/mF.U) in the Lineweaver–Burk plot (Tian, Bassit, Chau, & Li, 2010). Km and Vmax values were obtained from intersection of abscissa and ordinate, respectively. Graphs were plotted by using Sigma plot software (Version 12.0).

2.4.

In silico docking studies

For docking studies, the crystal structure (PDB ID: 2WJO) (Nicholls et al., 2010) used for target BACE1 and inhibitor was prepared from the PubChem database (CID 5280933 for GLA). Marvin was used for drawing, displaying, and characterizing the chemical structures [Marvin 5.11.4, 2012, ChemAxon (http:// www.chemaxon.com)]. Pck software was used to search the binding pocket residues of BACE1 and AutoDock Vina was used for protein-ligand docking simulations (Trott & Olson, 2010). The used parameters were as follows: xyz center coordinates of the binding pocket residues; search space (15 Å) in each dimension, and generation number (10) of each binding pocket residue. All docking structures were clustered and categorized by lowest energy and largest number of clusters.

2.5.

Statistical analysis

All experiments were performed in triplicate. Data of each experiment showed the mean ± SE. Significant differences were

journal of functional foods 10 (2014) 187–191

analyzed by Duncan’s multiple range tests using Statistical Analysis System (SAS) version 9.3.

3.

(A)

Results and discussion

BACE1 inhibitors have been recognized as the primary target for preventing AD by suppressing the proteolytic process of APP at its β-site in Aβ production. As shown in Fig. 1A, GLA inhibited BACE1 activity in a concentration-dependent manner (P < 0.05) with an IC50 value of 7.6 × 10−5 M. For further exploration of the inhibition kinetics of GLA, Dixon plot and Lineweaver–Burk plot were constructed. Increasing concentration of the substrate resulted in all fitted lines which declined and converged at an identical intercept on the x-axis, indicating that GLA is a noncompetitive inhibitor toward BACE1 (Ki value 3.5 × 10−5 M, Fig. 1B,C). As shown in Fig. 1D, GLA decreased the Vmax values without affecting the affinity of BACE1 toward the substrate (Km) which demonstrated that GLA exhibited pure noncompetitive inhibition against BACE1. In our previous study, saturated fatty acids such as myristic acid (14:0) and palmitic acid (16:0) exhibited no BACE1 inhibition (IC50 > 250, Youn et al., 2014). Stearic acid (18:0) did not show any BACE1 inhibitory effect in our preliminary study (IC50 > 250, data not shown). GLA displayed stronger BACE1 inhibition than linoleic acid (18:2, IC50 2.4 × 10−4, Youn et al., 2014) and α-linolenic acid (18:3, IC50 9.8 × 10−5, data not shown). However, GLA exhibited somewhat less BACE1 inhibitory potency compared with oleic acid (18:1, IC50 6.1 × 10−5 M, Youn et al., 2014). Further and detailed studies of various fatty acids on the inhibition of BACE1 are needed to clarify the structure–activity relationship. To confirm the enzyme specificity of GLA against BACE1, the inhibitory activities against α-secretase (TACE) and other serine proteases such as trypsin, chymotrypsin and elastase were compared with that of BACE1. As indicated in Table 1, GLA did not significantly inhibit TACE and neither trypsin, chymotrypsin nor elastase even at 100 μM, indicating that GLA was a specific and selective inhibitor of BACE1. In silico molecular modeling study was performed to clarify the coordination mode of GLA toward BACE1. Asp32 and Asp228 were verified to be the active catalytic centers where the substrate interacts with BACE1. On the contrary, GLA firmly interacted with the allosteric sites in the enzyme (Fig. 2A). The detailed graphical interactions including residues and interacting bonds are displayed in Fig. 2B. The binding sites of GLA were formed by following residues: LYS9, SER10, TYR14, LEU154, CYS155, GLN304, TYR305, ARG307, GLU339, CYS359, HIS360 and VAL361. The OH of CYS359 in BACE1 formed a hydrogen bond

Fig. 1 – (A) Concentration-dependent inhibition of BACE1 activity by γ-linolenic acid (GLA). (B) Dixon plot at three fixed substrate concentrations: (●) 250 nM; (○) 500 nM; (▼) 750 nM. (C) Lineweaver–Burk plot in the absence (●) and presence of 60 μM (○), 80 μM (▼), and 100 μM (▽) of GLA. (D) Km values as a function of the concentrations of the GLA. (Inset) Dependence of the values of Vmax on the concentration of GLA.

(B)

(C)

(D)

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Table 1 – Inhibitory activity of γ-linolenic acid (GLA) against α-secretase (TACE) and other serine proteases. Conc. (μM)

TACE

Trypsin

Chymotrypsin

Elastase

50 100

2.36 ± 0.19 4.83 ± 0.84

1.62 ± 0.18 1.30 ± 0.37

3.34 ± 1.18 0.21 ± 0.10

1.25 ± 0.03 1.08 ± 0.17

with the hydrogen atom of carboxylic acid moiety in GLA. In addition, the lowest energy conformation of the most proposed complex for GLA was −6.2 kcal/mol. Previous studies in designing inhibitors of BACE1 have focused on peptide-derived structures, which act as transitionstate analogues based on the amino acid sequences at the APP cleavage sites of BACE1. Even though peptidomimetic species

(A)

(B)

Fig. 2 – In silico docking poses for γ-linolenic acid (GLA). Representative binding mode of the most stable docking poses of GLA with human BACE1. (A) The complete view of the docking poses of GLA. Human BACE1 is expressed as a gray solid ribbon diagram and GLA as a green representation. Asp32 and Asp228 are the active catalytic center residues marked in red color. (B) The close-up figure of GLA-BACE1 docking modes. Hydrogen bond interactions between GLA and human BACE1 residues are displayed as a yellow dotted line. The structural ligands were performed by AutoDock Vina. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

displayed a nanomolar BACE1 inhibitory effect, low bioavailability, high hydrophilicity, large molecular mass and restricted brain penetration caused by binding affinity for transport protein have been their major obstacles and limitation to be BACE1 inhibitors (Kang et al., 2013). Based on efforts to discover natural molecules that controlled the generation of Aβ, several compounds have been proven to exhibit their neuroprotection through suppressing the amyloidogenic pathway. Rg1 from Panax notoginseng, green tea catechins, hispidin from Phellinus linteus, ellagic acid from Punica granatum, isoflavones from Ficus benjamina, loganin from Corni fructus, etc. (Dai, Shen, Yoshida, Parrish, & Williams, 2012; Jeon, Bae, Seong, & Song, 2003; Kwak et al., 2005; Park, Jeon, Lee, Kim, & Song, 2004; Wang & Du, 2009; Youn et al., 2013). Most of the previously discovered natural BACE1 inhibitors possess small molecular weight and have similarity in their structures with aromatic rings including biphenyl, pentacyclic, stilbene/stilbene-like, benzopyran/benzofuran etc. (Jeon et al., 2007; Lv et al., 2008). However, in the current study we found that GLA, an extraordinary n-6 PUFA possessed a principal inhibitory effect against BACE1. Considering the discovery of therapeutics for AD, the compounds must traverse the blood–brain capillary wall which forms blood–brain barrier (BBB). Only a limited class of compounds with low molecular weight less than 400–500 kDa with lipophilicility was demonstrated to cross BBB. The import mechanism of fatty acid into cells has been still controversial and only a few studies have investigated the mechanism of fatty acid transport across the BBB (Mitchell, Edmundson, Miller, & Hatch, 2009; Song et al., 2010). Indeed, recent encouraging study revealed that PUFA is available to pass through the BBB using transport proteins such as FATP4, FABP5 and CD36 (Mitchell, On, Del Bigio, Miller, & Hatch, 2011). In conclusion, our results demonstrated that GLA exerted a significant and specific inhibitory effect against BACE1, and firmly interacted with the allosteric sites in the enzyme. Even though comprehensive mechanistic studies need to be clarified, this study provides the scientific support for GLA as a potential BACE1 inhibitor, which furthermore might be a useful reagent for studying both enzyme properties of BACE1 and nonsynthetic BACE1 inhibitor in AD.

Acknowledgements This research was supported by the Agenda Program funded by Rural Development Administration (2012-PJ-008969), Republic of Korea. The work was also partially supported by the Brain Busan 21 Program. Dr. Jinhyuk Lee was supported by a grant from the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program.

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