D4 receptor agonist: Relevance to Parkinson's disease

Chemico-Biological Interactions 310 (2019) 108757

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Characterizing fucoxanthin as a selective dopamine D3/D4 receptor agonist: Relevance to Parkinson's disease

T

Pradeep Paudela, Su Hui Seonga, Hyun Ah Jungb,**, Jae Sue Choia,* a b

Department of Food and Life Science, Pukyong National University, Busan, 48513, Republic of Korea Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju, 54896, Republic of Korea

ARTICLE INFO

ABSTRACT

Keywords: Fucoxanthin Fucosterol GPCR-targeting Dopamine agonist Parkinson's disease

Fucoxanthin and fucosterol are archetypal lipid components of edible brown algae that provide several health benefits. Lately, their protective role in Aβ1-42-induced cognitive dysfunction in animal models has been reported (Alghazwi et al., 2019; Oh et al., 2018). However, their role in the aminergic system and as a prime treatment approach for multifactorial neurodegenerative diseases still requires exploration. The main aims of the present study are to characterize the role of fucoxanthin and fucosterol in the aminergic pathway via in vitro human monoamine oxidase (hMAO) inhibition and cell-based functional G-protein coupled receptor (GPCR) assays and to underline their possible mechanisms of action via in silico molecular docking studies. Fucoxanthin displayed weak inhibition with IC50 values of 197.41 ± 2.20 and 211.12 ± 1.17 μM over two isoenzymes hMAO-A and hMAO-B, respectively. Fucosterol remained inactive up to 500 μM. In functional assay results, fucoxanthin showed a concentration-dependent agonist effect on dopamine D3 and D4 receptors. The half maximal effective concentration (EC50) of fucoxanthin for dopamine D3 and D4 receptors was 16.87 ± 3.41 and 81.87 ± 6.11 μM, respectively. For dopamine as a reference agonist, the EC50 values for these two receptors were 3.7 and 24 nM, respectively. Fucosterol showed no agonist activity on any of the tested receptors. Similarly, fucoxanthin showed a mild antagonist effect on dopamine D1 and tachykinin (NK1) receptor with inhibition of control agonist response by approximately 40% at 100 μM. Fucosterol displayed mild antagonist effects only on dopamine D1 and D4 receptors. In silico studies revealed potential mechanisms by which fucoxanthin binds to dopamine receptors to exert its agonist effects, including low binding energy and H-bond interactions with Ser196 and Thr115 at the D3 receptor and with Ser196 and Asp115 at the D4 receptor. Our results collectively suggest that fucoxanthin is a potential D3/D4 agonist for the management of neurodegenerative diseases, such as Parkinson's disease.

1. Introduction Neurodegeneration is defined as a continuous loss of structural and functional properties of neurons that worsens with aging. Neurodegenerative disorders (NDs) signify a huge assembly of neurological disorders with varied pathological and clinical expressions, including cognitive decline due to neuronal loss (Alzheimer's disease; AD), movement disorder characterized by selective loss of dopaminergic neurons in the substantia nigra pars compacta (Parkinson's disease; PD), and loss of striatal projection neurons, abnormal energy

metabolism, and excitotoxicity (Huntington disease; HD) [1]. Based on the monoaminergic hypothesis of depression (imbalanced serotonergic and noradrenergic neurotransmission is at the core of the pathophysiology of depression), monoamine reuptake inhibitors have been developed as antidepressants and are now widely used in clinical practice [2]. Dopamine (DA), one of the major neurotransmitters, participates in a number of functions including motor coordination, emotions, memory, reward, and neuroendocrine regulation. Abnormalities in the dopaminergic system and its receptors in the basal ganglia structures are the basis of PD. However, DA also participates in other

Abbreviations: AD, Alzheimer's disease; ADT, AutoDockTools; CHO, Chinese hamster ovary; CIDs, compound identifiers; DA, dopamine; DMEM, Dulbecco's modified Eagle medium; GAs, genetic algorithms; GPCRs, G-protein coupled receptors; HBSS, Hank's balanced salt solution; HD, Huntington disease; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IBMX, 3-isobutyl-1-methylxanthine; MAO, monoamine oxidase; NCBI, National Center for Biotechnology Information; PD, Parkinson's disease; TM, transmembrane * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (H.A. Jung), [email protected] (J.S. Choi). https://doi.org/10.1016/j.cbi.2019.108757 Received 17 April 2019; Received in revised form 5 July 2019; Accepted 16 July 2019 Available online 16 July 2019 0009-2797/ © 2019 Elsevier B.V. All rights reserved.

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neurodegenerative disorders including HD, schizophrenia, and multiple sclerosis (MS) [3,4]. Hence, DA receptors are the main targets for psychostimulants and antipsychotics. Fucoxanthin (an orange carotenoid) and fucosterol are typical lipid components of edible brown algae that provide several health benefits. Fucoxanthin belongs to the class of non-pro-vitamin A carotenoids and has been reported to have remarkable biological properties, including antioxidant [5], antitumor [6], apoptotic-promoting [7], anti-inflammatory [8], radical scavenging [9], antidiabetic [10], and antiobesity [11] properties. We recently reported the anti-Parkinson's disease activity of fucoxanthin via monoamine oxidase inhibition [12]. More recently, fucoxanthin demonstrated neuroprotection against Aβ142-mediated toxicity in pheochromocytoma (PC-12) neuronal cells [13], protection of central nervous system neurons through anti-excitatory and anti-oxidative actions [14], protection of neurons from cerebral I/R injury via Nrf2/HO-1 signal activation [15], and reversal of LPS-induced anxiety-like behaviors in mice via AMPK-NF-κB signaling in the hippocampus, frontal cortex, and hypothalamus [16]. This study was designed to further investigate these reported activities and the higher content of fucoxanthin in brown algae. To the best of our knowledge, there are no reports of fucoxanthin action on the GPCRs that mediate multifactorial neurodegenerative diseases. Fucosterol is a phytosterol with diverse biological activities, including antioxidant, antidiabetic, anti-inflammatory, anticancer, hepatoprotective, and anti-Alzheimer's disease activity (as reviewed in our previous paper [17]). In our recent studies, fucosterol showed an antiobesity effect by inhibiting adipogenesis of 3T3-L1 preadipocytes through modulation of the FoxO signaling pathway [18]. Fucosterol also prevented soluble Aβ1-42-induced cognitive dysfunction in aging rats by downregulating glucose-regulated protein 78 (GRP78) expression and upregulating mature brain-derived neurotrophic factor (BDNF) expression in the dentate gyrus [19]. Similarly, fucosterol was reported as a potential candidate for the treatment of cervical cancer because it induced mitochondrial-mediated apoptosis, inhibited cell cycle migration, and downregulated the mTOR/PI3K/Akt signaling pathway, which is important in cancer tumorigenesis [20]. Furthermore, fucosterol exhibited antiproliferative effects on human lung cancer cells by inducing apoptosis, inducing cell cycle arrest, targeting the Raf/MEK/ ERK signaling pathway [21], protecting against amyloid β-induced neurotoxicity in SH-SY5Y cells [22], and alleviating acute liver injury induced by concanavalin A by inhibiting P38 MAPK/PPARγ/NF-κB signaling [23]. In a recent study, fucosterol inhibited cholinesterase enzyme activity and reduced the release of pro-inflammatory mediators in lipopolysaccharide and amyloid-induced microglial cells, highlighting itself as a potential candidate for multifactorial neurodegenerative disorders [24]. However, there are no reports of its effect on monoamine oxidase (MAO) enzyme activity, a potential drug target for neurologic disorders. There are not yet any reports on the effect of fucosterol in dopaminergic and non-dopaminergic pathways. The main purposes of this study were to delineate the effect of fucosterol in monoamine oxidase enzyme activity, accentuate its mechanism of inhibition via in silico molecular docking simulation, underline the modulatory effect of fucoxanthin and fucosterol in GPCRs via cell-based functional assays, and characterize their role in Parkinson's diseases.

Dulbecco's modified Eagle medium (DMEM) buffer, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer were obtained from Invitrogen (Carlsbad, CA, USA). Reference agonists and/or antagonists atropine sulphate salt, acetylcholine chloride, [Sar9, Met(O2) 11]-Substance P, [Arg8]-vasopressin (AVP), (+) butacamol, clozapine, dopamine, [d(CH2)5 1, Tyr(Me)2]-AVP, serotonin, SCH 2330, L733,060, (S)-WAY-100635, and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and reagents used were purchased from E. Merck, Fluka (Rupert-Mayer-Str., Munich, Germany), and Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated, and were of the highest grade available. 2.2. Isolation of compounds Fucoxanthin and fucosterol were isolated from Undaria pinnatifida and Eisenia bicyclis, respectively. The details of isolation and identification are reported in our previous works [10,25]. 2.3. Assay for hMAO inhibition To evaluate the human monoamine oxidase inhibition potential of fucosterol, a chemiluminescent assay was performed in a white opaque 96-well plate using the MAO-Glo kit (Promega, Madison, WI, USA). All experimental conditions and procedures followed herein were similar to those reported in our previous paper [12]. The percent of inhibition (%) was obtained by the following equation: % inhibition = (Ac - As)/ Ac × 100, where Ac is control fluorescence and As is sample absorbance. Deprenyl HCl was used as a reference control. 2.4. Functional GPCR assay A functional GPCR cell-based assay presents readouts of multiple second messengers including cAMP for Gi- and Gs-coupled receptors and IP1 and IP3/calcium flux for Gq-coupled receptors. Functional assays were conducted at Eurofins Cerep (Le Bois I'Eveque, France) using transfected cells expressing human cloned receptors following their inhouse functional assay protocol; experimental conditions are reported in our recent publication [26]. Stable cell lines expressing recombinant GPCRs were used in this study. 2.5. Measurement of cAMP level In brief, a plasmid containing the GPCR gene of interest (dopamine D1, D3, or D4) was transfected into Chinese hamster ovary (CHO) cells. The resulting stable transfectants (CHO-GPCR cells line) were suspended in HBSS buffer (Invitrogen, Carlsbad, CA, USA) supplemented with 20 mM HEPES buffer and 500 μM IBMX. These solutions were distributed into microplates at a density of 5 × 103 cells/well and incubated for 30 min at room temperature in the absence (control) or presence of fucoxanthin/fucosterol (12.5, 25, 50, and 100 μM) or reference agonist. Following incubation, cells were lysed and a fluorescence acceptor (D2-labeled cAMP) and fluorescence donor (anti-cAMP antibody with europium cryptate) were added. After 60 min at room temperature, fluorescence transfer was measured at λex = 337 nm and λem = 620 or 665 nm using a microplate reader (Envision, PerkinElmer, Waltham, MA, USA). Cyclic AMP concentration was determined by dividing the signal measured at 665 nm by that measured at 620 nm (ratio). Results are expressed as a percentage of the control response to dopamine for the agonist effect and as percent inhibition of the control response to dopamine. The standard reference control was dopamine, which was tested in each experiment at several concentrations to generate a concentration-response curve from which its EC50 value was calculated.

2. Materials and methods 2.1. Chemicals and reagents Human MAO isozymes were purchased from Sigma-Aldrich (St. Louis, MO, USA), and the MAO-Glo kit was purchased from Promega (Promega, Madison, WI, USA). Transfected Chinese hamster ovary (CHO) cells, rat basophil leukemia (RBL) cells, U373 cells, and BA/F3 cells were obtained from Eurofins Scientific (Le Bois I'Eveque, France). Various buffers, namely Hank's balanced salt solution (HBSS) buffer, 2

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2.6. Measurement of intracellular [Ca2+] level Depending on the receptor type, the method used to quantify intracellular [Ca2+] level varied slightly. In general, cells expressing different receptors were transfected with an expression vector encoding a receptor polypeptide and were allowed to grow for a time period sufficient for that receptor to be expressed. A fluorescent probe (Fluo8 Direct, Invitrogen, Carlsbad, CA, USA) mixed with probenecid in HBSS buffer (Invitrogen, Carlsbad, CA, USA) supplemented with 20 M HEPES (Invitrogen) (pH 7.4) was then added to each well and allowed to equilibrate with the cells for 60 min at 37 °C. Thereafter, assay plates were positioned in a microplate reader (CellLux, PerkinElmer, Waltham, MA, USA) and fucoxanthin/fucosterol (12.5, 25, 50, and 100 μM), reference agonist, or HBSS buffer (basal control) were added. Measurements were obtained for the change in fluorescence intensity, which varies proportionally to free cytosolic Ca2+ ion concentration. Standard reference controls (agonists and antagonists) were included in each experiment at several concentrations to generate a concentrationresponse curve from which EC50 values were calculated. Cellular agonist effect was calculated as the percentage of the control response to a known reference agonist for each target, and cellular antagonist effect was calculated as the percentage inhibition of the control reference agonist response for each target. Results are expressed as a percentage of the control agonist response or inverse agonist response (measured response/control response × 100) and as percent inhibition of control agonist response [100 − (measured response/control response × 100)] obtained in the presence of fucoxanthin or fucosterol.

Fig. 1. Chemical structures of fucoxanthin and fucosterol. Table 1 Inhibitory activity of fucoxanthin and fucosterol on human monoamine oxidases (hMAO-A & hMAO-B). Compounds

Fucoxanthinc Fucosterol Deprenyl HCld

2.7. Molecular docking study

50% inhibitory concentration (mean ± SD, μM)a hMAO-A

hMAO-B

197.41 ± 2.20 > 500 10.23 ± 0.82

211.12 ± 1.17 > 500 0.108 ± 0.034

SIb

0.93 ND 94.7

a

IC50 values were calculated from log-dose inhibition curves and expressed as means ± SDs of triplicate experiments. b Selectivity Index (hMAO-A/hMAO-B). c Values were extracted from our previous article [12]. d Reference control.

Docking of the target receptor with fucoxanthin and fucosterol was successfully simulated using AutoDock 4.2 [27]. X-ray crystallography of human dopamine D3 receptor (hD3R)- eticlopride complex (PDB ID: 3PBL) and human dopamine D4 receptor (hD4R)-nemonapride complex (PDB ID: 5WIU) were obtained from the RCSB Protein Data Bank (PDB); the resolution of these complexes is 2.89 and 1.96 Å, respectively [28,29]. 3D structures of fucoxanthin, fucosterol, dopamine, and clozapine were obtained from PubChem Compound (NCBI) with compound CIDs of 5281239, 5281328, 681, and 135398737, respectively. The energy was minimized using the Molecular Mechanics 2 force fields method. Automated docking simulations were performed using AutoDockTools (ADT) to assess appropriate binding orientations. For docking calculations, Gasteiger charges were added by default, rotatable bonds were set by ADT, and all torsions were allowed to rotate. Grid maps were generated by AutoGrid. The docking protocol for rigid and flexible ligand docking consisted of 10 independent genetic algorithms; the other parameters used were ADT defaults. The results were visualized and analyzed using Discovery Studio (v17.2, Accelrys, San Diego, CA, USA) and the University of California, San Francisco (UCSF)'s Chimera tool (http://www.cgl.ucsf.edu/chimera/).

oxidase isoforms (hMAO-A & hMAO-B) was evaluated fluorometrically, and deprenyl HCl was used as a reference drug. For fucoxanthin, the data were extracted from our recent publication [12] to compare with fucosterol. As shown in Table 1, fucosterol did not show any observable effect on either enzyme isoform (IC50 > 500 μM). However, fucoxanthin had IC50 values of 197.41 ± 2.20 μM and 211.12 ± 1.17 μM on hMAO-A and hMAO-B inhibition, respectively, and the selectivity index was 0.93. Deprenyl HCl was used as a reference compound to validate the study and showed potent inhibition of hMAO-A (IC50; 10.23 ± 0.82 μM) and hMAO-B (IC50; 0.108 ± 0.034 μM), with a selectivity index of 94.7. 3.2. Functional G-protein-coupled receptor (GPCR) assay The modulatory effect of fucoxanthin and fucosterol on various GPCRs involving neurodegenerative diseases was evaluated via cellbased functional assays. Tables 2 and 3 tabulate the results of agonist and antagonist effects of fucoxanthin, fucosterol, and reference compounds on various receptors, namely dopamine (D1, D3, and D4), muscarinic (M5), tachykinin (NK1), vasopressin (V1A), and serotonin (5HT1A) receptors. As shown in Table 2, fucoxanthin showed 71.05% of control agonist response on dopamine D3 receptor at 100 μM. Similarly, the control agonist response at 100 μM concentration was 59.90% on dopamine D4 receptor. However, it did not show any agonist effect on other receptors, except for a mild effect on the V1A receptor (35.46% of control agonist response at 100 μM). Interestingly, fucosterol did not show any agonist activity on all tested receptors. The concentrationdependent agonist effect of fucoxanthin, fucosterol, and dopamine on dopamine D3 and D4 receptors is presented in Fig. 2. The EC50 value calculated from a concentration-response curve demonstrated that fucoxanthin exhibited 50% of dopamine effect on dopamine D3 and D4

2.8. ADME prediction In the drug development process, pharmacokinetics serves as a useful tool in defining a drug's disposition characteristics and therapeutics. The pharmacokinetic parameters of fucoxanthin and fucosterol, such as absorption, distribution, metabolism, and excretion (ADME), were determined using the web-based software PreADMET (v2.0, YONSEI University, Seoul, Korea) [30]. 3. Results 3.1. hMAO inhibition activity The inhibitory potential of fucosterol (Fig. 1) on human monoamine 3

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Table 2 Agonist activity of fucoxanthin, fucosterol, and reference compounds on respective receptors. Receptors

Compounds

% of Control Agonist Response at 100 μM

EC50a (μM)

Reference agonists of respective target receptors EC50 (nM)b

D1 (h) Dopamine

Fucoxanthin Fucosterol Fucoxanthin 3.7 Fucoxanthin 24 Fucoxanthin Fucosterol Fucoxanthin Fucosterol Fucoxanthin Fucosterol Fucoxanthin Fucosterol

7.12 ± 3.27 0.83 ± 6.50 71.05 ± 4.09 1.37 ± 4.74 59.90 ± 2.05 −5.2 ± 11.78 11.56 ± 5.06 −0.40 ± 2.01 4.98 ± 3.07 −3.90 ± 1.84 35.46 ± 2.94 5.83 ± 0.14 16.96 ± 4.06 −0.87 ± 0.16

16.881.8-

dopamine

43

7 ± 3.41

dopamine

7 ± 6.11

dopamine

acetylcholine

1.3

[Sar9, Met (O2)11]-SP AVP

0.14

serotonin

2.5

D3 (h) Dopamine Fucosterol D4 (h) Dopamine Fucosterol M5 (h) Acetylcholine (Muscarinic) NK1 (h) Tachykinin V1A (h) Vasopressin/Oxytocin 5-HT1A (h) Serotonin a b

0.52

Concentration producing half-maximal agonist response. Concentration producing 50% agonist response for respective receptors, reported by Eurofins Panlab.

Table 3 Antagonist activity of fucoxanthin, fucosterol, and reference compounds against respective receptors. Receptors

Compounds

% Inhibition of Control Agonist Response at 100 μM

IC50a (μM)

Reference antagonists of respective target receptors IC50 (nM)b

D1 (h) Dopamine

Fucoxanthin Fucosterol Fucoxanthin Fucosterol Fucoxanthin Fucosterol Fucoxanthin Fucosterol Fucoxanthin Fucosterol Fucoxanthin Fucosterol Fucoxanthin Fucosterol

39.48 ± 4.58 24.85 ± 4.78 −10.8 ± 6.59 1.43 ± 5.72 3.18 ± 2.90 32.85 ± 2.64 14.62 ± 7.09 −2.75 ± 2.19 45.93 ± 3.11 −0.30 ± 0.85 29.13 ± 5.04 3.10 ± 2.71 −12.0 ± 3.89 −7.95 ± 2.90

-

SCH 23390

4.4

(+) butaclamol

31

clozapine

120

atropine

2.2

D3 (h) Dopamine D4 (h) Dopamine M5 (h) Acetylcholine (Muscarinic) NK1 (h) Tachykinin V1A (h) Vasopressin/Oxytocin 5-HT1A (h) Serotonin a b

L 733,060

1.6

1

5.5

[d(CH2)5 , Tyr (Me)2]-AVP (S)-WAY100635

1.8

Concentration producing half-maximal inhibition of control agonist response. Concentration producing 50% antagonist response for respective receptors, reported by Eurofins Panlab.

receptor at 16.87 ± 3.41 and 81.87 ± 6.11 μM, respectively. Dopamine itself had EC50 values of 3.7 and 24 nM on dopamine D3 and D4 receptors, respectively. The antagonist effects tabulated in Table 3 show that at 100 μM, fucoxanthin had a moderate antagonist effect on dopamine D1 (39.48%) and NK1 tachykinin (45.93%) receptors. However, fucosterol showed a mild antagonist effect on dopamine D1 (24.85%) and dopamine D4 (32.85%) receptors at 100 μM concentration. The

antagonist effect of fucoxanthin and fucosterol was null on the remaining receptors. 3.3. Molecular docking study Prediction of drug-biomolecule target interactions via computational technology is a cost-effective and reliable approach for rational

Fig. 2. Concentration-dependent percentage of control agonist effect of fucoxanthin and fucosterol on dopamine D3 (A) and D4 (B) receptors. 4

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Table 4 Binding sites and docking score of compounds in dopamine D3 receptor (D3R). Compounds

Binding energy (kcal/mol)

No. of Hbonds

H-bond interaction residues

Other interacting residues

Dopaminea

‒5.84

5

Val111, Cys114, Phe346

Eticlopridea Fucoxanthin

‒8.50b ‒10.61

2 3

Asp110 (Salt bridge), Val111 (O–H bond), Thr115 (O–H bond), Ser196 (O–H bond) Asp110 (Salt bridge, O–H bond) Val111 (O–H bond), Ser196 (O–H bond), Ser366 (O–H bond)

a b

Val111, Cys114, Val189, Phe346, His349, Ile183, Phe345 Ile183, His349, Pro362, Phe345, Phe346, Cys114, Val107, Asp110, Ser192, Thr115

Dopamine and eticlopride were used as positive ligands. RMSD value: 0.48 Å.

Table 5 Binding sites and docking score of compounds in dopamine D4 receptor (D4R). Compounds Dopaminea a

Binding energy (kcal/ mol)

No. of Hbonds

H-bond interaction residues

Other interacting residues

‒5.68

3

Asp115 (Salt bridge), Ser196 (O–H bond) Asp115 (Salt bridge, O–N bond), Ser196 (C–H bond), Tyr438 (O–H bond) Asp115 (Salt bridge), Cys185 (C–O bond) Leu90 (O–H bond) Asp115 (O–H bond), Ser196 (O–H bond)

Cys119, Phe410, Val116

b

Nemonapride

‒11.82

Clozapinea

‒9.49

1

Fucosterol Fucoxanthin

‒11.89 ‒7.15

1 3

a b

5

Val193, Leu111, Cys185, Phe91, Phe410, Val116, Cys119, Leu90, Phe91 Arg186, Leu187, Val193, Val116, Cys119, Phe411, His414, Val193, Val116, Phe410, His414, Leu187 Val87, Leu111, Met112, Cys185, Leu187, Val193, Cys119, Phe410, Phe411, His414 Val116, Cys119, Tyr192, Leu187, Val430, Phe411, Trp407, Phe410, Val193, His414, Val87, Cys185, Trp101, Leu111, Met112

Dopamine, nemonapride, and clozapine were used as positive ligands. RMSD value: 0.21 Å.

drug design and discovery. An in silico docking study was performed to gain insight into the basis of the hD3R/hD4R agonist activity of fucoxanthin, which was also compared with the action of inactive fucosterol. The docking study was validated using reference agonists and antagonists of each dopamine receptor. Binding sites and docking scores of fucoxanthin for hD3R and hD4R are tabulated in Tables 4 and 5, respectively. Fig. 3 shows putative positions of dopamine, eticlopride, and fucoxanthin in the active site cavity of hD3R, which are shown in red, black, and yellow, respectively. As tabulated in Table 4, fucoxanthin bound to the active site cavity of hD3R with −10.61 kcal/mol of binding energy. The binding energy of dopamine was −5.84 kcal/mol, while binding energy for the antagonist eticlopride was −8.50 kcal/ mol. Major H-bond interacting residues involved in forming the hD3Rfucoxanthin complex were Val111, Ser196, and Ser366 (shown by green dotted lines in Fig. 3C). In addition to H-bond interactions, other

observed interactions were via Ile183, His349, Pro362, Phe345, Phe346, Cys114, Val107, Asp110, Ser192, and Thr115 (shown by purple dotted lines in Fig. 3C). Similarly, for the hD3R-dopamine complex, Thr115, Ser196, Val111, and Asp110 were involved in Hbond interactions. Thr115 and Ser196 were common interacting residues involved in fucoxanthin and dopamine binding to hD3R, and these residues were not involved in binding of the antagonist eticlopride. Similarly, Fig. 4 shows the putative position of dopamine, clozapine, fucosterol, and fucoxanthin in the active site cavity of hD4R, which are shown in red, black, magenta, and yellow, respectively. As tabulated in Table 5, fucoxanthin bound to the active site cavity of hD4R with lower binding energy (−7.15 kcal/mol) by forming three H-bond interactions with Asp115 and Ser196 (shown by green dotted lines in Fig. 4E). Similarly, the same H-bond interacting residues (Asp115 and Ser196)

Fig. 3. Molecular docking of dopamine D3 receptor (D3R) binding with fucoxanthin along with positive controls (A). Chemical structures of dopamine, eticlopride, and fucoxanthin are shown in red, black, and yellow, respectively. Fucoxanthin (B and C) binding site showing D3R-ligand interaction.

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Fig. 4. Molecular docking of dopamine D4 receptor (D4R) binding with fucosterol and fucoxanthin along with positive controls (A). Chemical structures of dopamine, clozapine, fucosterol, and fucoxanthin are shown in red, black, magenta, and yellow, respectively. Fucosterol (B and C) and fucoxanthin (D and E) binding sites showing D4R-ligand interaction.

were observed in binding reference agonists dopamine and nemonapride to hD4R. Interestingly, interaction with Ser196 residue was not observed in the binding of reference antagonist clozapine and fucosterol. Fucosterol showed the lowest binding energy (−11.89 kcal/mol) with a single H-bond interacting residue Leu90.

4. Discussion Dopamine receptors activate numerous signal transduction pathways. However, activation or inhibition of the cAMP pathway and modulation of Ca2+ signaling are the best-reported effects. D1-type (D1 and D5) receptors are positive regulators of cAMP levels, and their stimulation activates protein kinase A, which phosphorylates cytoplasmic and nuclear proteins, regulates cellular metabolism and ion channel function, and desensitizes transmembrane receptors leading to the cellular response to neurotransmitter release [31]. Conversely, D2like (D2, D3, and D4) receptors inhibit adenylyl cyclase, leading to decreased cAMP levels. D3 receptors are more effective than D2. D2R stimulation also activates the β-arrestin2-mediated signaling pathway in a cAMP-independent manner [32]. Monoamine oxidase (MAO) is a catecholamine-degrading enzyme with a long-established therapeutic profile; MAO-A and MAO-B are its isoenzymes. MAO-B is the predominant form of the enzyme in the human brain, and dopamine and phenylethylamine are its preferred substrates. Norepinephrine and serotonin are the preferred substrates for MAO-A. MAO-B degrades dopamine in neural and glial cells, which generates free radicals that are believed to play an eminent role in the

3.4. Absorption, distribution, metabolism, and excretion (ADME) prediction As shown in Table 6, in silico pharmacokinetic parameter prediction by PreADMET indicated relatively higher log Po/w values of 8.62 and 7.53 for fucoxanthin and fucosterol, respectively. Plasma protein binding and human intestinal absorption for fucosterol was 100%, while it was 90.86 and 96.70%, respectively, for fucoxanthin. In addition, calculations of in vivo blood-brain barrier (BBB) penetration demonstrated higher absorption by the central nervous system (CNS) for both compounds. Interestingly, BBB penetration of fucosterol (19.54) was three times that of fucoxanthin (5.55). Favorable BBB permeability is a crucial factor in the development of CNS-active drugs. Together, these results indicate that fucoxanthin has favorable drug-like properties. Table 6 Drug-likeness and ADME characteristics as determined by PreADMET. Compounds

Fucoxanthin Fucosterol

Drug-likeness

ADME characteristics

MDDR-like rule

Lipinski's rule

Log Po/wa

PPBb

HIAc

In vitro Caco2 permeability (nm/sec)d

In vitro MDCK cell permeability (nm/s)e

In vivo BBB penetration ([brain]/[blood])f

Drug-like Mid-structure

Violated suitable

8.62 7.53

90.86 100.0

96.70 100.0

52.02 52.30

0.06 5.61

5.55 19.54

Lipinski's rule: An orally active drug has no more than one violation of H-bond donors (≤5), H-bond acceptors (≤10), molecular weight (≤500 Da), and logP (≤5). MDDR-like rule: The MDDR-like rule describes a molecule as drug-like or non-drug-like on the basis of the number of rings, rigid bonds, and rotatable bonds. a Log of coefficient of solvent partitioning between 1-octanol and water. b Plasma protein binding (PPB) (< 90% represents weak binding and > 90% represents strong binding). c Human intestinal absorption (HIA) (0–20% is poorly absorbed, 20–70% is moderately absorbed, and 70–100% is well-absorbed). d 0–10 nm/s is low permeability, 10–100 nm/s is medium permeability, and > 100 nm/s is high permeability. e Permeability across MDCK cells. f 0.1 is low absorption by central nervous system, 0.1–2.0 is middle absorption, and > 2.0 is high absorption.

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pathogenesis of PD [33]. However, MAO-B inhibitors stabilize dopamine concentrations in the synaptic cleft and prolong the effects of dopamine [34]. Hence, inhibition of MAO-B enzyme activity is one of several approaches to treat PD. To characterize the role of fucoxanthin and fucosterol in neurodegenerative disease, their effect on MAO inhibition and GPCR modulation was evaluated. Fucosterol was inactive in MAO inhibition even at 500 μM, however, fucoxanthin showed moderate inhibition of both of the isoenzymes with IC50 values of approximately 200 μM [12]. We previously reported details on the enzyme kinetics and molecular docking studies of fucoxanthin in MAO inhibition. Since these two lipid components are abundant in brown algae and had demonstrated protective roles in Aβ1-42-induced cognitive dysfunction in animal models, we hypothesized an inhibitory effect of fucosterol over MAO isoenzymes. However, our hypothesis was not supported because fucosterol did not show any noticeable effect up to 500 μM. Still, we were curious about the modulatory effect of these compounds over several GPCRs, which are well-established targets for neurodegeneration. The results of cell-based functional GPCR assays demonstrated fucoxanthin as a selective agonist of dopamine D3/D4 receptor. Fucoxanthin was 5 times more selective for D3 over D4, as revealed by its EC50 values. In addition, the binding energy of fucoxanthin at dopamine D3 receptor was lower (−10.61 kcal/mol) than that at dopamine D4 receptor (−7.15 kcal/mol). Interestingly, fucosterol had the lowest binding energy among fucoxanthin and reference ligands at the dopamine D4 receptor. However, it did not show any modulatory effect in functional assay results. Results of the computational study revealed that fucosterol could not bind with the main interacting residues, leading to conformational changes that exhibit a modulatory effect. Fucoxanthin involved interactions with determinant interacting residues of dopamine D3 (Val111, Thr115, and Ser196) and D4 receptors (Asp115 and Ser196). These results affirm the importance of modulatory residues in attaining maximal agonist activity on dopamine D3/D4 receptors. Unlike fucosterol, the structure of fucoxanthin enables it to reach the core active site cavity of dopamine D3 and D4 receptors where it binds to conserved interacting residues and leads to conformational change. The activation mechanism of dopamine for transmitting extracellular ligand binding phenomena through transmembrane (TM) helices to cytoplasmic G proteins at the atomic level remains unclear. In the absence of ligands, GPCRs are believed to exist in dynamic equilibrium between the inactive and active states. Agonist binding is speculated to increase the probability of the receptor resuming its active state [35]. Antagonists restrain the receptor in its inactive state and block agonist binding. In a recent study [36], Weng et al. simulated conformational alterations in dopamine D3 receptor's active and inactive forms during microsecond-timescale molecular dynamic simulations by constructing a complete dopamine D3R with a homologymodeled N-terminus. They proposed a complete activation mechanism for D3R. When an agonist binds to D3R, the N-terminal conformational change induces transmembrane molecular switches to form an internal water channel that increases the volume of the cytoplasmic side, which is suitable for G protein binding. The continuous water channel of activated D3R enables downstream signal transduction. Selective dopamine receptor D4 antagonists developed for schizophrenia in the early 1990s failed in clinical trials. Since then, D4 receptors have not been intensively studied as therapeutic targets. However, recent studies demonstrating the role of D4 receptor against addiction [37], Parkinson's disease (PD), dyskinesia, and cancer has revived the interest in researching D4 receptors. Fucosterol displayed a mild dopamine D4 antagonist effect by inhibiting the agonist effect of dopamine by 32% at 100 μM. In previous reports, fucoxanthin at 3 μM concentration potently protected SH-SY5Y cells against H2O2-induced neurotoxicity via concurrently activating the PI3K/Akt cascade and inhibiting the ERK pathway [38] and at 100–200 mg/kg doses significantly reversed Aβ1-42 oligomer-induced impairments of spatial memory in mice [39].

Similarly, in an in vivo middle cerebral artery occlusion (MCAO) model, fucoxanthin at 90 mg/kg dose attenuated cerebral ischemic/reperfusion (I/R) injury [15]. Furthermore, 100 mg/kg dose of fucoxanthin reduced traumatic brain injury (TBI)-induced apoptosis in mice model [40], 10 mg/kg dose ameliorated bleomycin-induced pulmonary fibrosis in mice, and also inhibited the transforming growth factor-beta1-induced expression of α-smooth muscle actin, type 1 collagen, fibronectin, and IL-6 in human pulmonary fibroblasts (HPFs) [41]. Collectively, previous in vivo neuroprotective effect of fucoxanthin and the dopamine D3/D4 receptor agonist effect of the present study provides an interesting insight on fucoxanthins’ role in neurodegenerative diseases. The findings of this study must be considered in light of some limitations that should be addressed in future research. This study employed in vitro cell-based functional assays and in silico molecular simulation to characterize fucoxanthin as a potent dopamine D3R/D4R agonist for management of neurodegenerative disease, particularly PD. Therefore, in vivo studies in dopamine receptor knockout or knockdown mice models along with detailed studies on signal transduction pathways are warranted. 5. Conclusion Fucoxanthin and fucosterol are typical lipid components of edible brown algae with numerous biological activities. Fucoxanthin displayed good inhibition on two isoenzymes, hMAO-A, and hMAO-B. However, fucosterol remained inactive up to 500 μM. In functional assay results, fucoxanthin showed a concentration-dependent agonist effect on dopamine D3 and D4 receptors. The results of the computational study reveal the pivotal role of Thr115 and Ser196 amino acid residues in D3R-fucoxanthin binding and Asp115 and Ser196 residues in D4R-fucoxanthin binding. Overall, the present study characterizes fucoxanthin as a potent dopamine D3/D4 agonist that might be useful in the management of neurodegenerative diseases, especially PD. 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). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbi.2019.108757. Conflicts of interest The authors declare that there are no conflicts of interest. Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References [1] R. Rajput, R. Kaur, R. Chadha, S. Mani, R. Rachana, H. Kaur, M. Singh, The aging brain: from physiology to neurodegeneration, Handbook of Research on Critical Examinations of Neurodegenerative Disorders, IGI Global, PA, USA, 2019, pp. 1–23. [2] L. Perez-Caballero, S. Torres-Sanchez, C. Romero-López-Alberca, F. González-Saiz, J.A. Mico, E. Berrocoso, Monoaminergic system and depression, Cell Tissue Res. (2019), https://doi.org/10.1007/s00441-018-2978-8. [3] C. Rangel-Barajas, I. Coronel, B. Florán, Dopamine receptors and neurodegeneration, Aging Dis. 6 (2015) 349–368. [4] K.L. Davis, R.S. Kahn, G. Ko, M. Davidson, Dopamine in schizophrenia: a review and reconceptualization, Am. J. Psychiatry 148 (1991) 1474–1486. [5] N.M. Sachindra, E. Sato, H. Maeda, M. Hosokawa, Y. Niwano, M. Kohno,

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