Effect of the Biphenyl Neolignan Honokiol on AÎ²42-Induced Toxicity in

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Effect of the biphenyl neolignan honokiol on A#42induced toxicity in Caenorhabditis elegans, A#42 fibrillation, cholinesterase activity, DPPH radicals, and iron(II) chelation Srinivas Kantham, Stephen Chan, Gawain McColl, Jared Miles, Suresh Kumar Veliyath, Girdhar Singh Deora, Satish N Dighe, Samira Khabbazi, Marie-Odile Parat, and Benjamin P. Ross ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00071 • Publication Date (Web): 26 Jun 2017 Downloaded from http://pubs.acs.org on June 27, 2017

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ACS Chemical Neuroscience

Effect of the biphenyl neolignan honokiol on Aβ42-induced toxicity in Caenorhabditis elegans, Aβ42 fibrillation, cholinesterase activity, DPPH radicals, and iron(II) chelation Srinivas Kantham,‡ Stephen Chan,‡ Gawain McColl,§ Jared A. Miles,‡ Suresh Kumar Veliyath,‡ Girdhar Singh Deora,‡ Satish N. Dighe,‡ Samira Khabbazi,‡ Marie-Odile Parat‡ and Benjamin P. Ross*,‡ ‡

The University of Queensland, School of Pharmacy, Brisbane, Queensland 4072, Australia.

§

The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Victoria 3010, Australia.

ABSTRACT: The biphenyl neolignan honokiol is a neuroprotectant which has been proposed as a treatment for central nervous system disorders such as Alzheimer’s disease (AD). The death of cholinergic neurons in AD is attributed to multiple factors, including: accumulation and fibrillation of amyloid beta peptide (Aβ) within the brain; metal ion toxicity; and oxidative stress. In this study, we used a transgenic Caenorhabditis elegans model expressing full length Aβ42 as a convenient in vivo system for examining the effect of honokiol against Aβ-induced toxicity. Furthermore, honokiol was evaluated for its ability to: inhibit Aβ42 oligomerization and fibrillation; inhibit acetylcholinesterase and butyrylcholinesterase; scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals; and chelate iron(II). Honokiol displayed similar activity to resveratrol and (−)-epigallocatechin gallate (EGCG) in delaying Aβ42-induced paralysis in C. elegans, and it exhibited moderate-to-weak ability to inhibit Aβ42 on-pathway aggregation, inhibit cholinesterases, scavenge DPPH radicals, and chelate iron(II). Moreover, honokiol was found to be chemically stable relative to EGCG, which was highly unstable. Together with its good drug-likeness and brain availability, these results suggest that honokiol may be amenable to drug development, and the synthesis of honokiol analogues to optimize these properties should be considered. KEYWORDS: Alzheimer’s disease, amyloid beta peptide, Caenorhabditis elegans, cholinesterases, honokiol

INTRODUCTION The bark of various Magnolia species, called “Houpo” in Chinese and “Koboku” in Japanese, is used in traditional Chinese and Japanese medicine to treat various brain disorders and gastrointestinal diseases.1, 2 Honokiol (Figure 1) is a biphenyl neolignan present in the stem bark of Magnolia officinalis Rehder et Wilson (Chinese Magnolia) and Magnolia obovata Thunberg (Japanese Magnolia), family Magnoliaceae.1, 3 Honokiol is reported to possess activities against inflammation,4 depression,5 anxiety,6 and oxidative stress.7 Moreover, honokiol: protects neurons from focal cerebral ischemia-reperfusion injury;8 can reverse glucose deprivation-induced neuronal damage and N-methyl-D-aspartate (NMDA)induced mitochondrial dysfunction;9 and, protects neurons against amyloid beta peptide (Aβ)-induced toxicity.10 Honokiol can also scavenge free radicals, and it protects DNA against Cu2+/glutathione and 2,2′-azobis(2amidinopropane hydrochloride)-induced oxidation.11 Although more than 98% of existing small-molecule drugs do not cross the blood-brain barrier (BBB),12 recent studies suggest that honokiol can cross both the BBB and blood-cerebrospinal fluid barrier.13 The ability of honokiol to enter the central nervous system (CNS), in addition to its reported pharmacological activities, makes honokiol a promising molecule for the development of

drugs to treat CNS disorders such as Alzheimer’s disease (AD).

Figure 1. The chemical structures of honokiol, (-)epigallocatechin gallate (EGCG), and resveratrol.

AD is an irreversible neurodegenerative disorder and the leading cause of dementia.14 Across the globe, there were more than 44.4 million people with AD and related dementia in 2013, and this number is expected to increase to 75.6 million by 2030, and 135.5 million by 2050. In AD, the disruption in cholinergic neurotransmission15 and overstimulation of NMDA receptors16 triggers a gradual decline in memory and cognitive functions that can be temporarily alleviated by cholinesterase (ChE) inhibitors, and NMDA receptor antagonists.17 However, there is no disease-modifying treatment that can halt or slow the progression of AD. Although AD involves multiple pathogenic mechanisms18, it is widely acknowledged that the primary event in AD pathogenesis is the accumu-

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lation and fibrillation of Aβ within the brain, in accordance with the amyloid cascade hypothesis.14 Aβ oligomers formed during the fibrillation process are the main neurotoxic species, via mechanisms such as cell membrane perturbation and oxidative stress.19 Aβ contains metal binding sites, and metals such as copper, zinc and iron are found at elevated concentrations in areas of the brain containing Aβ plaques.19 Metals can accelerate Aβ fibrillation, affect Aβ aggregate morphology, and enhance the neurotoxicity of Aβ.20 Additionally, the redox potential of iron and copper facilitates the generation of reactive oxygen species via Fenton chemistry, and these reactive species cause oxidative stress and subsequent neuronal damage.21 The multiple pathogenic mechanisms present in AD means that multifunctional compounds may be beneficial as disease-modifying drugs for AD treatment.18, 22 Also, some of the fundamental properties of honokiol that are relevant to AD treatment have not been examined. Therefore, in this study, honokiol was subjected to in vitro assays to determine its ability to: inhibit Aβ42 onpathway aggregation; inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE); scavenge 2,2diphenyl-1-picrylhydrazyl (DPPH) radicals; and chelate iron(II). Furthermore, we evaluated the in vivo protective effect of honokiol against Aβ-induced toxicity using transgenic Caenorhabditis elegans. In this animal model, the human Aβ42 peptide is expressed in body wall muscle cells, where Aβ42 oligomerizes, aggregates, and results in severe, and fully penetrant, age progressive-paralysis.23 The phenols resveratrol and (−)-epigallocatechin gallate (EGCG) were included in the study for comparison with honokiol (Figure 1).

RESULTS AND DISCUSSION Inhibition of Aβ42 fibrillation. The inhibition of Aβ42 fibrillation by honokiol was evaluated using a thioflavin T (ThT) fluorescence assay under quiescent conditions at pH 7.4 and 37˚C.24, 25 The ThT fluorescence signal is enhanced upon binding to amyloid fibrils, hence ThT fluorescence can be used to monitor Aβ fibrillation.26 Aβ42 (27 µM) alone, and when incubated with honokiol (10, 100, and 1000 µM) or resveratrol (100 µM), exhibited a characteristic sigmoidal kinetic growth curve (Figure 2A).27, 28 The degree of fibrillation was estimated from the amplitude of each sample’s sigmoidal curve, expressed relative to Aβ42 control amplitude which represented 100% fibrillation (Figure 2B). Figure 2B clearly shows that honokiol (10, 100, and 1000 µM) caused a dose-dependent decrease in Aβ42 fibrillation. Interestingly, EGCG (100 µM) prevented the formation of a sigmoidal curve (Figure 2A), with final fluorescence intensity (FI) less than the initial FI,29, 30 suggesting complete inhibition of Aβ42 fibrillation and the remodeling of preformed fibrils31 that were present at the start of the experiment.

Figure 2. A) Aβ42 fibril formation monitored by in situ ThT fluorescence. FI readings (λex 440 nm, λem 480 nm) were recorded every 5 min. The graph shows representative kinetic plots, corrected for the fluorescence of ThT alone (20 µM). Aβ42 (27 µM) was incubated with honokiol (10, 100, and 1000 µM), resveratrol (100 µM), or EGCG (100 µM) in phosphate buffer (20 mM, pH 7.4, I 0.17 M, containing 20 µM ThT) at 37˚C under quiescent conditions. Honokiol and resveratrol samples contained DMSO (0.1% v/v). The extent of fibrillation is depicted in B) as percentage of the Aβ42 control amplitude. Values are the mean + SEM of three independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test with: **p < 0.01 and ****p < 0.0001 c.f. Aβ42 + DMSO 0.1%; and ^^^^p < 0.0001 c.f. Aβ42 control.

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There were no significant differences in half-life between groups (Figure S1). C) Aβ42 fibrillation estimated by ProteoStat® fluorescence. Aβ42 (27 µM) was incubated with honokiol (10, 100, and 1000 µM), resveratrol (10, 100, and 1000 µM) or EGCG (10 and 100 µM) in phosphate buffer (20 mM, pH 7.4, I 0.17 M) at 37˚C under quiescent conditions. Honokiol, resveratrol, and EGCG samples contained DMSO (0.1% v/v). After 21 h, ProteoStat® dye was added and FI was recorded (λex 544 nm, λem 590 nm). The extent of fibrillation is depicted as percentage of the Aβ42 control amplitude. Values are the mean + SEM of three independent experiments with *p < 0.05 and ****p < 0.0001 c.f. Aβ42 + DMSO 0.1%, determined by one-way ANOVA followed by Tukey’s multiple comparisons test.

To confirm the results of the ThT assay, the extent of fibrillation at equilibrium was also estimated with ProteoStat® dye (Figure 2C), which just like ThT, exhibits enhanced fluorescence emission when it binds to Aβ fibrils.32 However, compared to ThT,33 ProteoStat® dye is more sensitive, and it is less susceptible to spectroscopic interference because it has longer wavelength excitation and emission maxima.32 The ProteoStat® assay results were aligned with the outcome of the ThT assay, and showed that honokiol, resveratrol and EGCG dosedependently decrease Aβ42 fibrillation. Overall, considering the results of both of the fluorometric assays, the order of activity for inhibition of Aβ42 fibrillation was EGCG25, 29, 30 > resveratrol25 ≥ honokiol. Honokiol (100 µM) was recently shown to completely inhibit the fibrillation of Aβ42 (10 µM),34 although in our assay honokiol was only a moderate inhibitor relative to the extensively studied potent inhibitor EGCG.24, 25, 29-31, 35 Inhibition of Aβ42 oligomer formation. Intermediates in the fibrillation process, Aβ oligomers, are highly toxic,36 therefore compounds that inhibit oligomer formation, or decrease levels of preformed oligomers, may be more beneficial than compounds that only affect mature fibrils. Hence, the oligomer-specific antibody A1137 was used in a dot blot assay to analyze the effect of honokiol on Aβ42 oligomer formation (Figure 3A). The A11 immunoreactivity of Aβ42 control was positive on day 0, and increased slightly by day 1, after which it remained fairly constant. This indicates that substantial oligomer formation occurred prior to the first sample being applied to the membrane, and maximal levels of oligomers were attained by day 1. EGCG showed strong inhibition of Aβ42 oligomer formation,38 evident from day 0, consistent with its ability to promote the formation of offpathway protein aggregates from prefibrillar and fibrillar Aβ42.30, 31 The immunoreactivity of Aβ42 incubated with 1000 µM honokiol peaked on day 1 and then decreased, signifying that honokiol can decrease the levels of Aβ42 oligomers, albeit slowly. This activity was similar to 100 µM resveratrol. A control membrane was probed using antibody 6E10,39 which recognises all forms of Aβ via its residues 3-8, and this experiment displayed immunore-

activity for all samples at all time points thus confirming the presence of Aβ42 (Figure 3B).

Figure 3. Dot blot assay showing Aβ42 oligomer levels over 7 days. Aβ42 (27 µM) was incubated with resveratrol (100 µM), honokiol (10, 100, and 1000 µM), or EGCG (100 µM), in phosphate buffer (20 mM, pH 7.4, I 0.17 M) at 37˚C under quiescent conditions. Honokiol and resveratrol samples contained DMSO (0.1% v/v). Aliquots were removed at various time points, spotted onto nitrocellulose membranes, and probed with: (A) oligomer-specific A11 antibody; and (B) 6E10 antibody that recognizes all forms of Aβ via its residues 3-8.

Morphology of Aβ42 aggregates. Transmission electron microscopy (TEM) was used to study the influence of honokiol, resveratrol and EGCG on the abundance and morphology of Aβ42 aggregates on day 1 and day 7 (Figure 4). Aβ42 control exhibited abundant fibrils in large dense clusters and there were clearly more fibrils on day 7 compared to day 1. Honokiol (1000 µM), resveratrol (100 µM), and EGCG (100 µM) decreased the abundance and density of fibrils relative to the control, and many non-fibrillar aggregates were observed. These results are consistent with published TEM images.25, 30, 31, 34

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Figure 4. Effect of honokiol, resveratrol and EGCG on the morphology of Aβ42 aggregates. Aβ42 (27 µM) was incubated with honokiol (1000 µM), resveratrol (100 µM) and EGCG (100 µM) in phosphate buffer (20 mM, pH 7.4, I 0.17 M) at 37˚C under quiescent conditions. TEM images of: (A) Aβ42 control on day 1; (B) Aβ42 control on day 7; (C) Aβ42 + honokiol on day 1; (D) Aβ42 + honokiol on day 7; (E) Aβ42 + resveratrol on day 1; (F) Aβ42 + resveratrol on day 7; (G) Aβ42 + EGCG on day 1; (H) Aβ42 + EGCG on day 7. Images of Aβ42 + 0.1% v/v DMSO (Figure S2) were similar to (A). The scale bar represents 500 nm.

Molecular docking studies. Many studies have investigated the mechanism of the Aβ self-assembly process leading to the formation of neurotoxic Aβ oligomers and subsequently Aβ fibrils.40-43 Aβ42 misfolding and assembly into β-sheet rich aggregates is promoted by the formation of an intramolecular salt bridge Asp23 and Lys28 which stabilises a β-turn that connects two betastrands.40, 44 Furthermore, Lys16 residues participate in both hydrophobic and electrostatic interactions, which contribute to the stability of β-sheets.40, 44 Interestingly, the substitution of Lys16 and Lys28 with Ala in Aβ42 sup-

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pressed the toxicity of Aβ42 towards rat pheochromocytoma (PC12) cells,41 which suggests that lysine and aspartic acid residues are key target residues for the development of Aβ fibrillation inhibitors. Compounds that interact with these residues are proven to interfere with Aβ42 fibrillation and reduce the toxic effects of Aβ42.41, 42 Therefore, in this study, molecular docking studies of honokiol, resveratrol and EGCG were performed to probe their proposed binding interactions with Aβ42 monomer (PDB ID: 1IYT),45 Aβ16-21 (KLVFFA) fibre (PDB ID: 3OVJ),46 and monomorphic Aβ42 amyloid fibrils (PDB ID: 5KK3)47 using the Schrödinger molecular modelling suite. The docking models are shown in Figure 5 and the 2Dinteraction diagrams are provided in supplementary information (Figures S3-S11). Despite the differences in inhibition of Aβ42 fibrillation, as discussed above, all three compounds (EGCG, honokiol and resveratrol) were predicted to bind to the same region of the Aβ42 monomer (Figures 5A-C and S3-S5), forming hydrogen bonds with the key amino acid residues Lys16 and Asp23. Similarly, Hyung et al. found that EGCG docked into Aβ40 monomer (PDB ID: 2LFM)48 occupied the region spanning residues His13 to Asp23.49 The docking models for binding interactions with the Aβ16-21 (KLVFFA) fibre predicted that both honokiol and EGCG form hydrogen bonds with two lysine residues with EGCG showing additional hydrogen bonding interactions with the amide backbone of residues Leu17, and Phe19 (Figures 5D, 5F, S6, and S8). In contrast, resveratrol, formed a hydrogen bond with the amide backbone of Phe19 and displayed π-π stacking with two Phe20 residues (Figures 5E and S7). Monomorphic Aβ42 amyloid fibrils possess a hydrophobic groove composed of Val18 and Ala21, which is gated by Glu22 and Lys16, and also a hydrophobic patch composed of Val40 and Ala42.47 The interface between the monomers within monomorphic Aβ42 amyloid fibrils shows intermolecular contacts between Met35 of one molecule and Lys17 and Gln15 of the second molecule.47 As shown in Figures 5G-I, honokiol, resveratrol and EGCG occupied the hydrophobic groove on the monomorphic Aβ42 fibril surface. Honokiol made hydrogen bonds with Lys16 and Ala21 (Figures 5G and S9). Resveratrol participated in Hbonding with Glu22 and two Lys16 residues, and also made a π-cation interaction with Lys16 (Figures 5H and S10). EGCG interacted with Ala21 and multiple Lys 16 and Glu22 residues (Figures 5I and S11) via hydrogen bonding. Although the catalytic surface for secondary nucleation can be anywhere on the fibril, it is likely to involve a major fraction of the fibril surface.27, 47, 50 Therefore, honokiol, resveratrol and EGCG may inhibit secondary nucleation because they interact with hydrophobic grooves on the fibril surface. Overall, relative to resveratrol and honokiol, EGCG showed a superior network of interactions with all forms of Aβ, which is consistent with its strong inhibition of Aβ fibrillation.

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Figure 5. Docking model of honokiol, resveratrol and EGCG with Aβ42 monomer (PDB ID: 1IYT), Aβ16-21 (KLVFFA) fibre (PDB ID: 46 47 3OVJ), and monomorphic Aβ42 amyloid fibrils (PDB ID: 5KK3) developed using the Schrödinger molecular modelling suite. Colour scheme: yellow, honokiol; orange, resveratrol; and green, EGCG. A) Proposed binding interactions of honokiol with Aβ42 monomer: H-bonding (dashed line) with Lys16 and Asp23; B) Proposed binding interactions of resveratrol with Aβ42 monomer: H-bonding with Lys16 and Asp23; C) Proposed binding interactions of EGCG with Aβ42 monomer: H-bonding with Lys16, Asp23 and Gln15; D) Proposed binding interactions of honokiol with Aβ16-21 (KLVFFA) fibre: H-bonding with Lys16; E) Proposed binding interactions of resveratrol with Aβ16-21 (KLVFFA) fibre: H-bonding with the amide backbone of Phe19, and π-π stacking with two Phe20 residues; F) Proposed binding interactions of EGCG with Aβ16-21 (KLVFFA) fibre: H-bonding with Lys16 and the amide backbone of residues Leu17 and Phe19; G) Proposed binding interactions of honokiol with monomorphic Aβ42 amyloid fibrils: H-bonding with Lys16 and Ala21; H) Proposed binding interactions of resveratrol with monomorphic Aβ42 amyloid fibrils: Hbonding with Lys16 and Glu22; and I) Proposed binding interactions of EGCG with monomorphic Aβ42 amyloid fibrils: Hbonding with Lys16, Ala21 and Glu22.

Inhibition of cholinesterases. Honokiol is known to prevent age-related memory and learning deficits through preservation of cholinergic neurons in the forebrain.51 In addition, honokiol promotes potassiuminduced release of acetylcholine in rat hippocampal slice.52 The inhibition of AChE and BuChE enzymes could hypothetically provide additional enhancement to these acetylcholine increasing effects. In this study, the extent

of AChE and BuChE inhibition by honokiol was elucidated using Ellman’s assay. The ChE inhibitor drugs rivastigmine, donepezil and tacrine, and the phenols resveratrol and EGCG, were used as reference compounds. The concentration-response curve enabled estimation of IC50 values (Table 1). Micromolar concentrations of honokiol inhibited both Electrophorus electricus AChE (EeAChE) and BuChE from equine serum (eqBuChE), with negligible (1.33-fold) selectivity towards EeAChE. This activity was

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superior to that of resveratrol and EGCG, but inferior to that of the AChE and BuChE inhibitor drugs. These drugs contain a basic nitrogen atom, which facilitates their entry into and binding within the active site of the enzyme through cation-π interactions with electronegative subsites.53 The synthesis of analogues that incorporate a basic nitrogen atom might be a suitable strategy to enhance the anti-cholinesterase activity of honokiol. Molecular docking simulations were performed to gain insight into the inhibition of AChE and BuChE by honokiol. Honokiol is a small molecule, and it easily fits within the deep active site pocket of both human AChE (hAChE) (Figure 6A) and human BuChE (hBuChE) (Figure 6C). The docking model for honokiol-hAChE indicated that the 2'hydroxyl group of honokiol forms a hydrogen bond with the amide backbone of Phe295, and the A-ring of honokiol has π-π interactions with Phe338 and Tyr341 (Figure 6B). Similarly, the honokiol-hBuChE docking model showed hydrogen bonds between the 2'- and 4hydroxyl groups of honokiol, and the amide backbone of catalytic residue His438 and Glh197, respectively. In addition, the A-ring of honokiol exhibited parallel π-π stacking with Trp82 within the choline binding pocket (Figure 6D). The 2D-interaction diagrams of honokiol with hAChE and hBuChE are provided in Figures S12 and S13, respectively.

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Figure 6. Docking models of honokiol within the active sites of human cholinesterase enzymes. Honokiol was docked 54 into the active sites of hAChE (PDB ID: 4EY7) and hBuChE 55 (PDB ID: 4TPK) using the Schrödinger molecular modelling suite. (A) Top view of the hAChE binding pocket, showing bound honokiol as a green stick-structure; (B) Proposed binding interactions of honokiol with hAChE: H-bonding with the amide backbone of Phe295 and π-π stacking with Phe338 and Tyr341; (C) Top view of the hBuChE binding pocket, showing bound honokiol as a green stick-structure; (D) Proposed binding interactions of honokiol with hBuChE: H-bonding with Glh197 and the amide backbone of His438, and π-π stacking with Trp82.

Table 1. Inhibition of ChEs, and radical scavenging activities of honokiol and reference compounds. Compound

EeAChE-Inhibition IC50 (µM)

eqBuChE-Inhibition IC50 (µM)

Stoichiometry and reactivity with b the DPPH radical Stoichiometric c factor (n)

Honokiol Resveratrol EGCG Rivastigmine Donepezil Tacrine

87.0 ± 2.6 133.4 ± 2.8 121.2 ± 6.1 73.9 ± 3.2 0.116 ± 0.008 0.081 ± 0.009

107.3 ± 0.1 > 250 > 250 0.414 ± 0.011 7.15 ± 0.37 0.015 ± 0.0001

a

2.9 ± 0.0 25 2.8 ± 0.1 25 14.8 ± 0.3 nd nd nd

Bimolecular rate constant -1 -1 d kb (M s ) 49.4 ± 3.9 25 91.4 ± 3.8 25 3569 ± 249 nd nd nd

a

The phenols resveratrol and EGCG were included for comparison with honokiol. The cholinesterase inhibitor drugs rivastigmine, donepezil, and tacrine were included as positive control reference compounds. Values are the mean ± SEM of three independent experiments. b Methanol was used as the solvent. c Number of DPPH molecules scavenged by one molecule of antioxidant in 24 h. d 2 A plot of (dA/dt)0 vs A0[AO] gives the overall bimolecular rate constant (kb) as the gradient of the linear fit; r values of plots of (dA/dt)0 vs A0[AO] were between 0.97 and 0.99 (Figure S14). nd- Not determined

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Scavenging of DPPH radicals. Oxidative stress may cause neuronal death in AD, hence antioxidants may be beneficial as disease-modifying drugs.21, 25 The radical scavenging potential of honokiol11 was studied by monitoring its ability to reduce the deep violet DPPH free radical to yellow DPPH-H. Two parameters were considered: a) the stoichiometric factor (n), which is the number of DPPH radicals scavenged by one molecule of antioxidant; and b) the reactivity, quantified by the bimolecular rate constant (kb) for the reaction between the antioxidant and DPPH (Table 1). Each molecule of honokiol scavenged 2.9 DPPH radicals. Honokiol contains two phenolic hydroxyl groups, which are a source of transferrable hydrogen atoms, hence honokiol would be expected to scavenge two DPPH radicals. The ability of honokiol to scavenge one more DPPH radical than expected, could possibly be attributed to a previously described solvent effect, where the methanol solvent can participate in nucleophilic addition to the oxidized forms of the antioxidant.56 The reference phenols, resveratrol and EGCG, were previously shown to scavenge 2.8 and 14.8 DPPH radicals, respectively.25 Honokiol and resveratrol25 displayed similar, moderate reactivity, as illustrated by the bleaching of DPPH absorbance at 515 nm over time (Figure S15) and quantified by the kb values (Table 1). In contrast, EGCG is a highly reactive radical scavenger with a kb value of 3569 M-1s-1.25 Metal chelation activity. Metal chelation therapy is a potential clinical strategy to address metal-associated toxicity in AD.57 The ability of honokiol and resveratrol to chelate iron(II) was measured using the ferrozine assay (Table S1). Compounds with ortho or peri phenolic groups are known to be good chelators of iron,58, 59 however honokiol and resveratrol, which do not contain those substitution patterns, were only weak chelators of iron(II), with honokiol slightly more potent than resveratrol.25 EGCG, which contains multiple ortho phenolic groups, was previously shown to be a strong iron(II) chelator.25 Protection against Aβ42-induced toxicity in C. elegans. The amyloid cascade hypothesis states that accumulation and fibrillation of Aβ in the brain is central to AD pathogenesis.14 To study the protective effect of honokiol, resveratrol and EGCG against Aβ-induced toxicity in vivo, we used the transgenic GMC101 strain of C. elegans in which the expression of full length Aβ42 induces paralysis within 48 h at 25°C.23 Strain CL2122 was used as a transgenic control and does not express Aβ.23, 60 Figure 7 shows the fraction of nematodes not paralysed after 16, 20 and 24 h at 25°C, for untreated strains GMC101 and CL2122, and GMC101 grown on media that was pretreated with 100 µL of 2 mM of honokiol, resveratrol or EGCG. The control strain CL2122 exhibited no paralysis over the time course of the experiment. In contrast, C. elegans GMC101 expressing Aβ42 showed a timedependent paralysis, which was delayed by treatment

with honokiol, resveratrol, or EGCG. The protective effect of EGCG was significant at all three time points, while resveratrol and honokiol exhibited significant activity at only the first or last two time points, respectively. Despite the observed differences in the in vitro activities of honokiol, resveratrol and EGCG which were discussed earlier in this report, the three compounds had similar protective effect against Aβ-induced toxicity in vivo. Thus, further studies may be necessary to clarify the protective mechanisms of these compounds in vivo. EGCG61, 62 and resveratrol63 were previously found to protect against Aβ3-42-induced paralysis in the transgenic C. elegans strain CL2006,64 with EGCG demonstrating inhibition of Aβ oligomerization. EGCG also protects C. elegans against ROS-mediated62 and age-dependent behavioural declines.65 Chemical stability and bioavailability. Chemical stability was assessed by incubating honokiol in phosphate buffer (20 mM, pH 7.4, I 0.17 M containing 0.1% v/v DMSO) for 7 days at 37˚C. The UV-Visible absorbance spectra of honokiol (10 µM, 100 µM, and 1000 µM) did not change over the 7 day incubation period (Figures S17, S18, and S19), which suggests that honokiol is stable under those conditions. Furthermore, RP-HPLC was used to monitor the stability of honokiol, resveratrol and EGCG (100 µM) incubated over 48 h in phosphate buffer (20 mM, pH 7.4, I 0.17 M containing 0.1% v/v DMSO) at 37°C under quiescent conditions. Samples were collected at various time points [0 min (immediately after preparation of sample), 30 min, 1, 2, 4, 6, 8, 10, 12, 18, 24, and 48 h] and an aliquot (10 µL) was injected into the RPHPLC system with the results expressed as the percentage of compound remaining. Honokiol was stable over the entire 48 h incubation period (Figures 8 and S20), signified by a consistent retention time and constant peak area, whereas resveratrol displayed a small amount of degradation after 8 h of incubation (Figures 8 and S21). In contrast, EGCG degraded rapidly, with 55% degradation after 1 h, and complete degradation by 2 h (Figures 8 and S22). EGCG is known to auto-oxidise to oquinones, which polymerise to higher molecular weight compounds.66 These auto-oxidation products may contribute to the observed disruption of Aβ fibrillation, due to interactions with hydrophobic binding sites in amyloid fibrils, and because of the formation of quinoprotein adducts by Michael addition of o-quinones with the amino groups of Aβ lysine residues.35, 43 As demonstrated by the latter mechanism, o-quinones are strong electrophiles which have the potential to react with a variety of nucleophiles in vivo causing toxicity which may hinder the development of EGCG as a drug.66 Furthermore, the inherent instability of EGCG presents a challenge to the development of stable EGCG dosage forms.

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Figure 7. Protection against Aβ-induced paralysis in C. elegans at 25˚C. Values are the mean with SEM from three independent experiments. A) Paralysis curves for untreated strains GMC101 and CL2122, and treated GMC101, grown in Petri dishes (30 mm x 15 mm) containing media that was pre-spotted with 100 μL of OP50 culture alone or in combination with 100 µL of 2 mM honokiol, resveratrol and EGCG. B) Data at 16, 20 and 24 h plotted as bar graphs to illustrate statistical significance, determined by two-way ANOVA, followed by Holm-Sidak’s multiple comparisons test, with * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001, c.f. untreated GMC101.

Ultimately, for honokiol to be developed into a drug for AD, it must be absorbed via the oral route, cross the BBB, and attain concentrations in the brain that can exert a therapeutic effect. The predictive models of Asteris (www.asteris-app.com) were used to estimate the ADME and physicochemical properties of honokiol, resveratrol and EGCG (Table S2). The calculated values indicate that honokiol has drug-like and/or lead-like properties, including: log P, 4.36; MW, 266.3 g/mole; 2 hydrogen bond acceptors and 2 hydrogen bond donors; and topological polar surface area, 40.46 Å2. Honokiol was predicted to have good passive intestinal absorption and the ability to cross the BBB; this is consistent with experimental data which shows that oral honokiol prevents oxidative stress in the mouse brain7 and inhibits subcutaneous tumor growth in mice.67 Furthermore, intravenous honokiol protects the rat brain from cerebral ischemia-reperfusion injury8 and inhibits brain tumor growth in rats.13 Intrave-

nous administration of 20 mg/kg (75 µmol/kg) of honokiol to rats, resulted in honokiol levels in brain tissue of 44.9, 23.0, 10.1, and 5.52 nmol/g at 5, 30, 60, and 120 min, respectively13 Despite these promising results, a limitation of honokiol is low aqueous solubility, although apparently this can be improved using drug delivery systems.68, 69 In comparison, resveratrol had similar calculated properties to honokiol, including predicted good passive intestinal absorption, however it was predicted to not cross the BBB. Indeed, oral resveratrol is well absorbed in humans, however extensive metabolism results in poor systemic availability.70 Intravenous administration of 15 mg/kg (65 µmol/kg) of resveratrol to rats, resulted in brain tissue levels of 0.17 nmol/g of resveratrol and 0.04 nmol/g of resveratrol sulfate at 90 min.71 The calculated properties of EGCG indicates that it is not drug-like, with poor passive intestinal absorption and an inability to cross the BBB predicted by Asteris. This is

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supported by experimental data showing poor systemic and brain availability of oral EGCG in humans and rats.72, 73 Intravenous administration of 50 mg/kg (109 µmol/kg) of EGCG to rats, resulted in an EGCG level in the cortex of 0.014 nmol/g at 15 min.74 A 500 mg/kg (1090 µmol/kg) oral dose of EGCG resulted in 0.5 nmol/g at 60 min in the brain tissue of rats.73 Hence, although honokiol was less potent than EGCG in some of the assays discussed above, the superior stability, drug-likeness, and brain availability of honokiol is an advantage for drug development.

Figure 8. Time course showing the chemical stability of 100 μM of honokiol, resveratrol and EGCG incubated over 48 h in phosphate buffer (20 mM, pH 7.4, I 0.17 M containing 0.1% v/v DMSO) at 37°C under quiescent conditions.

CONCLUSIONS In conclusion, honokiol had similar in vivo potency to resveratrol and EGCG by protecting against Aβ42-induced toxicity in transgenic C. elegans. Furthermore, honokiol exhibited moderate-to-weak in vitro activity in terms of inhibition of Aβ42 on-pathway aggregation, inhibition of cholinesterases, scavenging of DPPH radicals, and chelation of iron(II). Awareness of these activities might facilitate the interpretation of other studies involving honokiol, and the synthesis of honokiol analogues to optimise these properties should be considered. Furthermore, these results in conjunction with other known neuroprotective mechanisms of honokiol7, 8, 10 and its good chemical stability and brain availability,13 makes honokiol a promising agent with potential for development into a disease-modifying drug for AD.

METHODS General. All experiments were conducted at room temperature (23˚C) unless otherwise specified. Commercial chemicals and reagents of at least analytical grade were used. Type 1 ultrapure water was used throughout the study. Acetylthiocholine iodide (ATCI) (T3516), dithionitrobenzoic acid (DTNB) (E4143), acetylcholinesterase from Electrophorus electricus (EeAChE) (C3389), butyrylcholinesterase from equine serum (eqBuChE) (C7512), bovine serum albumin (BSA) (A7906), 2,2-diphenyl-1-

picrylhydrazyl (DPPH) (D9132), (−)-epigallocatechin gallate (EGCG) (E4143), ferrozine (160601), honokiol (H4914), iron(II) sulfate heptahydrate (215422), resveratrol (R5010), tween 20 (443314), and thioflavin T (ThT) (T3516) were purchased from Sigma-Aldrich. Aβ42 with purity > 95% was purchased from Mimotopes and stored at –80°C. ProteoStat® Protein aggregation assay kit (ENZ51023-KP002) was purchased from Enzo Life Sciences. Methanol (HPLC grade) was purchased from Burdick and Jackson. Protein LoBind Eppendorf tubes were used to minimize Aβ42 binding to surfaces. Copper grids, 200 mesh square for transmission electron microscopy (TEM), were purchased from ProSciTech. Bacteriological peptone (LP0037), yeast extract (LP0021), and tryptone (LP0042) were purchased from Thermo Fisher Scientific. Antibodies used in the dot blot assay included: rabbit (polyclonal) anti-oligomer antibody (A11) (AB_2536236, AHB0052, Thermo Fisher Scientific); mouse (monoclonal) Aβ1-16 antibody (6E10) (AB_10175637, SIG-39300-1000, Covance); goat anti-rabbit IgG, (H+L), horseradish peroxidase conjugated (AB_228341, 31460, Thermo Fisher Scientific); and goat anti-mouse IgG (H+L), horseradish peroxidase conjugated (AB_228307, 31430, Thermo Fisher Scientific). Blocker™ Casein in PBS (37528) was obtained from Thermo Fisher Scientific. Clarity™ Western ECL Substrate (170-5060) was purchased from Bio-Rad. Amersham Protran 0.2 μm nitrocellulose blotting membrane (10600001) was purchased from GE Healthcare. Buffers were prepared by reference to ChemBuddy Buffer Maker software (version 1.0.1.55), using: disodium hydrogen phosphate (S9763) and sodium dihydrogen phosphate dihydrate (175698) (Sigma-Aldrich); HEPES (22278) and HEPES sodium salt (151376) (Sigma-Aldrich); or tris HCl (84470) and tris base (6257) (Amresco). The buffer ionic strength (I) was adjusted with NaCl for use at 37°C. Solution pH was monitored with an Orion™ double junction refillable combination pH electrode with glass body semimicro tip (9110DJWP) linked to a Navi Horiba F51 pH meter. All the buffer solutions were filtered through a 0.45 µm regenerated cellulose membrane before use. Corning polystyrene assay plates (96 well clear flat bottom polystyrene microplates) were used for metal chelation and antioxidant studies. For the ThT and ProteoStat® assays, Corning non-binding surface assay plates (96 well half area black with clear flat bottom polystyrene NBSTM microplate) were used. To minimise evaporation, the plates were sealed with Corning non-sterile aluminium sealing tape. FI was measured using a BMG FLUOstar Omega microplate reader. Absorbance was measured using a BMG SPECTROstar Nano microplate reader. Regression and statistical analysis was performed using GraphPad Prism 6 (Version 6.05). Thioflavin T assay. The method used was based on our published protocols.25 The final concentrations of test compounds were: honokiol, 10, 100, and 1000 µM;

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resveratrol, 100 µM; and EGCG 100 µM. DMSO (final concentration 0.1% v/v) was present in honokiol and resveratrol samples as a cosolvent to ensure complete dissolution. Amplitude and half-life values are reported as the mean with SEM of three independent experiments, and statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. ProteoStat® assay. ProteoStat® solutions were handled and stored away from light to prevent photobleaching. The manufacturer’s ProteoStat® stock solution (3.6 µL) was diluted with the manufacturer’s 10× buffer (7.2 µL) and water (61.2 µL). This solution was distributed into aliquots of 6.5 µL and stored in low-binding microtubes wrapped in foil at -80 oC until required. On the day of assay, solutions of test compounds, and low molecular weight (LMW) Aβ42 solution rich in monomers and small oligomers, were prepared in the same manner as for the ThT assay, according to our published protocol.25 Before plating, solutions were mixed in Eppendorf tubes in the following order of addition in equal parts: 1) test compound in phosphate buffer (20 mM, pH 7.4, I 0.225 M) containing 0.3% v/v DMSO; 2) phosphate buffer (20 mM, pH 7.4, I 0.225 M); and 3) Aβ42 in phosphate buffer (20 mM, pH 7.4). The final concentration of components were: 10-1000 µM test compound; and 27 µM Aβ42; in phosphate buffer (20.0 mM, pH 7.4, I 0.17 M) with DMSO 0.1% v/v. A ProteoStat® control was prepared using only phosphate buffer (20.0 mM, pH 7.4, I 0.17 M). An Aβ42 control was prepared by replacing the test compound solution with phosphate buffer (20 mM, pH 7.4, I 0.225 M). The tubes were placed on ice to inhibit fibrillation, and their contents were transferred to the wells of a microplate such that each well contained a total sample volume of 90 µL. The plate was sealed with aluminium sealing tape, inserted into a microplate reader, and incubated at 37 oC for 21 h without recording FI. Near the end of the incubation period, an aliquot of ProteoStat® solution (6.5 µL) was thawed, and mixed with 13.1 µL of phosphate buffer (20.0 mM, pH 7.4, I 0.17 M) to obtain a ProteoStat® working solution of 19.6 µL. At the endpoint of incubation, the sealing tape was carefully removed from the microplate, and ProteoStat® working solution (2 µL) was added to the wells. The plate was resealed with aluminium sealing tape, inserted into the microplate reader, and FI was recorded (λex 544 nm, λem 590 nm). The extent of fibrillation was estimated using equation 1, % Fibrillation =

FI − FI × 100 FI − FI

where FI is the fluorescence intensity of the sample or control, and PS is ProteoStat®. Dot blot assay. LMW Aβ42 rich in monomers and small oligomers was prepared in the same manner as for the ThT assay, according to our published protocol.25 The following method was adapted from the method of

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Kayed et al.37 One part of test compound in phosphate buffer (20 mM, pH 7.4, I 0.225 M) and 1 part of buffer, were added to an Eppendorf tube, followed by the addition of 1 part of LMW Aβ42 stock solution [81 µM in phosphate buffer (20 mM, pH 7.4)]. The final concentrations of components were: test compounds, 10 or 100 or 1000 µM; and Aβ42, 27 µM; in phosphate buffer (20 mM, pH 7.4, I 0.17 M). DMSO (final concentration 0.1% v/v) was present in honokiol and resveratrol samples as a cosolvent to ensure complete dissolution. The resulting solutions were incubated at 37°C in a water bath for 7 days. Two μL of each sample was applied to a nitrocellulose membrane at the following time intervals: day 0 (immediately after preparation of the samples); day 1; day 3; day 5; and day 7. Two sets of nitrocellulose membrane were prepared. The non-specific sites were blocked with casein blocking solution, at room temperature for 1 h. One membrane was incubated overnight at 4˚C with anti-oligomer antibody [A11-antibody, diluted 1:1000 in 3% BSA in tris buffer (100 mM, pH 7.4, I 0.087 M) containing 0.001% Tween 20 (TBS-T)]. The membrane was washed three times for 5 min each with TBS-T, incubated with the secondary antibody [horseradish peroxidase-conjugated anti-rabbit IgG [diluted 1:10,000 in 1% BSA/TBS-T)] for 1 h at room temperature, and further washed three times for 5 min each with TBS-T. The second membrane was incubated overnight at 4˚C with Aβ 6E10 antibody (diluted 1:1000 in 3% BSA/TBS-T). The membrane was washed three times for 5 min each with TBS-T, incubated with the secondary antibody, horseradish peroxidase-conjugated anti-mouse IgG (diluted 1:10,000 in 1% BSA/TBST) for 1 h at room temperature and washed. Membranes were developed with ECL chemiluminescence reagents and visualized using a BioRad ChemiDoc™ Touch Imaging System. Molecular docking studies with Aβ. Molecular modelling simulations of test compounds were performed using Maestro (version 9.8), implemented from the Schrödinger Molecular Modelling Suite-2014. Honokiol, resveratrol, and EGCG were sketched in 3D format using the build panel of Maestro and energy minimized using OPLS-2005 force field to produce low-energy conformers. Aβ42 monomer (PDB ID: 1IYT),45 Aβ16-21 (KLVFFA) fibre (PDB ID: 3OVJ),46 and monomorphic Aβ42 amyloid fibrils (PDB ID: 5KK3)47 were obtained from the RCSB Protein Data Bank (PDB; www.rcsb.org). Protein preparation was performed on the raw PBD protein structure by giving preliminary treatment including adding hydrogen atoms and finally minimized by using OPLS-2005 force field. The grids for docking simulations were generated using the structural coordinates of the whole protein molecule, for Aβ16-21 (KLVFFA) fibre and monomorphic Aβ42 amyloid fibrils, or using the region spanning residues 5-36, for Aβ42 monomer. Molecules were docked using Glide module in extra-precision (XP) mode, with up to ten poses saved per molecule. The ligands were kept flexible, whereas, the protein was kept rigid throughout the docking stud-

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ies. The lowest energy conformations were selected and the ligand interactions with the target protein were determined. AChE/BuChE inhibitory assay. AChE and BuChE activities were estimated using a modified Ellman’s spectrophotometric method. Assay solutions were prepared in Tris buffer

(100 mM, pH 7.4) unless indicated otherwise. Tris buffer containing NaCl (100 mM) and MgCl2 (20 mM) was used for the preparation of dithionitrobenzoic acid (DTNB, 3.0 mM) stock solution. Acetylthiocholine iodide (ATCI, 9 mM) stock solution was prepared in water. Stock solutions of EeAChE (0.15 U/mL) and eqBuChE (0.15 U/mL) were prepared in tris buffer. Compounds were dissolved in a small volume of DMSO then diluted with Tris buffer such that the final concentration of DMSO was 0.3% v/v. To a half-area 96-well plate, the following components were added: 20 µL of test compound; 60 µL of DTNB; and 20 µL of EeAChE or eqBuChE. The plate was incubated at room temperature for 30 min, then the absorbance was measured at 415 nm every 1 min for 10 min. Then 20 µL of ATCI was added, and again the absorbance was measured at 415 nm every 1 min for 10 min. The final concentrations of the components were: DTNB (1.5 mM), enzyme (0.025 U/mL); and ATCI (1.5 mM); with the concentration of compound varied to attain a suitable concentration-response plot. The reaction velocity (V) was obtained by fitting equation 2, At = Vt + A0, to a plot of absorbance (A) versus time (t) by linear regression. The % inhibition at each concentration was estimated by comparing the velocities for the sample to the blank, using following equation 3, (&' − &( ) *+ % Inhibition = 100 − $ . 100 (&' − &( ) ,-

where: V0 is the reaction velocity calculated before addition of ATCI; and V1 is the reaction velocity after addition of ATCI. The IC50 of the compound was calculated by fitting a sigmoidal dose-response (variable slope) equation to a plot of % inhibition versus log compound concentration by non-linear regression. Three independent experiments were conducted. Molecular docking studies with AChE/BuChE. Molecular modelling simulations of honokiol and reference molecules were performed using Maestro (version 9.8), implemented from the Schrödinger Molecular Modelling Suite-2014. All molecules were sketched in 3D format using the build panel of Maestro and energy minimized using OPLS-2005 force field to produce low-energy conformers. The crystal structures of hAChE and hBuChE were taken from the PBD with PDB IDs 4EY754 and 4TPK,55 respectively. Raw PBD protein structures were prepared by giving preliminary treatment including adding hydrogen atoms and finally minimized by using OPLS2005 force field. The grids for docking simulations were generated using the structural coordinates of bound co-

crystallized molecules (donepezil in 4EY7 and N-((1-(2,3dihydro-1H-inden-2-yl)piperidin-3-yl)methyl)-N-(2methoxyethyl)-2-naphthamide in 4TPK).54, 55 Molecules were docked using Glide module in extra-precision (XP) mode, with up to three poses saved per molecule. The ligands were kept flexible, whereas, the receptor was kept rigid throughout the docking studies. The lowest energy conformations were selected and the ligand interactions with target proteins were determined. DPPH radical scavenging assay. The DPPH assays were 25 performed according to our published method. Solutions

of honokiol were prepared in methanol immediately prior to use. The stoichiometric factor (n) was estimated using a final concentration of 20 µM honokiol. The reactivity, quantified by the bimolecular rate constant (kb), was estimated using the following final concentrations of honokiol: 10, 25, 50, 75, and 100 µM. Three independent experiments were conducted. Chelation of iron (II). The method used was based on our published ferrozine assay protocol.25 Honokiol, resveratrol, or EDTA were incubated with iron(II) solution for 30 min to allow complex formation, then ferrozine was added to determine the concentration free iron(II). The final concentrations of test compounds were: honokiol, 0, 25, 50, 100, 125, 250, 500, 750, and 1000 μM; resveratrol, 0, 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 μM; and EDTA, 0, 0.01, 0.1, 1, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25 and 30 µM. DMSO (final concentration 0.1% v/v) was present in honokiol samples as a cosolvent to ensure complete dissolution. Ferrozine reacts rapidly with free iron (Figure S16) to form a watersoluble purple complex with absorbance maximum at 560 nm. The % chelation was estimated using equation 4, % Chelation = 11 −

23(44)567897:; 100 23(44)52= − 23(44)

where: A is the absorbance at 560 nm of a solution containing one or more of the following solutes: Fe(II); honokiol, resveratrol, or EDTA (compound); and ferrozine (FZ, indicator ligand). The concentration of compound for 50% chelation was estimated by fitting an exponential one-phase association equation by nonlinear regression to a plot of chelation (%) versus compound concentration. The 50% chelation values are reported as the mean ± SEM of three independent experiments. Paralysis assay. The paralysis assay was performed on nematode growth agar (NGA) media plates spotted with E. coli strain OP50 grown in Luria broth. NGA solution containing 3.0 g NaCl, 2.5 g peptone and 17.0 g agar in 975 mL of double distilled water was autoclaved, followed by the addition of 25.0 mL of 1 M K2PO4, 1.0 mL of 1 M MgSO4, 1.0 mL of 1 M CaCl2 and 1.0 mL of cholesterol (5 mg/mL in absolute ethanol). An aliquot (4.0 mL) of liquid NGA was transferred into each 30 mm x 15 mm Petri dish, and allowed to solidify overnight at room

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temperature. Luria broth was made by dissolving 5.0 g NaCl, 5.0 g yeast extract and 10.0 g tryptone in 1.0 L of double distilled water. E. coli strain OP50 was grown with shaking in LB media overnight. Each plate containing solidified NGA was spotted with 100 μL of OP50 culture and allowed to dry overnight at room temperature. Plates for treatment groups were then spotted with 100 µL of 2 mM of honokiol, resveratrol or EGCG in 0.1% v/v DMSO/water, and allowed to dry overnight at room temperature. Strain GMC101, dvIs100 [pCL354(unc54:DA-Aß1-42) + pCL26 (mtl-2:GFP)] was used to study Aβ42-induced paralysis, while strain CL2122, dvIs15(mtl2:GFP) was used as transgenic control. Eggs were transferred to the plates followed by incubation for 72 h at 20°C. The plates were then incubated at 25°C, a temperature at which Aβ42 production increases, and observed for the protective effect of honokiol, resveratrol, and EGCG at 16 h, 20 h, and 24 h. Nematodes that did not move, or only moved the head, or failed to complete full body movement (i.e. a point of inflection traversing the entire body length), under a gentle touch with a loop were scored as paralyzed.23 The investigator (S. Kantham) was blinded to the treatment conditions and the scores were verified by another blinded investigator (S.C.). Results were reported as the mean with SEM based on data from three independent experiments. Statistical significance was determined by two-way ANOVA (factors: time and treatment) followed by Holm-Sidak’s multiple comparisons test. The number of nematodes per group in each experiment was (group, experiment 1, experiment 2, experiment 3): CL2122, 230, 119, 138; GMC101, 98, 169, 411; GMC101 + Honokiol, 43, 59, 98; GMC101 + Resveratrol, 47, 68, 86; and GMC101 + EGCG, 11, 65, 61. Strain CL2122 was also grown on media that was treated with honokiol, resveratrol or EGCG (as described above), and no paralysis was observed during the incubation period (data not shown). All nematodes of strain GMC101 (untreated and treated with compounds) eventually progressed to complete paralysis (data not shown). Stability studies. Honokiol, resveratrol and EGCG (100 µM) were incubated over 48 h in phosphate buffer (20 mM, pH 7.4, I 0.17 M containing 0.1% v/v DMSO) at 37°C under quiescent conditions. Samples were collected at various time points [0 min (immediately after preparation of sample), 30 min, 1, 2, 4, 6, 8, 10, 12, 18, 24, and 48 h] and an aliquot (10 µL) was analysed on a ShimadzuNexera-i LC-2040C-3D HPLC system connected to a photodiode array (PDA) detector. An Agilent ZORBAX SB-C18 column was used as the stationary phase (see Table S3 for column specifications) and the PDA was set to record a wavelength range of 200-400 nm with the absorbance at 280 nm used in the final analysis. As shown in Table S3, a gradient elution was applied by varying the proportion of solvent from A to B with a flow rate of 1 mL/min. The percentage of compound remaining (CR) was calculated using equation 5, CR = (At/A0)*100%, where At and

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A0 are the RP-HPLC peak areas at time t and time 0, respectively.

ASSOCIATED CONTENT The Supporting Information associated with this article is available free of charge on the ACS Publications website at DOI: 10.xx. Graph showing half-life of Aβ42 fibrillation monitored by in situ ThT fluorescence. TEM images showing the morphology of Aβ42 aggregates in the presence of 0.1% v/v DMSO. 2D interactions diagrams for the compounds with: Aβ42 monomer; Aβ16-21 (KLVFFA) fibre; monomorphic Aβ42 amyloid fibrils; hAChE; and hBuChE. Plots of (dA/dt)0 vs A0[AO] for honokiol. Absorbance-time plots of the bleaching of DPPH by honokiol and resveratrol. Absorbancetime plot showing the rapid reaction of ferrozine with Fe(II). Absorbance spectra of honokiol incubated in phosphate buffer at 37˚C for 7 days. Iron(II) chelation activities of honokiol and reference compounds. Physicochemical properties of the compounds predicted in silico by Asteris. Description of the RP-HPLC analytical method. RP-HPLC chromatograms of the compounds incubated over 48 h in phosphate buffer at 37°C. Additional references.

AUTHOR INFORMATION Corresponding Author *Benjamin P. Ross. Tel.: +61 7 33461900; fax: +61 7 33461999; email: [email protected]

Author Contributions S. Kantham and B.P.R. designed the study. S. Kantham carried out molecular docking studies, paralysis studies in C. elegans, chemical stability, ThT assay, dot-blot assay, cholinesterase inhibition assay, DPPH radical scavenging assay and the metal chelation assay. S.C. acquired the TEM images. G.M. contributed to the paralysis studies in C. elegans. J.A.M. and S.N.D. contributed to the cholinesterase inhibition assay. S.K.V. conducted ProteoStat® assay. G.S.D. contributed to the molecular docking studies. S. Khabbazi and M.O.P. contributed to the dot blot assay. S. Kantham and B.P.R. wrote the manuscript. All authors read and approved the final manuscript.

Funding Sources Funding for this work was provided by The University of Queensland.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS S. Kantham thanks the Australian Government – Endeavour Fellowships and Scholarships Program, and UQ, for PhD scholarships. S.C., J.A.M., S.K.V., G.S.D., S.N.D., and S. Khabbazi thank UQ for PhD scholarships. The authors thank Dr. Kathryn Green and Rick Webb for assistance with TEM. Some C. elegans strains where provided by the Caenorhabditis Genetics Center (CGC), supported by the US National Institutes of Health-Office of Research Infrastructure Programs (P40 OD010440). The authors acknowledge the facilities, and the scientific and technical assistance, of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy and Microanalysis, UQ.

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ABBREVIATIONS AD, Alzheimer’s disease; Aβ, amyloid beta peptide; AChE, acetylcholinesterase; ADME, absorption, distribution, metabolism, and excretion; A11, anti-oligomer antibody; ATCI, acetylthiocholine iodide; BBB, blood-brain barrier; BuChE, butyrylcholinesterase; 6E10, beta amyloid (Aβ) 1-16 antibody; CNS, central nervous system; ChE, cholinesterase; DPPH, 2,2-diphenyl-1-picrylhydrazyl; DTNB, dithionitrobenzoic acid; EGCG, (−)-epigallocatechin gallate; EeAChE, Electrophorus electricus AChE; eqBuChE, butyrylcholinesterase from equine serum; LWM, low molecular weight; NMDA, N-methyl-D-aspartate; PDB, protein data bank; ThT, thioflavin T; and TEM, transmission electron microscopy.

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Figure 1. The chemical structures of honokiol, (-)-epigallocatechin gallate (EGCG), and resveratrol. 52x17mm (600 x 600 DPI)



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Figure 2. A) Aβ42 fibril formation monitored by in situ ThT fluorescence. FI readings (λex 440 nm, λem 480 nm) were recorded every 5 min. The graph shows representative kinetic plots, corrected for the fluorescence of ThT alone (20 µM). Aβ42 (27 µM) was incubated with honokiol (10, 100, and 1000 µM), resveratrol (100 µM), or EGCG (100 µM) in phosphate buffer (20 mM, pH 7.4, I 0.17 M, containing 20 µM ThT) at 37˚C under quiescent conditions. Honokiol and resveratrol samples contained DMSO (0.1% v/v). The extent of fibrillation is depicted in B) as percentage of the Aβ42 control amplitude. Values are the mean + SEM of three independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test with: **p < 0.01 and ****p < 0.0001 c.f. Aβ42 + DMSO 0.1%; and ^^^^p < 0.0001 c.f. Aβ42 control. There were no significant differences in half-life between groups (Figure S1). C) Aβ42 fibrillation estimated by ProteoStat® fluorescence. Aβ42 (27 µM) was incubated with honokiol (10, 100, and 1000 µM), resveratrol (10, 100, and 1000 µM) or EGCG (10 and 100 µM) in phosphate buffer (20 mM, pH 7.4, I 0.17 M) at 37˚C under quiescent conditions. Honokiol, resveratrol, and EGCG samples contained DMSO (0.1% v/v). After 21 h, ProteoStat® dye was added and FI was recorded (λex 544 nm,


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λem 590 nm). The extent of fibrillation is depicted as percentage of the Aβ42 control amplitude. Values are the mean + SEM of three independent experiments with *p < 0.05 and ****p < 0.0001 c.f. Aβ42 + DMSO 0.1%, determined by one-way ANOVA followed by Tukey’s multiple comparisons test. 169x529mm (300 x 300 DPI)



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Figure 3. Dot blot assay showing Aβ42 oligomer levels over 7 days. Aβ42 (27 µM) was incubated with resveratrol (100 µM), honokiol (10, 100, and 1000 µM), or EGCG (100 µM), in phosphate buffer (20 mM, pH 7.4, I 0.17 M) at 37˚C under quiescent conditions. Honokiol and resveratrol samples contained DMSO (0.1% v/v). Aliquots were removed at various time points, spotted onto nitrocellulose membranes, and probed with: (A) oligomer-specific A11 antibody; and (B) 6E10 antibody that recognizes all forms of Aβ via its residues 3-8. 106x142mm (600 x 600 DPI)


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Figure 4. Effect of honokiol, resveratrol and EGCG on the morphology of Aβ42 aggregates. Aβ42 (27 µM) was incubated with honokiol (1000 µM), resveratrol (100 µM) and EGCG (100 µM) in phosphate buffer (20 mM, pH 7.4, I 0.17 M) at 37˚C under quiescent conditions. TEM images of: (A) Aβ42 control on day 1; (B) Aβ42 control on day 7; (C) Aβ42 + honokiol on day 1; (D) Aβ42 + honokiol on day 7; (E) Aβ42 + resveratrol on day 1; (F) Aβ42 + resveratrol on day 7; (G) Aβ42 + EGCG on day 1; (H) Aβ42 + EGCG on day 7. Images of Aβ42 + 0.1% v/v DMSO (Figure S2) were similar to (A). The scale bar represents 500 nm. 141x286mm (300 x 300 DPI)



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Figure 5. Docking model of honokiol, resveratrol and EGCG with Aβ42 monomer (PDB ID: 1IYT),45 Aβ16-21 (KLVFFA) fibre (PDB ID: 3OVJ),46 and monomorphic Aβ42 amyloid fibrils (PDB ID: 5KK3)47 developed using the Schrödinger molecular modelling suite. Colour scheme: yellow, honokiol; orange, resveratrol; and green, EGCG. A) Proposed binding interactions of honokiol with Aβ42 monomer: H-bonding (dashed line) with Lys16 and Asp23; B) Proposed binding interactions of resveratrol with Aβ42 monomer: H-bonding with Lys16 and Asp23; C) Proposed binding interactions of EGCG with Aβ42 monomer: H-bonding with Lys16, Asp23 and Gln15; D) Proposed binding interactions of honokiol with Aβ16-21 (KLVFFA) fibre: H-bonding with Lys16; E) Proposed binding interactions of resveratrol with Aβ16-21 (KLVFFA) fibre: H-bonding with the amide backbone of Phe19, and π-π stacking with two Phe20 residues; F) Proposed binding interactions of EGCG with Aβ16-21 (KLVFFA) fibre: H-bonding with Lys16 and the amide backbone of residues Leu17 and Phe19; G) Proposed binding interactions of honokiol with monomorphic Aβ42 amyloid fibrils: H-bonding with Lys16 and Ala21; H) Proposed binding interactions of resveratrol with monomorphic Aβ42 amyloid fibrils: Hbonding with Lys16 and Glu22; and I) Proposed binding interactions of EGCG with monomorphic Aβ42 amyloid fibrils: H-bonding with Lys16, Ala21 and Glu22. 169x158mm (300 x 300 DPI)


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Figure 6. Docking models of honokiol within the active sites of human cholinesterase enzymes. Honokiol was docked into the active sites of hAChE (PDB ID: 4EY7)54 and hBuChE (PDB ID: 4TPK)55 using the Schrödinger molecular modelling suite. (A) Top view of the hAChE binding pocket, showing bound honokiol as a green stick-structure; (B) Proposed binding interactions of honokiol with hAChE: H-bonding with the amide backbone of Phe295 and π-π stacking with Phe338 and Tyr341; (C) Top view of the hBuChE binding pocket, showing bound honokiol as a green stick-structure; (D) Proposed binding interactions of honokiol with hBuChE: H-bonding with Glh197 and the amide backbone of His438, and π-π stacking with Trp82. 142x120mm (300 x 300 DPI)



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Figure 7. Protection against Aβ-induced paralysis in C. elegans at 25˚C. Values are the mean with SEM from three independent experiments. A) Paralysis curves for untreated strains GMC101 and CL2122, and treated GMC101, grown in Petri dishes (30 mm x 15 mm) containing media that was pre-spotted with 100 µL of OP50 culture alone or in combination with 100 µL of 2 mM honokiol, resveratrol and EGCG. B) Data at 16, 20 and 24 h plotted as bar graphs to illustrate statistical significance, determined by two-way ANOVA, followed by Holm-Sidak’s multiple comparisons test, with * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001, c.f. untreated GMC101. 129x129mm (300 x 300 DPI)


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Figure 8. Time course showing the chemical stability of 100 µM of honokiol, resveratrol and EGCG incubated over 48 h in phosphate buffer (20 mM, pH 7.4, I 0.225 M containing 0.1% v/v DMSO) at 37°C under quiescent conditions. 53x35mm (600 x 600 DPI)



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TOC graphic 80x26mm (300 x 300 DPI)


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Effect of the Biphenyl Neolignan Honokiol on AÎ²42-Induced Toxicity in

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