Inhibitory Activity of Curcumin Derivatives Towards Metal-Free and Metal-Induced Amyloid-β Aggregation - PDF

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Send rders for Reprints to Current Alzheimer Research, 25, 2, Inhibitory Activity of Curcumin Derivatives Towards Metal-Free and Metal-Induced Amyloid-β Aggregation Akiko Kochi,2, Hyuck Jin Lee,2, Sashiprabha M. Vithanarachchi 3, Vediappen Padmini 4, Matthew J. Allen 3, * and Mi Hee Lim,5, * Department of Chemistry, Ulsan ational Institute of Science and Technology (UIST), Ulsan , Korea; 2 Department of Chemistry, University of Michigan, Ann Arbor, MI 489, USA; 3 Department of Chemistry, Wayne State University, Detroit, MI 4822, USA; 4 Department of rganic Chemistry, Madurai Kamaraj University, Madurai-2.Tamil nadu, India; 5 Life Sciences Institute, University of Michigan, Ann Arbor, MI 489, USA Abstract: When Alzheimer s disease (AD) progresses, several pathological features arise including accumulation of misfolded protein aggregates [e.g., amyloid-β (Aβ) plaques], metal ion dyshomeostasis, and oxidative stress. These characteristics are recently suggested to be interconnected through a potential factor, metal-associated Aβ (metal Aβ) species. The role of metal Aβ species in AD pathogenesis remains unclear, however. To elucidate the contribution of metal Aβ species to AD pathology, as well as to develop small molecules as chemical tools and/or theranostic (therapeutic and diagnostic) agents for this disease, curcumin (Cur), a natural product from turmeric, and its derivatives have been studied towards both metal-free and metal-induced Aβ aggregation. Although Cur has indicated anti-amyloidogenic activities and antioxidant properties, its biological use has been hindered due to low solubility and stability in physiologically relevant conditions. Herein, we report the reactivity of Cur and its derivatives (Gd-Cur, a potential multimodal Aβ imaging agent; Cur-S, a water soluble derivative of Cur that has substitution at the phenolic hydroxyls) with metal-free Aβ and metal Aβ species. ur results and observations indicate that Gd-Cur could modulate Cu(II)-triggered Aβ aggregation more noticeably over metal-free or Zn(II)-induced analogues; however, Cur-S was not observed to noticeably modulate Aβ aggregation with and without metal ions. verall, our studies present information that could aid in optimizing the molecular scaffold of Cur for the development of chemical tools or theranostics for metal Aβ species. Keywords: Alzheimer s disease, amyloid-β, metal-associated Aβ, Aβ aggregation control, water-soluble curcumin derivatives, chemical reagents. ITRDUCTI Alzheimer s disease (AD) is the most common, incurable neurodegenerative disease and is currently projected to affect 3.8 million Americans by 25 []. Several pathological features are thought to be related to AD development, including accumulation of amyloid-β (Aβ) peptide aggregates, metal ion dyshomeostasis, and increased oxidative stress [2-9]. An inter-relationship between these factors has been implied (e.g., metal binding to Aβ has been shown to facilitate peptide aggregation and to generate reactive oxygen species (RS) in vitro), making the etiology of the disease elusive and complex [2-5, 7-9]. These multiple pathological components have been investigated for gaining a fundamental understanding of the disease onset and progression and have been targeted for AD theranostic (diagnostic and therapeutic) purposes. *Address correspondence to these authors at the Department of Chemistry, Ulsan ational Institute of Science and Technology (UIST), Ulsan, , Korea; Tel: ; Fax: ; and Department of Chemistry, Wayne State University, 5 Cass Avenue, Detroit, MI, 4822, USA; Tel: ; Fax: ; To modulate the reactivity (i.e., peptide aggregation and RS scavenging) of the potential factors [specifically, Aβ and metal-associated Aβ (metal Aβ)] that lead to AD pathogenesis, small molecules screened from natural products such as curcumin (Cur; Fig. ) have been explored [-9]. Cur is a naturally occuring phenol in turmeric, a popular Indian spice [2, 2]. Cur has been observed to inhibit the formation of Aβ oligomers and fibrils [, 22], aid in the prevention of neuronal damage [23], and diminish oxidative stress [24-26] and amyloid accumulation [] in an AD transgenic mouse model. Although Cur displays beneficial effects towards AD pathogenic factors (vide supra), its application as a chemical tool or theranostic agent has been limited due to its low solubility and stability in physiological environments [27-29]. Therefore, water-soluble Cur derivatives would be valuable to be investigated for inhibitory reactivity towards metal-free and metal-induced Aβ aggregation. ne of the articles in this thematic issue by Huang and co-worker also reported one kind of Cur derivatives as effective inhibitors of Aβ aggregation [3]. Besides, a number of different types of Aβ aggregation inhibitors have been explored up to now [3], including organic small molecules [32], short peptides, nanoparticles, and metal complexes [33, 34] /5 $ Bentham Science Publishers 46 Current Alzheimer Research, 25, Vol. 2, o. 5 Kochi et al. Gd 2- H CH 3 H t-bu H H CH 3 H Gd H Cur CH 3 H Cur-L CH 3 2- Gd S H H H CH 3 H a CH 3 H Gd-Cur H Fig. (). Chemical structures of curcumin derivatives. Gd: Gd(III)(diethylenetriaminepentaacetate); curcumin (Cur): (E,4Z,6E)-5- hydroxy-,7-bis(4-hydroxy-3-methoxyphenyl)hepta-,4,6-trien-3-one; curcumin-linker (Cur-L): tert-butyl(2-(2-(4-((e,4z,6e)-5-hydroxy- 7-(4-hydroxy-3-methoxyphenyl)-3-oxohepta-,4,6-trien--yl)-2-methoxyphen-oxy)acetoamido)ethyl)carbamate; Gd(III)(DTPA)-conjugated curcumin (Gd-Cur): Gd(III)(diethylenetriaminepentaacetate)-linked-2-(4-((E,4Z,6E)-5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-3- oxohepta-,4,6-trien--yl)-2-methoxyphenoxy)--(2-(3-(p-tolyl)thioureido)ethyl)acetamide; and watersoluble curcumin (Cur-S): 2,2 - ((((E,3Z,6E)-3-hydroxy-5-oxohepta-,3,6-triene-,7-diyl)-bis(2-methoxy-4,-phenylene))bis(oxy))diacetate. CH 3 a Cur-S CH 3 Gd-Cur (Fig. ), prepared by linkage of Gd(III)(DTPA) (Gd; Fig. ; DTPA = diethylenetriamine pentaacetate; a clinically approved contrast agent) with Cur, was reported asa potential multimodal contrast agent to detect Aβ plaques in vitro, applicable to both magnetic resonance imaging (MRI) and fluorescence microscopy [35]. The ability of Gd-Cur to control metal-free and metal-induced Aβ aggregation pathways has not been reported, however. From our results and observations presented herein, together with the previous work [35], Gd-Cur, in comparison to Cur-S, is demonstrated to be a viable molecular scaffold that could be a foundation for the development of a theranostic agent, capable of indicating Aβ plaques and redirecting metal-triggered Aβ aggregation. MATERIAL AD METHDS All reagents were purchased from commercial suppliers and used as received unless otherwise stated. The compounds Gd(III)(diethylenetriaminepentaacetate)-linked-2-(4- ((E,4Z,6E)-5-hydroxy-7-(4-hydroxy-3-methoxy-phenyl)-3- oxohepta-,4,6-trien--yl)-2-methoxy-phenoxy)--(2-(3-(ptolyl)thioureido)ethyl)acetamide (Gd-Cur) [35] and 2,2 - ((((E,3Z,6E)-3-hydroxy-5-oxohepta-,3,6-triene-,7-diyl) bis(2-methoxy-4,-phenylene))bis(oxy))diacetate (Cur-S) [36] were prepared following previously reported methods. Aβ 4 (DAEFRHDSGYEVHHQKLVFFAEDVGSKGAI- IGLMVGGVV) and Aβ 42 (DAEFRHDSGYEVHHQKLVFF AEDVGSKGAIIGLMVGGVVIA) were purchased from AnaSpec (Fremont, CA, USA). All ddh 2 used during experiments was obtained from a Milli-Q Direct 6 system (Merck KGaA, Darmstadt, Germany). ptical spectra were recorded on an Agilent 8453 UV-visible (UV-vis) spectrophotometer. Transmission electron microscopy (TEM) images were recorded with a Philips CM- transmission electron microscope (Microscopy and Image Analysis Laboratory, University of Michigan, Ann Arbor, MI, USA) and a JEL JEM-2 Transmission Electron Microscope (UIST Central Research Facilities, UIST, Ulsan, Korea). A SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA) was employed for measurements of absorbance for the Trolox equivalent antioxidant capacity (TEAC), the MTT (MTT = 3-[4,5-dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide), and the Parallel Artificial Membrane Permeability Assay adapated for bloodbrain barrier (PAMPA-BBB) assays. Inhibitory Activity of Curcumin Derivatives Current Alzheimer Research, 25, Vol. 2, o Amyloid-β (Aβ) Peptide Experiments Aβ experiments were performed according to previously published procedures [5, 37-4]. Aβ 4 or Aβ 42 was dissolved in ammonium hydroxide (H 4 H, % v/v, aq), aliquoted, freeze dried overnight, and stored at 8 C. A stock solution was prepared by dissolving Aβ in % H 4 H followed by dilution with ddh 2. For the inhibition experiment, Aβ (25 µm) was treated with or without metal ions (CuCl 2 or ZnCl 2 ; 25 µm) for 2 min, followed by addition of a compound (5 µm; % v/v final dimethyl sulfoxide (DMS) concentration). The resulting Aβ samples were incubated for 24 h at 37 C with constant agitation. For the disaggregation experiment, Aβ (25 µm) with and without metal ions (CuCl 2 or ZnCl 2, 25 µm) was first incubated for 24 h at 37 C with constant agitation. Afterward, a compound (5 µm, % v/v final DMS concentration) was treated with preincubated Aβ samples. The resulting solution was incubated for 24 h at 37 C with constant agitation. Both studies were performed using a buffered solution [2-[4-(2- hydroxyethyl)piperazin--yl]-ethanesulfonic acid (HEPES) (2 µm), ph 6.6 (for Cu(II) samples) or ph 7.4 (metal-free and Zn(II) samples), acl (5 µm)]. Gel Electrophoresis with Western Blotting The resulting Aβ 4 /Aβ 42 species from both inhibition and disaggregation experiments were analyzed using gel electrophoresis followed by Western blotting with an anti- Aβ antibody (6E, Covance, Princeton, J, USA) [5, 37-4]. Each sample ( µl) was separated using a 2% tris-tricine gel (Invitrogen, Grand Island, Y, USA). The proteins were transferred onto a nitrocellulose membrane and blocked with bovine serum albumin (BSA, 3% w/v, Sigma- Aldrich, St. Louis, M, USA) in tris-buffered saline (TBS) containing.% Tween-2 (TBS-T) for 2 or 3 h at ambient temperature. The membranes were treated with an anti-aβ antibody (6E; :2,) in 2% BSA (w/v in TBS-T) overnight at 4 C. After washing, the membranes were probed with the horseradish peroxidase-conjugated goat anti-mouse antibody (:5,) in 2% BSA for h at ambient temperature. Thermo Scientific Supersignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA) was used to visualize protein bands. Transmission Electron Microscopy (TEM) TEM samples were prepared following a previously reported method [5, 37-4]. Glow-discharged grids (Formar/Carbon 3-mesh, Electron Microscopy Sciences, Hatfield, PA, USA) were treated with the samples (5 µl) from either the inhibition or disaggregation experiment for 2 min at ambient temperature. Excess sample was removed with filter paper. The grids were washed three times with ddh 2, stained with uranyl acetate (% w/v, ddh 2, 5 µl) for min, and dried for 5 min at ambient temperature. Metal Binding Studies The interactions of Cur, Cur-L, Gd-Cur, and Cur-S with Cu(II) and Zn(II) were investigated by UV-vis according to previously reported procedures [5, 37-4]. Solutions of Cur, Cur-L, Cur-S ( µm for Cu(II) binding studies; 2 µm for Zn(II) binding studies), and Gd-Cur ( µm for either Cu(II) or Zn(II) binding studies) in EtH were treated with to 2 equiv of CuCl 2 or ZnCl 2 followed by incubation for 5 min at ambient temperature prior to acquiring spectra. Trolox Equivalent Antioxidant Capacity (TEAC) Assay The antioxidant activity of compounds was determined by the TEAC assay employing cell lysates following the protocol of the antioxidant assay kit purchased from Cayman Chemical Company (Ann Arbor, MI, USA) with modifications [39]. Murine euro-2a (2a) cells were used for this assay. This cell line purchased from ATCC (Manassas, VA, USA) was maintained in media containing 5% Dulbecco s modified Eagle s medium (DMEM) and 5% PTI-MEM (GIBC, Grand Island, Y, USA) supplemented with % fetal bovine serum (FBS, Sigma), % non-essential amino acids (EAA, GIBC), 2 mm glutamine, units/ml penicillin, and mg/ml streptomycin (GIBC). Cells were grown and maintained at 37 C in a humidified atmosphere with 5% C 2. For the antioxidant assay using cell lysates, cells were seeded in a 6 well plate and grown to approximately 8 9% confluence. Cell lysates were prepared according to the previously reported method with modifications [42]. 2a cells were washed once with cold (4 C) phosphate buffered saline (PBS, ph 7.4, GIBC) and harvested by gently pipetting off adherent cells with cold PBS. The resulting PBS solution was collected and the cell pellet was generated by centrifugation (2, g for min at 4 C). Cell pellets were sonicated on ice (5 sec pulses, 5 with 2 sec intervals between each pulse) in 2 ml of cold (4 C) assay buffer (5 mm potassium phosphate, ph 7.4,.9% acl, and.% glucose). Cell lysates were centrifuged at 5, g for min at 4 C. The supernatant was removed and stored on ice until use. To standard and sample in a 96 well plate was added µl of the supernatant of cell lysates followed by compound, metmyoglobin, ABTS (2 2-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid)), and H 2 2 in sequential order. After 5 min incubation at ambient temperature on a shaker, the values of absorbance at 75 nm were recorded. Six concentrations (.45,.9,.35,.8,.225, and.33 mm) of compounds and Trolox (Sigma-Aldrich; Trolox = 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; dissolved in DMS) were used. The percent inhibition was calculated according to the measured absorbance (% inhibition = (A A)/A, where A is the absorbance of the supernatant of cell lysates) and was plotted as a function of compound concentration. The TEAC value of ligands was calculated as a ratio of the slope of the standard curve of the compound to that of Trolox. The measurements were conducted in triplicate. Cell Viability (MTT Assay) The 2a cell line was maintained in media containing 5% DMEM and 5% PTI-MEM (GIBC) supplemented with % FBS (Sigma), % EAA (GIBC), 2 mm glutamine, units/ml penicillin, and mg/ml streptomycin (GIBC). The cells were grown and maintained at 37 C in a humidified atmosphere with 5% C 2. Cell viability upon treatment with compounds was determined using the MTT assay. 2a cells were seeded in a 96 well plate (5, cells in µl per well). Cells were treated with compound only, compound with a metal chloride salt ([compound]:[m(ii)] = 48 Current Alzheimer Research, 25, Vol. 2, o. 5 Kochi et al. (:); [compound] = 2.5, 5,, 25, or 5 µm), or Aβ (2 µm) with or without CuCl 2 or ZnCl 2 (2 µm), followed by the addition of Gd-Cur or Cur-S (2 µm). After 24 h incubation, MTT (25 µl of 5 mg/ml in PBS (ph 7.4, GIBC)) was added to each well, and the plate was incubated for 4 h at 37 C. Formazan produced by the cells was solubilized using an acidic solution of,-dimethylformamide (5%, v/v aq) and sodium dodecyl sulfate (SDS, 2%, w/v) overnight at ambient temperature in the dark. Absorbance was measured at 6 nm using a microplate reader. Viability of cells was calculated relative to that with an equivalent a- mount of a buffer solution [HEPES (2 µm), ph 6.6 (for Cu(II) samples) or ph 7.4 (metal-free and Zn(II) samples), acl (5 µm)]. Parallel Artificial Membrane Permeability Assay Adapted for Blood-Brain Barrier (PAMPA-BBB) Previously reported protocols with modification using the PAMPA Explorer kit (Pion, Inc. Billerica, MA, USA) were applied to our PAMPA-BBB experiment [38-4, 43-45]. Each stock solution of compounds was diluted to a final concentration of µm (% v/v final DMS concentration) with ph 7.4 Prisma HT buffer (Pion). The resulting solution (2 µl) was added to each of the wells of the donor plate (number of replicates per sample = 2). The BBB- lipid (Pion formulation, 5 µl) was used to coat the polyvinylidene fluoride (PVDF,.45 µm) filter membrane on the acceptor plate. The acceptor plate was placed on the top of the donor plate generating a sandwich and each well of the acceptor plate was filled with brain sink buffer (2 µl, Pion). Sandwiches were incubated at ambient temperature for 4 h without stirring. A microplate reader was used to obtain the optical spectra (25 5 nm) of the solutions in the reference, acceptor, and donor plates. The logp e for each compound was calculated using the PAMPA Explorer software c. 3.5 (Pion). CS+/ assignment was determined in comparison to compounds identified previously [43-45]. Compounds categorized as CS+ likely have the ability to permeate through the BBB and be available in the CS. Compounds assigned as CS likely have poor permeability through the BBB and, therefore, their bioavailability into the CS is considered to be minimal. RESULTS Design Rationale As shown in (Fig. ), the conjugate Gd-Cur was developed based on a rational structure-based approach, where a clinically approved MRI contrast agent (Gd) was appended onto Cur [35], a nontoxic natural product that has been shown to have anti-amyloidogenic activity [, 22]. Although Cur has multiple advantageous aspects, including anti-inflammatory effects and mediation of AD neuropathogenic factors, its biological applications are hindered due to its low water solubility and stability [27-29]. Thus, to overcome these limitations, water soluble Cur derivatives, Gd- Cur and Cur-S, were investigated to determine whether the beneficial characteristics (i.e., nontoxicity, antioxidant activity, and control of Aβ aggregation) of Cur are retained after structural modification to enhance biological use through increased solubility. Either linkage of Gd to Cur (Gd-Cur; Fig. ) or substitution of the phenolic hydroxyls to sodium acetate (Cur-S; Fig. ) vastly improved the solubility in buffered solution (ca. mm M in aqueous media). Influence on Metal-Free and Metal-Triggered Aβ Aggregation In Vitro The control of Gd, Cur, Cur-L, Gd-Cur, and Cur-S towards metal-free and metal-induced Aβ aggregation in vitro was examined through two experiments: inhibition (modulation of Aβ aggregate formation) and disaggregation (transformation of preformed Aβ aggregates) (Figs. 2 and 3) [5, 37-4]. The Aβ species generated from both experiments were monitored via gel electrophoresis followed by Western blot (gel/western blot) with an anti-aβ antibody (6E) for analyzing the distribution of molecular weight (MW), as well as transmission electron microscopy (TEM) for visualizing morphological changes [5, 37-4]. The inhibition studies, as depicted in (Fig. 2), demonstrated different reactivity of Gd, Cur, Cur-L, Gd-Cur, and Cur-S towards the formation of metal-free and metalinduced Aβ aggregates composed of either Aβ 4 or Aβ 42. Through the studies by gel/western blot, in the case of samples containing Cu(II) and Aβ 4, noticeable reactivity was only observed upon treatment of peptide with Cur-L (Fig. 2A, lane 3, left) and some reactivity with Cur and Gd-Cur (Fig. 2A, lanes 2 and 4, left). Gd or Cur-S was not able to modulate both metal-free and metal-triggered Aβ 4 aggregation (Fig. 2A, lanes and 5, left). Any significant difference between compound-free and compound-treated samples was not indicated from metal-free Aβ 4 and Zn(II) Aβ 4 samples. Upon incubation of Cu(II)-treated Aβ 4 with Cur, Cur-L, or Gd-Cur, smaller amorphous Aβ 4 aggregates were observed by TEM; however, Gd and Cur-S presented similar size and morphology of Aβ 4 species compared to compound-free conditions (Fig. 2B). In the absence and presence of metal ions, the addition of small molecules to Aβ 42 showed overall similar reactivity patterns relative to Aβ 4 samples. The distinct distribution of MW, in reference to compound-free Aβ 42, was observed only in Cu(II)-added samples incubated with Cur, Cur-L, or Gd-Cur (Fig. 2A, lanes 2 4, right). From these samples, TEM images revealed shorter, thinner, and less dense fibrils upon Cur, Cur-L
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