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Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

Acetylcholinesterase and Aβ Aggregation Inhibition by Heterometallic Ruthenium(II)−Platinum(II) Polypyridyl Complexes Nilima A. Vyas,⊥,† Sushma B. Singh,⊥,† Avinash S. Kumbhar,*,† Dnyanesh S. Ranade,‡ Gulshan R. Walke,‡ Prasad P. Kulkarni,‡ Vinod Jani,§ Uddhavesh B. Sonavane,§ Rajendra R. Joshi,§ and Srikanth Rapole∥ †

Department of Chemistry, Savitribai Phule Pune University, Pune-411007, India Bioprospecting Group, Agharkar Research Institute, Savitribai Phule Pune University, Pune-411004, India § Centre for Development of Advanced Computing (C-DAC), Savitribai Phule Pune University, Pune-411007, India ∥ Proteomics Laboratory, National Centre for Cell Sciences, Savitribai Phule Pune University, Pune-411007, India ‡

S Supporting Information *

ABSTRACT: Two heteronuclear ruthenium(II)−platinum(II) complexes [Ru(bpy)2(BPIMBp)PtCl2]2+ (3) and [Ru(phen)2(BPIMBp)PtCl2]2+ (4), where bpy = 2,2′-bipyridine, phen = 1,10-phenanthroline, and BPIMBp = 1,4′-bis[(2-pyridin2-yl)-1H-imidazol-1-ylmethyl]-1,1′-biphenyl, have been designed and synthesized from their mononuclear precursors [Ru(bpy)2(BPIMBp)]2+ (1) and [Ru(phen)2(BPIMBp)]2+ (2) as multitarget molecules for Alzheimer’s disease (AD). The inclusion of the cis-PtCl2 moiety facilitates the covalent interaction of Ru(II) polypyridyl complexes with amyloid β (Aβ) peptide. These multifunctional complexes act as inhibitors of acetylcholinesterase (AChE), Aβ aggregation, and Cu-induced oxidative stress and protect neuronal cells against Aβ-toxicity. The study highlights the design of metal based anti-Alzheimer’s disease (AD) systems.

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amyloid morphology.11 Intracellular release of zinc leads to several signaling cascades which finally result in ROS production.12 Copper and iron are primarily involved in the toxicity of Aβ by an oxidative mechanism. It is observed that Cu(II)−Aβ complexes are redox active and generate reactive oxygen species.13−16 The higher level of complexity in AD is thus a result of all these concurrent and interlinked processes. Therefore, targeting more than one cascade becomes a novel approach for the development of multifunctional compounds able to address the complexity and multifactorial nature of AD.4 In this respect, organic compounds have been effectively developed to target more than one AD cascade. Recently, Storr et al. have reported a series of multitarget directed ligands possessing a phenol-triazole framework.17 Development of metal compounds as potential agents capable of modifying and inhibiting Aβ aggregation is an emerging area in metal-based therapeutic drug design.18−20 A recent pioneering study by Barnham et al. on Aβ aggregation inhibition by covalent binding platinum complexes triggered new research, and several Pt, Ru, Rh, and Ir complexes have been developed, principally targeting Aβ aggregation and the Cu−Aβ redox process.21 These complexes are able to bind Aβ covalently, interfere with the Aβ−Cu redox process, inhibit Aβ aggregation, and/or act as luminescent Aβ sensors. However,

INTRODUCTION The development and progress of Alzheimer’s disease (AD) is a result of several concurrent abnormalities such as low levels of choline, amyloid beta (Aβ) aggregation, tau-phosphorylation, altered metal levels, and oxidative stress.1−3 Though these parallel pathological cascades have their own mechanism and effect in the pathogenesis of AD, they are linked with each other and escalate the complexity of AD.4 Current AD treatment is aimed at enhancing the choline level with the use of acetylcholine esterase (AChE) inhibitors. This oldest and widely studied cholinergic strategy still remains a promising approach for AD drug development, as among the five approved AD drugs, four are AChE inhibitors.5−7 Apart from its primary role in the hydrolysis of the neurotransmitter acetylcholine, AChE also plays a key role in the Aβ aggregation process in which stable AChE−Aβ complexes are formed, which are responsible for the acceleration of Aβ aggregation. AChE serves as an important cofactor for Aβ aggregation, inducing conformational and biochemical changes of Aβ in solution and accelerating its fibrillogenesis. Also AChE−Aβ complexes exhibit considerably enhanced neurotoxicity compared to that of free Aβ.8,9 Formation and aggregation of Aβ peptides finally results in deposition of Aβ plaques, which is an important hallmark of AD pathogenesis. Aβ pathogenesis is also greatly influenced by dyshomeostasis of transition metal ions, especially Cu(II), Fe(II), and Zn(II).10 Zinc plays a major role in self-aggregation and precipitation of Aβ and also regulates © XXXX American Chemical Society

Received: January 10, 2018

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DOI: 10.1021/acs.inorgchem.8b00091 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry

Scheme 1. (a) Structures and (b) Design Approach of Complexes in the Present Study Representing Proposed AChE and Aβ Binding Sites

these studies target only the Aβ cascade of AD pathogenesis. Recently we have explored the potential of covalently saturated Ru(II) polypyridyl complexes to act as bifunctional inhibitors of AChE, as well as inhibitors of Aβ aggregation, thus targeting simultaneously the cholinergic and amyloid strategy of AD. These studies show that with the appropriate modification of phenanthroline ligand, Ru(II) polypyridyl complexes can be effectively developed as potent inhibitors of AChE as well as Aβ aggregation.22,23 Similarly, R. Carlos and co-workers have also reported multifunctional polypyridyl Ru(II) complexes of aminopyridine exhibiting AChE inhibitory activity and acting as Aβ luminescent imaging agents.24 Considering the paramount importance of AChE inhibitors in AD treatment and recent promising results on interaction of platinoid complexes with Aβ, here we have designed multifunctional complexes via the incorporation of Aβ binding site, i.e. cis-PtCl2 moiety, onto Ru(II) polypyridyl moiety as cisplatin binds to Aβ peptide.25 Here our strategy is to develop Ru(II) polypyridyl complexes that would primarily target AChE and Aβ aggregation (Scheme 1). Platinoid complexes, mainly Ru and Pt, offer a potential option for development of multitargeting compounds on account of their inertness and stability in a biological medium, possible versatile derivatization and recognition of the target by planar ligands, and further

establishment of aromatic interactions with Aβ, thus minimizing other side reactions.26 These complexes exhibit significantly high potential of AChE inhibition, almost equal to AChE inhibitor drug tacrine. Complexes are able to inhibit Aβ aggregation as revealed by ThT fluorescence, turbidity measurements, and confocal microscopy. These complexes are also able to inhibit Aβmetal mediated H2O2 production and Cu(II) mediated Aβ oxidation. Further, complexes 1−3 exhibit moderate toxicity toward human neuroblastoma cells and have potential to rescue these cells from Aβ induced toxicity to a smaller extent.

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EXPERIMENTAL SECTION

Synthesis and Characterization. Synthesis of Bridging Ligand [BPIMBp]. The bridging ligand was synthesized by using a reported procedure.27 Preparation of Complexes 1−4. Complexes 1−4 were synthesized according to a reported procedure.28,29 a. [Ru(bpy)2(BPIMBp)](PF6)2 (1). Complex 1 was synthesized using a modified reported procedure.28 To a solution of bridging ligand (BPIMBp) (324 mg, 0.69 mM) in 40 mL of methanol:water (2:1) was added cis-[Ru(bpy)2Cl2]·2H2O (120 mg, 0.23 mM) and stirred for 30 min under nitrogen. The resulting purple solution was heated to 80 °C for 4 h. The color of the mixture changed from purple to red. After the completion of reaction, the resulting mixture was cooled and evaporated under vacuum and red solid residue was obtained. The B


Article

Inorganic Chemistry

AChE activity. The reaction took place in a final volume of 200 μL of 0.1 M phosphate-buffered solution, pH 8.0, containing 20 μL of AChE (0.47 μg/mL in 10 mM phosphate buffer of pH 7.0), 20 μL of a 3.3 mM solution of 5,5′-dithiobis(2-nitrobenzoic) acid (DTNB) in 10 mM phosphate buffer of pH 7.0 containing 6 mM NaHCO3 and 20 μL of a solution of inhibitor (10 to 12 concentrations ranging from 0.05 μM to 200 μM). After 20 min incubation period at 37 °C, 20 μL of acetylthiocholine iodide (3.0 mM aqueous solution) was added. Hydrolysis of substrate was followed by measuring the variation of the absorbance at 405 nm for 5 min. Absorbance measurements for enzyme assay were performed on a Thermo-Fischer Multiskan FC plate reader. Self-hydrolysis of substrate was verified by running a blank containing no inhibitor and no enzyme in the reaction mixture. Inhibitor controls were also run separately under the same conditions to eliminate inhibitor contribution in the readings. IC50 value that is concentration of inhibitor that reduces 50% enzyme activity was calculated by nonlinear regression of the response−log (concentration) curve. Data are expressed as the mean ± SD of at least three different experiments in triplicate. Kinetic Analysis of AChE Inhibition. To obtain estimates of the mechanism of action of these compounds, reciprocal plots of 1/V vs 1/[S] were constructed at different substrate concentrations (0.1 to 1 mM) by the method of Ellman. The final reaction mixture volume 200 μL (0.1 M phosphate-buffered solution, pH 8.0) contained 20 μL of enzyme (0.47 U/mL in 10 mM phosphate buffer of pH 7.0), 20 μL of a 3.3 mM solution of 5,5′-dithiobis(2-nitrobenzoic) acid (DTNB), and 20 μL of inhibitor to give the final desired concentration. Progress curves were monitored at 405 nm for 3 min with and without inhibitor concentrations. The plots were assessed by a weighted least-squares analysis. Slopes of the reciprocal plots were plotted against inhibitor concentration for the estimation of Ki, as the x-axis intercept. Molecular Docking Studies. The molecular docking studies were carried out using crystal structure coordinates of AChE from Torpedo californica (TcAChE) PDB ID 1QTI. The crystal structure of TcAChE is used due to its similarity to EeAChE sequence. Amber ff99SB force field was applied to parametrize protein. All water molecules are removed. Protein preparation was carried out using chimera software.31 Ruthenium complexes were docked into the active site of the enzyme. Prior to docking the ruthenium complexes were energy optimized through density functional theory (DFT) method using gaussian03.32 The partial atomic charges for the compounds were derived using Guassian 03 program package using Becke’s three parameter hybrid functional (B3LYP) method and LANL2DZ basis set for the ruthenium atom and 6-31G(d,p) basis set for the other atoms. Docking was carried out using DOCK6 software.33 Each compound was subjected to multiple docking runs. Analyses of docking results were carried out using chimera by visual inspection along with the docking scores. Rigid docking was carried out for these complexes. The relative binding affinities of the best poses of the complexes were determined by using gridscore. Further, molecular dynamics simulations were run using the AMBER 11 suite of programs for complex 2-AChE system to check the stability of the binding mode obtained by docking. Protein-2 complex was subjected to a short minimization of 5000 steps comprising the initial 2500 steps of steepest descent and remaining 2500 steps of conjugate gradient. This minimized structure was further heated until 300 K and then equilibrated for 1 ns at the same temperature. The equilibrated structure was then subjected to production run for 5 ns. Protein was allowed to move with 2 restrained. The Root Mean Square Deviation (RMSD) for the residues belonging to the CAS and PAS region is calculated by the “ptraj” module of AMBER. Thioflavin T Assay. Inhibitory activities of complexes against Aβ (1−42) aggregation were determined by the Thioflavin T (ThT) fluorescence method.34 It is the most commonly and widely accepted method. ThT is an organic fluorescent dye which is able to interact with amyloid fibrils and used to monitor their aggregation. It exhibits no fluorescence with Aβ monomers; however, it shows enhanced fluorescence when it binds to β sheet rich amyloid fibrils.35 Monomeric 20 μM Aβ (1−42) peptide was mixed with an equimolar concentration of complexes, 20 μM ThT, and incubated at 4 °C for 15

residue was redissolved in water and the unreacted bridging ligand was filtered off. Saturated solution of NH4PF6 was added to the solution, and the orange red precipitate formed was immediately filtered off. The crude product was purified on aluminum oxide using acetonitrile and methanol as eluent. The fraction was collected and concentrated under vacuum to get pure red solid. Yield: 90 mg (35%); 1H NMR (300 MHz, DMSO-d6, δ, ppm) 8.83 (d, J = 5.4 Hz, 2H), 8.18 (qd, J = 15.4, 7.7 Hz, 6H), 8.01−7.91 (m, 3H), 7.90−7.80 (m, 4H), 7.77 (d, J = 5.2 Hz, 1H), 7.73−7.60 (m, 9H), 7.51 (dd, J = 13.0, 6.5 Hz, 4H), 7.31 (d, J = 8.4 Hz, 4H), 7.10 (t, J = 7.8 Hz, 2H), 6.68 (s, 1H), 5.95 (d, J = 18.8 Hz, 4H); IR (ν, cm−1) 3366 (H2O), 3099, 3063 (Ar−H), 2977, 2868 (CH2), 1641, 1598, 1468 (CC, CN); elemental analysis calculated (%) for C50H40N10RuP2F12: C 51.32, H 3.93, N 11.97; found C 51.11, H 4.052, N 12.10; ESI-MS (m/z, positive mode): ([M − PF6]+) = 1026.9, ([M − 2PF6]2+) = 443.4. b. [Ru(phen)2(BPIMBp)](PF6)2 (2). The synthesis and purification of complex 2 were similar to those of complex 1 using precursor cis[Ru(phen)2Cl2] (150 mg, 0.26 mM) and BPIMBp (370 mg, 0.79 mM). Yield: 120 mg (38%); 1H NMR (400 MHz, DMSO-d6, δ, ppm) 8.88−8.82 (m, 2H), 8.71 (dd, J = 7.9, 4.4 Hz, 2H), 8.60 (d, J = 4.7 Hz, 2H), 8.43−8.32 (m, 6H), 8.26−8.20 (m, 2H), 8.18−8.13 (m, 2H), 8.11−8.06 (m, 2H), 8.00 (dd, J = 11.2, 6.4 Hz, 2H), 7.88 (d, J = 7.9 Hz, 1H), 7.80 (d, J = 8.7 Hz, 1H), 7.74−7.70 (m, 2H), 7.67−7.57 (m, 4H), 7.47 (s, 1H), 7.40−7.32 (m, 1H), 7.29−7.11 (m, 4H), 6.61 (d, J = 7.9 Hz, 2H), 6.04−5.93 (m, 4H); IR (ν, cm−1) 3396.73 (H2O), 3143.15, 3093.20 (Ar−H), 2913.06, 2869.91 (CH2), 1635.38, 1557.85, 1498.37 (CC, CN); elemental analysis calculated (%) for C54H40N10RuP2F12: C 53.25, H 3.3, N 11.5; found C 53.498, H 3.475, N 11.3; ESI-MS (m/z, positive mode): ([M − PF6]+) = 1075.21, ([M − 2PF6]2+) = 465.12. c. [Ru(bpy)2(BPIMBp)PtCl2](PF6)2 (3). Complex 3 was synthesized using a modified reported procedure.29 A mixture of complex 1 (90 mg, 0.076 mM) and cis-[Pt(DMSO)2Cl2]30 (37 mg, 0.084 mM) in ethanol was refluxed for 4 h. After the completion of reaction, the resulting mixture was cooled and evaporated under vacuum and red residue was obtained. The crude product was purified on aluminum oxide using acetonitrile and methanol as eluent. The fraction was collected and concentrated under vacuum to get pure red solid. Yield: 80 mg (73%); 1H NMR (500 MHz, DMSO-d6, δ, ppm) 9.40 (d, J = 4.9 Hz, 2H), 8.83 (t, J = 6.8 Hz, 6H), 8.27−8.09 (m, 6H), 7.94 (ddd, J = 10.8, 6.2, 4.8 Hz, 4H), 7.90−7.76 (m, 4H), 7.73−7.61 (m, 4H), 7.57−7.48 (m, 3H), 7.38−7.27 (m, 3H), 7.10 (t, J = 10.4 Hz, 2H), 6.69 (d, J = 1.3 Hz, 2H), 6.07−5.90 (d, 4H); 195Pt-NMR (δ, ppm): −2355.91; IR (ν, cm−1) 3395.15 (H2O), 3135.56, 3085.24 (Ar−H), 2933.11, 2904.18, 2861.24 (CH2), 1605.34, 1540.99, 1468.31 (CC, CN), elemental analysis calculated (%) for C50H40N10Cl2RuPtP2F12· 2H2O: C 40.8, H 3.0, N 9.51; found C 40.934, H 3.2, N 9.6; ESI-MS (m/z, positive mode): ([M − 2PF6]2+) = 574.2. d. [Ru(phen)2(BPIMBp)PtCl2](PF6)2 (4). The synthesis and purification of complex 4 were similar to those for complex 3 using precursor complex 2 (115 mg, 0.09 mM) and cis-[Pt(DMSO)2Cl2] (44 mg, 0.1 mM). Yield: 80 mg (57%); 1H-NMR (500 MHz, DMSO-d6, δ, ppm) 9.41 (d, J = 4.9 Hz, 2H), 8.84 (dt, J = 8.0, 3.9 Hz, 2H), 8.76−8.69 (m, 2H), 8.44−8.32 (m, 5H), 8.28−8.20 (m, 2H), 8.14 (d, J = 8.3 Hz, 1H), 8.10−8.05 (m, 2H), 8.03−7.89 (m, 5H), 7.84−7.79 (m, 1H), 7.75−7.66 (m, 6H), 7.63−7.58 (m, 1H), 7.56 (d, J = 1.5 Hz, 1H), 7.34 (dd, J = 21.2, 8.3 Hz, 2H), 7.24−7.12 (m, 3H), 6.62 (dd, J = 6.3, 3.0 Hz, 1H), 6.03−5.91 (m, 4H); 195Pt-NMR (δ, ppm): −2358.89; IR (ν, cm−1) 3396.73 (H2O), 3150.56, 3086.39 (Ar−H), 2905.49, 2855.53 (CH2), 1606.61, 1512.75, 1477.18 (CC, CN); elemental analysis calculated (%) for C54H40N10Cl2RuPtP2F12.2CH3OH: C 43.4, H 3.1, N 9.0; found C 43.2, H 3.12, N 8.74; ESI-MS (m/z, positive mode): ([M − PF6]+) = 1341.1 ([M − 2PF6]2+) = 598.07 Biological Activity. Acetylcholinesterase Inhibition Studies. The concentration of AChE solution in 10 mM phosphate buffer of pH 7.0 was calculated from its known extinction coefficient at 280 nm (Extinction Coefficient: E1% = 18.0). Stock solutions of tested complexes were prepared in DMSO and then diluted with phosphate buffer of pH 7.0 to give final DMSO concentration less than 0.15% in the assay reaction mixture. This DMSO concentration did not affect C


Article

Inorganic Chemistry

absorbance of ascorbate was monitored at λ = 265 nm as a function of the time in 100 mM ammonium acetate buffer (pH-7.4). The Aβ (1− 16) was incubated in the presence of Cu(II) for 6 h at 4 °C in ammonium acetate buffer (10 μL). Similarly, Aβ (1−16) was incubated in the presence of Cu(II) for 3 h at 4 °C in ammonium acetate buffer with subsequent addition of complexes followed by 3 h incubation at 4 °C (10 μL) to form a reaction mixture of Aβ (1−16)Cu(II)-complexes. The kinetics of ascorbate (190 μL) was measured for each 30 s of interval for 5 min. Then, the ascorbate consumption was started by addition of reaction mixtures Aβ (1−16)-Cu(II) and Aβ (1−16)-Cu(II)- complexes. The final concentrations of Aβ (1−16), Cu(II), and complexes were 25 μM and the ascorbate concentration was 250 μM each in the final volume of 200 μL. Mass Spectrometry of Copper Catalyzed Oxidation Reactions of Aβ(1−16) Preincubated with Complexes 3 and 4. MALDI-MS was used to study the copper catalyzed oxidation products of Aβ peptide. The copper catalyzed oxidation of Aβ peptide was initiated by the addition of ascorbic acid. This reaction was stopped by the addition of 1% glacial acetic acid which removed copper from copper bound Aβ. Then the oxidized products of Aβ peptide were desalted and immediately analyzed by MALDI-MS. Mass spectra were collected on a AB Sciex 4800 MALDI-TOF/TOF mass spectrometer (AB Sciex, Framingham, MA) linked to 4000 series explorer software (v.3.5.3). Samples were then allowed to dry and loaded on MALDI. Mass spectra were recorded within mass range 800 to 4000 Da using a Nd:YAG 355 nm laser. The acceleration voltage used was 20 kV and extraction voltage was 18 kV. The instrument was calibrated using 6 peptide standard mix that was purchased from AB Sciex. MS spectra were obtained in the reflector mode using 900 laser shots with 4000 laser intensity. Further MS/MS spectra were acquired with a total accumulation of 1500 laser shots and collision energy of 2 kV. Cytotoxicity Studies. Human neuroblastoma cells SH-SY5Y were grown in DMEM & Ham’s F-12 medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic solution (penicillin and streptomycin) at 37 °C in a humidified chamber under 5% CO2. The compound solutions (40 μM or 20 μM in phosphate buffer of pH 7.0 containing 1% DMSO) were passed through 0.22 μm syringe filters for sterilization before treatment. Cell viability was measured in 96-well plates by quantitative colorimetric assay using MTT. Briefly, 105 cells per mL were seeded in 96-well plates for assay. The cells were treated with compounds in media without FBS. At 24 h after the treatment, media was removed and MTT was added to the wells at the final concentration as 5 mg/mL and the cells were incubated at 37 °C for another 3 h. The MTT solution was removed and the colored formazan crystals in each well were dissolved in 150 μL dimethyl sulfoxide. Absorbance at 595 nm was measured using a μQuant, Biotek Instruments microplate reader. The effect of complexes on Aβ (1−42) mediated toxicity in neuronal cell culture (Human neuroblastoma cells SH-SY5Y) was determined by MTT assay. Cells were incubated with Aβ (1−42) (20 μM) in the presence and in the absence of complexes for 3 days. After this, 20 mL MTT (5 mg/mL) was added to the cells and incubated for 3 h. Cell viability was measured after dissolving the crystals in DMSO and reading the absorbance at 570 nm as mentioned above. Reagents and Materials. All reagents and solvents were purchased commercially and were used as received unless specified. Doubly distilled water is used to prepare buffer solution. All other chemicals and reagents were of analytical grade and used without further purification. Acetylthiocholine iodide, 5,5′-dithiobis(2-nitrobenzoic) acid (DTNB), propidium iodide, 1,1,1,3,3,3-hexafluoro-2propanol (HFIP), and Thioflavin T (ThT) were purchased from Sigma-Aldrich USA. Sodium chloride, potassium hydrogen phosphate (K2HPO4), and potassium dihydrogen phosphate (KH2PO4) used for buffer preparation were of molecular biology grade and obtained from SRL (India). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Spectrochem Pvt. Ltd., Mumbai. Dulbecco’s Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), and Phosphate Buffered Saline for cell culture were purchased from HiMedia, Mumbai. Acetylcholinesterase from Electrophoricuselectricus (EeAChE) Type V−S and human amyloid-β peptide (Aβ 1−40)

min. After incubation, 1% HFIP (v/v) was added to Aβ peptide solution in order to initiate rapid aggregation. This reaction was carried out at 37 °C in a 96 well plate and ThT fluorescence was continuously monitored with excitation and emission of 440 and 495 nm, respectively, using a Hitachi F-2500, spectrofluorimeter for 30 min. Plateau fluorescence values were taken as representative of aggregate formation. The experiment was performed in triplicate, and standard error was calculated (n = 3) Preparation of Monomeric Aβ (1−42) Solution. We have adapted the protocol used by Nichols et al. for the preparation of monomeric Aβ (1−42) peptide solution.36 Lyophilized Aβ (1−42) peptide was dissolved in HFIP and stored at −80 °C. Further HFIP was removed by lyophilization, and peptide was then stored at −80 °C. Prior to use Aβ (1−42) peptide was reconstituted in 60 mM NaOH and diluted to a final concentration of 20 μM in phosphate buffered saline, pH 7.4, and the concentration of Aβ (1−42) peptide was determined by BCA method. Confocal Fluorescence Imaging. The samples of Aβ (1−42) alone and Aβ (1−42) with complexes were incubated for 10 days at 37 °C. Then, the samples were poured on glass slides and dried in air and covered with a coverslip. The confocal images were obtained using a FITC fluorescence cube at 494 nm excitation and 518 nm emission on a Nikon AIR instrument. ESI-MS of Aβ (1−16) with Complexes. Ten μM Aβ (1−16) peptide was mixed with complexes 3 and 4 in a 1:1 ratio and incubated in water at room temperature for 15 min. After incubation, the ESI-MS of the samples was carried out on a Bruker, Impact HD ESI-Q TOF mass spectrometer, at Central Instrumentation Facility (CIF), SPPU, Pune. 1 H NMR of Aβ (1−16) with Complexes. 1H NMR spectra of Aβ (1−16) (50 μM) in the absence and presence of complex were recorded on a Bruker Avance Liquid State 500 MHz equipped with AV III HD one bay console in D2O solvent at pH 7.4 and 296 K at Central Instrumentation Facility (CIF), SPPU, Pune. Spectra were processed with Topspin 3.5 software. Five mM stock solutions of complexes in DMSO-d6 were used so that the DMSO-d6 proportion never exceeded 5% (v/v). Turbidity Assay. Twenty μM Aβ (1−42) peptide was combined with complexes in a (1:1) ratio and incubated at 4 °C for 15 min. After incubation 1% HFIP (v/v) was added to each solution to induce rapid aggregation.37 The solutions were added to individual wells in a 96well plate, and the turbidity was measured at 405 nm for 40 min time intervals on a plate reader (Biotek SYNERGY HT, microplatereader). Molecular Docking with Aβ. Molecular docking studies were carried out on full length Aβ (1−42). Initial coordinates for simulation were taken from pdb structure with pdb id 1IYT. Initially peptide was solvated with water, and then counterions were added to the system to neutralize the system. Amber ff99SB force field was applied to parametrize protein. Protein preparation was carried out using chimera software.31,38 Blind docking was carried out. Prior to docking the complex energy was optimized through the DFT method using gaussian03.32,39 The partial atomic charges for the compounds were derived using the Gaussian 03 program package using Becke’s three parameter hybrid functional (B3LYP) method and the LANL2DZ basis set for the ruthenium atom and the 6-31G(d,p) basis set for the other atoms. Standard parameters were used for the docking. Docking was carried out using DOCK6.33 Analyses of docking results were carried out using chimera by visual inspection along with the docking scores. Rigid docking was carried out. The relative binding affinities of the best poses of the complex were determined by using gridscore. Molecular dynamics simulations were run using a gromacs 4.5.6. Amberff03 force field. A solvated neutral system was subjected to a short minimization of 5000 steps. This minimized structure was subjected to NVT simulation for 100 ps and was followed by NPT simulation for 1 ns. The equilibrated structure was then simulated for 700 ns. The analyses were carried out using gromacs tools. After analyzing the trajectory the 700th ns snapshot was taken for docking study. Ascorbate Consumption Assay. The traces of kinetics of ascorbate consumption were recorded on a BioTek SYNERGY-HT microplate reader, at 25 °C, in a 96 UV transparent well plate (Corning). The D


Article

Inorganic Chemistry were purchased from Sigma-Aldrich. Aβ (1−42) peptide was purchased from Invitrogen. Aβ1−16 peptide was obtained from BioResource Biotech Pvt. Ltd. India with >95% purity. The solvents methanol, ethanol, dimethylformamide, diethyl ether, dichloromethane, chloroform, and acetonitrile were of analytical grade and used except for purification procedure or unless otherwise noted. The deuterated solvents CDCl3 (99.9%), DMSO-d6 (99.5%), and D2O (99.9%) were obtained from Aldrich Chemical Co. (U.S.A.). Water used for the biochemical studies was triply distilled or Milli-Q grade.

The active site of AChE (Catalytic Active Site, CAS) is located at the bottom of the active gorge, at the entrance of which Peripheral Anionic Site (PAS) is present. PAS is also involved in Aβ binding and further aggregation, that is AChE induced Aβ aggregation.42−44 Bifunctional or also generally called as dual AChE inhibitors are able to bind at both CAS and PAS and have attracted great interest.45 Binding of dual inhibitor to CAS results in inhibition of AChE activity whereas by occupying PAS region, dual inhibitor inhibits AChE induced Aβ aggregation making PAS unavailable for Aβ binding. Mixed type of enzyme inhibition exhibited by almost all dual AChE inhibitors is a key indication of their binding at both CAS and PAS. Thus, to determine AChE inhibition mode exhibited by complexes 1−4, Lineweaver−Burk reciprocal plots were obtained by kinetic measurements in the presence of different substrate and inhibitor concentrations. Lineweaver−Burk reciprocal plots of complexes 1 (Figure S1-a in Supporting Information) and 2 (Figure 1-a) displayed a decrease in Vmax (increase in y-axis intercept) and increase in Km values with increase in inhibitor concentration. This indicates mixed-type of inhibition and suggests their binding at both CAS and PAS of AChE. Further, their appreciably low values of inhibition constants (Ki) indicate their high affinity and strong binding to AChE. Thus, their mixed type of inhibition and strong binding reflect their strong potential of AChE inhibition. Interestingly for mixed Ru(II)−Pt(II) complexes 3 (Figure S2-b in the Supporting Information) and 4 (Figure 1-b), a distinct inhibition pattern was observed. Their Lineweaver− Burk reciprocal plots exhibited a decrease in Vmax as well as Km with an increase in inhibitor concentration. This type of inhibition is the mixed or uncompetitive type of inhibition. Mixed type of inhibition suggests their dual binding, as observed for complexes 1 and 2. Their uncompetitive inhibition could be a result of the presence of a different binding mode or of a different binding site. It can be proposed that, here in the case of complexes 3 and 4, there is a simultaneous existence of different binding patterns which could arise due to the presence of two different binding metal centers.46 All complexes exhibited nearly 50 times higher potential than parent noncompetitive [Ru(phen)3]2+, and complex 4 displayed an IC50 value similar to the well-known AChE inhibitor drug tacrine.47 Molecular Docking Studies. Molecular docking is one of the most commonly used methods to predict the conforma-

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RESULTS AND DISCUSSION AChE Inhibition. Complexes 1−4 were evaluated for their activity against AChE (Electrophorus electricus) following the

Table 1. AChE Inhibition Data for Complexes [Ru(phen)3]2+ and 1−4 compound

IC50

Kia

type of Inhibition

[Ru(phen)3]2+b 1 2 3 4 Tacrinec

9.23 0.32 0.20 0.26 0.18 0.18

4.10 0.05 0.04

Noncompetitive Mixed Mixed Mixed or uncompetitive Mixed or uncompetitive

a

Estimates of Ki were obtained from replots of the slopes of Lineweaver−Burk plots vs inhibitor concentration. bData taken from ref 22. cData taken from ref 47.

method of Ellman.40 Previous studies of inhibition of AChE by ruthenium polypyridyl complexes report activity of complexes in comparison with parent [Ru(phen)3]2+.41 In our previous work, we found [Ru(phen)3]2+ as the most appropriate compound to compare activities with the aim to evaluate the effect of phenanthroline modification on AChE inhibition.22,23 Therefore, in the present work also we have used [Ru(phen)3]2+ as parent compound. The IC50 curves against AChE are shown in Figure S1 in the Supporting Information, and AChE inhibition data for complexes 1−4 and [Ru(phen)3]2+ are given in Table 1. All complexes exhibited good potential of AChE inhibition with IC50 values in the submicromolar range. The IC50 values for complexes 1−4 range between 0.18 μM to 0.32 μM indicating that Pt linking to Ru(II) polypyridyl moiety does not result in any significant enhancement in their inhibition activity.

Figure 1. Steady state inhibition of AChE hydrolysis of acetylthiocholine (ATCh). Lineweaver−Burk reciprocal plots of initial velocity and substrate concentration (0.1 to 1.0 mM) for 2 (a) and 4 (b). E


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Figure 2. Top scored poses of 3 (a) and 4 (b) for Orientation-I in the AChE active site (PDB id 1QTI). Positions of 3 (c) and 4 (d) observed in Orientation-II. PAS amino acids and internal gorge with catalytic site of the enzyme are highlighted in yellow and red, respectively.

(Top scored poses are shown in Figure S4 in the Supporting Information). Ancillary ligand is able to span the whole active gorge whereas polypyridyl coligands participate in interactions with PAS amino acids. Trp84, Ser200, and His440 from CAS show CH−π or π−π interactions with ancillary ligand. Polypyridyl coligands participate in interactions with Trp279, Tyr70, and Tyr121 of PAS regions. These results explain their mixed type of inhibition observed in kinetic results. For heteronuclear complexes 3 and 4, interestingly, we could observe two different orientations with comparable energies. The grid score for complexes with AChE is given in Table S2 in the Supporting Information. Figure 2 shows interactions of AChE active gorge amino acid residues with complexes 3 and 4 in orientations-I and -II. Orientation-I is similar to mononuclear Ru(II) complexes, where AChE active gorge can accommodate ancillary ligand effectively, which behaves as bridging ligand in these complexes. Pt center with chloride ligands shows strong interaction with catalytic triad amino acids His440 and Glu327. Aromatic rings of bridging ligand establish π−π and hydrophobic interactions with the whole active gorge. Phenanthroline and bipyridine polypyridyl ligands surrounding the Ru(II) center bind to Trp279, Tyr70, and Tyr121 of the PAS region. All interactions are essentially π−π and hydrophobic interactions. Additionally, electrostatic interactions also stabilize the binding of complex to AChE. Interactions of aqua complexes formed by removal of labile chloride ligands cannot be ruled out; however, complexity due to the bimetallic system

Figure 3. Turbidity assay showing inhibition of Aβ (1−42) aggregation by complexes 1−4 (1:1 ratio) in PBS, pH 7.4. Experiment was performed in triplicate (n = 3), and standard deviation was calculated.

tions and detail binding of ligands with proteins. Though structural complexity of inorganic metal complexes makes its use difficult, we have performed molecular docking studies to elucidate binding behavior of Ru(II)−Pt(II) complexes. Figure S3 in the Supporting Information shows the orientation of mononuclear complexes 1 and 2 in the AChE active gorge and molecular interactions with amino acids of AChE. Both complexes interact with AChE by binding at PAS and CAS F


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Figure 4. Confocal images of ThT alone (a) and (b) Aβ (1−42) using ThT after 10 days of incubation; Aβ (1−42) alone (c) and in the presence of complexes 1 (d), 2 (f), 3 (g), and 4 (h); complex alone 1 (h), 2 (i), 3 (j), 4 (k) at 37 °C, pH 7.0. Aβ (1−42)−complex ratio used was 1:1 (20 μM). Green: FITC emission (494−518 nm); Scale size: (a) 100 μm and (b to k) 10 μm.

restricted these studies to primary docking and further molecular dynamic simulations to study detail interactions could not be performed. Whereas in orientation-II, complete opposite positions of complexes 3 and 4 are observed, in which the Pt center interacts with the PAS region and the Ru center along with coligands shows binding with the CAS region. Overall, complexes are able to interact with the entire active gorge. Though such orientation is also possible for complexes 1 and 2, considering their energies and explaining the mode of inhibition are not considered here. Orientation-I as well as Orientation-II confirm mixed type of inhibition observed for complexes 3 and 4. Binding of Pt at PAS (Orientation- II) provides a possible explanation for their uncompetitive mode of inhibition as suggested for cisplatin, which is also able to inhibit AChE uncompetitively by binding at PAS.46 For complexes 3 and 4, the presence of orientations-I and -II could be a reason for observed mixed and uncompetitive inhibition of AChE by these complexes. Aβ Aggregation Inhibition Studies. Thioflavin T Fluorescence Method. Inhibitory activities of complexes 1−4 against Aβ (1−42) aggregation was determined by the Thioflavin T (ThT) fluorescence method.22,23 Here experiments were performed in the presence of dilute Hexafluoro-2propanol (HFIP) to induce rapid Aβ (1−42) aggregation and to abolish the lag phase of the aggregation process.48 Control Aβ (1−42) showed the increase in the fluorescence, and in the period of 30 min of incubation fluorescence intensity reached to 100% indicating complete Aβ aggregation (Figure S5 in the Supporting Information), whereas Aβ (1−42) coincubated with complexes 1−4 showed the significant decrease in the ThT fluorescence. Among these, complex 1 exhibited the highest inhibitory potential with 80% inhibition of aggregation while complex 2 showed a relatively low reduction in fluorescence

intensity with 53% inhibition. Ru−Pt mixed complexes 3 and 4 indicated nearly similar decrease in fluorescence which accounts for 70% inhibition. Though any trend could not be defined across the complexes, yet it is clear that all complexes are potent inhibitors of Aβ (1−42) aggregation. Turbidity Measurements. Formation of Aβ aggregates can also be monitored by turbidometry. This spectroscopic technique correlates turbidity or absorbance of Aβ solution at 405 nm with fibrils present in the solution.48 The increase in the turbidity can be correlated with the increase in the Aβ aggregation. Figure 3 shows the effect of complexes 1−4 on Aβ aggregation monitored by turbidity measurements at 405 nm. Compared to control Aβ (1−42) turbidity, turbidity for Aβ (1− 42) incubated with complexes 1−4 showed the significant reduction, indicating the presence of significantly reduced fibrils. The turbidity results indicate that complexes 1−4 act as potent inhibitors of Aβ aggregation. The reduction of Aβaggregation by these complexes is further confirmed by confocal fluorescence imaging experiments. Confocal Fluorescence Imaging. Recently, R. Carlos and co-workers effectively demonstrated the use of luminescent confocal imaging experiments to monitor real time conformational changes of Aβ aggregation in the presence of Ru(II) polypyridyl complexes.24 These complexes are able to detect early stages of Aβ aggregation where wormlike structures have been identified as precursors of the protofibril, and U-shaped or globular structures of Aβ for short peptide. Here we have used confocal fluorescence imaging of Aβ using ThT to examine the Aβ fibril formation in the presence and in the absence of complexes after 10 days of incubation. Figure 4 displays images of Aβ stained with ThT. Control Aβ (1−42) shows the presence of a thick network of long Aβ fibers indicating nearly complete Aβ aggregation, whereas Aβ (1−42) coincubated with G


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complexes shows protofibrils and globular structures. These protofibrils are able to bind with ThT and hence the complete inhibition of Aβ-aggregation is not observed in both ThT as well as turbidity assays. No fluorescence was observed for only ThT or complexes (Figure 4(b) and (h−k)). These results suggest that the complexes possibly inhibit Aβ aggregation at early stages of fibril formation. Further detailed interactions of complexes with Aβ (1−42) were elucidated by molecular docking studies (Figure S6 in the Supporting Information). All complexes were found to be positioned in the hydrophobic region and C-terminal region of Aβ (1−42). Involvement of hydrophobic core LVFF(17−20) residues in interactions with ligands possibly leads to interference in intermolecular interactions of Aβ (1−42), resulting in reduction in fibril formation. Aβ Binding Studies of 3 and 4 by Mass Spectrometry. To explore the covalent binding ability of cis-PtCl2 moiety of complexes 3 and 4 with Aβ, ESI-MS experiments were performed. Mass spectrometry has been routinely used to explore the binding mode of metal complexes to Aβ.21,49−53 Due to their high rate of aggregation, Aβ (1−40) and Aβ (1− 42) are difficult to handle for such studies. Therefore, Aβ (1− 16) is a commonly used Aβ fragment as it contains the metal binding site of Aβ and represent a good model for Aβ metal binding studies avoiding complexity due to aggregation.49,54 ESI-MS spectra for Aβ (1−16) and coincubated Aβ (1−16) with complexes 3 and 4 are shown in Figure 5. Assignments of the observed peaks in the ESI-MS spectra are summarized in Table 2. ESI-MS of Aβ (1−16) shows intense peaks corresponding to multiple charged species. Peptide treatment with complexes 3 resulted in the appearance of new peaks at m/ z 1010.3 and 757.7. In the presence of complex 4, the appearance of new peaks at m/z 770.0 and 1026.6 is observed. In both cases these new peaks are consistent with Aβ (1−16)complex adduct formed by loss of two chlorides from platinum. Thus, these results indicate that complexes 3 and 4 bind to Aβ (1−16) covalently through platinum coordination. Aβ Binding Studies of 3 and 4 by 1H NMR. The covalent binding interaction of complex 3 and 4 with Aβ (1−16) peptide was further confirmed by 1H NMR experiment. Figure 6 and Figure S7 in the Supporting Information show changes in 1H NMR of Aβ (1−16) after increments of complexes 4 and 3, respectively. These spectra show almost complete disappearance of aromatic signals of three histidines accompanied by downfield shift on addition of complex 4 and upshift in the presence of complex 3. These observations indicate changes in the chemical environment of corresponding nuclei upon complex−Aβ (1−16) interaction and strongly suggest His binding to the Pt center. However, no noticeable changes were observed in NMR spectra of Aβ (1−16) in the presence of complex 1 or 2, consistent with their structural inactivity for Aβ (1−16) covalent binding (Figure S8 in the Supporting Information) The proton assignments were carried out as per recent reports by Faller et al.55,56 Inhibition of Cu-Aβ Induced ROS Production. The binding of complexes 3 and 4 to Aβ (1−16) encouraged us to study the inhibition of Cu-induced oxidation of Aβ-peptide by 3 and 4. Cu(II)−Aβ complexes are redox active and produce reactive oxygen species (ROS) significantly contributing to oxidative stress development in AD. This catalytic process promotes Fenton-type chemistry resulting in H2O2 production. Here the potential of complexes 1−4 to inhibit Cu(II)-Aβ induced H2O2 production is investigated by ascorbate consumption assay.25,57

Figure 5. ESI-MS spectra of (a) Aβ (1−16) (10 μM) and coincubated Aβ (1−16) with complexes (b) 3 and (c) 4. Aβ (1−16): complex ratio used was 1:1.

Table 2. Assignments of the Peaks in the ESI-MS Spectra for the Reaction of Aβ (1−16) with Complexes 3 and 4 species [Aβ [Aβ [Aβ [Aβ [Aβ [Aβ

(1−16) (1−16) (1−16) (1−16) (1−16) (1−16)

+ + + + + +

3H]3+ 2H]2+ (3-2Cl-2PF6) ]4+ (3-2Cl-2PF6)]3+ (4-2Cl-2PF6) ]4+ (4-2Cl-2PF6)]3+

observed m/z

calculated m/z

652.6 978.4 757.7 1010.3 770.0 1026.6

652.6 978.5 758.0 1010.7 770.0 1026.7 H


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Figure 6. 1H NMR spectra of Aβ (1−16) control and Aβ (1−16) after addition of different equivalents of complex 4 in D2O (pH 7.4), T = 296 K. Proton signals of the coordinated imidazole ring are labeled with a δ and ε referred to the N(δ) or N(ε) imine nitrogens of the imidazole ring.

Oxidation or consumption of ascorbic acid by Cu(II)-Aβ can be followed by the changes occurring in ascorbate absorbance at 265 nm (Figure S9 in the Supporting Information). The rate of ascorbate oxidation is found to be much higher for Cu(II)− Aβ(1−16) than Cu(II)−Aβ(1−16) with complexes 1−4. Inhibition of Cu(II)−Aβ(1−16) triggered H2O2 production by all complexes was observed which may suggest their potential to act as ROS regulators. Further copper-catalyzed oxidation of Aβ(1−16) in the presence and absence of complexes 1−4 was carried out, and the products were analyzed using matrix-assisted laser desorption ionization (MALDI) MS (Figure 7). MS spectrum of alone Cu(II)−Aβ (1−16) showed oxidation peaks at m/z 1971, 1987, and 2003 and additional three peaks at m/z 1941, 1925, and 1910 are observed (Figure 7(a)). These peaks correspond to deamination, decarboxylation, and decarboxylation/deamination of the Asp1 residue of Aβ (1−16) respectively. However, in the presence of complexes 1−4 the significant reduction in these peak intensities was observed, indicating that the complexes are able to reduce coppercatalyzed oxidation of Aβ (1−16). Ascorbate oxidation and MALDI−MS results clearly indicate that dinuclear Ru−Pt complexes 3 and 4 interfere in Cu(II) binding to Aβ (1−16), resulting in decrease in oxidation of Aβ(1−16) and thus consequently aggregation inhibition. For Ru−Pt complexes 3 and 4, it can be postulated that Pt coordination to Aβ (1−16) leads to a protective effect of these complexes against copper catalyzed oxidation whereas inhibition of copper-catalyzed H2O2 production and oxidation of Aβ (1−16) by complexes 1 and 2 could be a result of Cu(II) chelation by ancillary ligand of complexes 1 and 2 (Figure S10 and S11 in the Supporting Information). We further confirmed the interaction of complexes 3 and 4 with Aβ (1−16) in the presence of copper by MS/MS spectra. We carried out MS/MS analysis of m/z 1971 ion to understand

the structural rearrangement of the complexes during redox cycling of Aβ (1−16)-CuII in the presence of complex 3 and 4. In the absence of complexes 3 and 4, the MS/MS analysis of m/z 1971 showed the peak at m/z 872 corresponds to b7 ion (Figure S12 in the Supporting Information). Therefore, all the peaks are expressed as a relative percentage assuming 100% for b7 peaks. The spectrum showed a peak at m/z 772 corresponding to b6* which indicated the oxidation of His6 for both the complexes 3 and 4. Also the peak at m/z 1696 corresponds to b14*, indicating the oxidation of His14. In the presence of complexes, an overall decrease in the intensities of the oxidation peaks was observed as compared to that in the absence of complexes. For complexes 3 and 4, the ratio of b6*/b6 is decreased from 0.76 to 0.66 containing His6 residues indicating the lowering of oxidation reaction. Additionally, for complex 3, the ratio of b5*/b5 encompassing Arg5 residues decreased by 0.35 encompassing Arg5. These results indicate that binding of complexes 3 and 4 to Aβ (1−16)-CuI decreased the oxidation of His6. MS data indicate the partial protection of His6 against oxidation. The results also suggest the different binding mode of complexes 3 and 4 as compared to cisplatin as cisplatin binds with Asp1 and His6 residues of Aβ peptide.21,25,56 Inhibition of Aβ Induced Cytotoxicity. The ability of these complexes to protect neuronal SH-SY5Y cells from Aβ (1−42) toxicity was examined using neurotoxicity assay.58 The effect of complexes 1−4 on the Aβ (1−42)-induced cytotoxicity is shown in Figure 8. Cells treated with Aβ (1−42) alone showed 58% cell viability. When cells were treated with Aβ (1−42) coincubated with complex 1 and 2, the cell survival showed a marginal protection from Aβ (1−42)-induced cytotoxicity. Complex 4 itself shows slight toxicity at this concentration and hence is unable to rescue cells from Aβ (1−42) toxicity. Interestingly, the cell viability in the presence of complex 2 raises from 58% to 77%, suggesting its potential to act as I


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Figure 8. Effects of complexes 1−4 on Aβ (1−42) induced toxicity in neuronal cell culture. Twenty μM of Aβ (1−42) peptide combined with complexes in a 1:1 ratio. Experiment was performed in triplicate. Results were expressed as mean % cell viability ± standard deviation.

the parent [Ru(phen)3]2+ (IC50 = 9.23 μM), all complexes are nearly 50 times more potent than [Ru(phen)3]2+. Detailed enzyme kinetic studies and molecular docking revealed their dual binding mode. ThT fluorescence and turbidity measurements indicated that complexes 1−4 inhibit Aβ (1−42) aggregation significantly. In addition to inhibiting Aβ (1−42) aggregation, these complexes also modulate Cu(II)-Aβ redox chemistry by acting as potent inhibitors of Cu(II)-Aβ induced H2O2 generation. The effect on these complexes altering Cu(II)-Aβ interaction can be correlated with their structural features. ESI-MS spectrometry indicated complexes 3 and 4 form covalent adducts with Aβ (1−16) through platinum coordination and possibly block the Cu(II) binding site. 1H NMR studies of Aβ (1−16) in the presence of complexes revealed covalent binding of complexes 3 and 4 through Pt coordination at the His residue. Further, except complex 4, other complexes are moderately toxic to human neuroblastoma SH-SY5Y cells and also exhibit moderate potential to inhibit Aβ(1−42) induced toxicity to neuronal SH-SY5Y cells. Thus, we developed multifunctional complexes combining two well identified functional metal based groups viz. Ru(II) poypyridyl center as AChE and/or Aβ inhibitor and cisplatin as Aβ binding moiety. Though no definite effect on activity of complexes is observed by structural variations and insertion of cisplatin to Ru(II) polypyridyl moiety has resulted only in different binding modes of these complexes with Aβ, these studies explore the use of metal complexes in the development of multipotent compounds that are able to target different stages in AD pathogenesis.

Figure 7. MS (MALDI) spectra of oxidized Aβ(1−16)-CuII (a) and Aβ(1−16)-CuII in the presence of complex 3 (b) and 4 (c).

inhibitor of Aβ (1−42) induced toxicity. However, it has been observed that complex 2 itself promotes the cell growth at 20 μM (Figure S14). Thus, the protective effect observed for complex 2 is a combined effect of increased cell growth and protection against Aβ (1−42) induced toxicity. Overall, all complexes exhibited moderate toxicity toward SH-SY5Y with very high IC50 values (Figure S14 and Table S3 in the Supporting Information).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b00091. AChE steady state inhibition and docking, ThT Fluorescenece, Aβ (1−42) docking, ascorbate consumption experiment, MALDI spectra of oxidized Aβ (1−16)CuII, Cytotoxicity evaluation of complexes against human neuroblastoma cells and stability of complexes by UV− visible spectroscopy. (PDF)

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CONCLUSIONS Two mononuclear Ru(II) polypyridyl complexes (1 and 2) and two mixed Ru(II)−Pt(II) complexes (3 and 4) have been synthesized and characterized in detail. Their IC50 values for AChE inhibition range in the 0.18 to 0.32 μM. Comparing with J


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(14) Hureau, C.; Faller, P. Aβ-mediated ROS production by Cu ions: structural insights, mechanisms and relevance to Alzheimer’s disease. Biochimie 2009, 91, 1212−1217. (15) Barnham, K. J.; Masters, C. L.; Bush, A. I. Neurodegenerative diseases and oxidative stress. Nat. Rev. Drug Discovery 2004, 3, 205− 214. (16) Lynch, T.; Cherny, R.; Bush, A. Oxidative processes in Alzheimer’s disease: the role of Aβ-metal interactions. Exp. Gerontol. 2000, 35, 445−451. (17) Jones, M. R.; Mathieu, E.; Dyrager, C.; Faissner, S.; Vaillancourt, Z. A.; Korshavn, K.; Lim, M. H.; Ramamoorthy, A.; Yong, V. W.; Tsutsui, S.; Stys, P. K.; Storr, T. Multi-target-Directed Phenol-Triazole Ligands as Therapeutic Agents for Alzheimer’s Disease. Chem. Sci. 2017, 8, 5636−5643. (18) Liu, H.; Qu, Y.; Wang, X. Amyloid β-targeted metal complexes for potential applications in Alzheimer’s disease. Future Med. Chem. 2018, 10, 679−701. (19) Miller, J. J.; Gomes, L. M.; Storr, T.; Casini, A. The Interaction of Metal Compounds with Protein Targets: New Tools in Medicinal Chemistry and Chemical Biology. EIBC 2017, 1. (20) Hickey, J. L.; Donnelly, P. S. Diagnostic imaging of Alzheimer’s disease with copper and technetium complexes. Coord. Chem. Rev. 2012, 256, 2367−2380. (21) Streltsov, V. A.; Epa, V. C.; James, S. A.; Churches, Q. I.; Caine, J. M.; Kenche, V. B.; Barnham, K. J. Structural insights into the interaction of platinum-based inhibitors with the Alzheimer’s disease amyloid-β peptide. Chem. Commun. 2013, 49, 11364−11366. (22) Vyas, N. A.; Bhat, S. S.; Kumbhar, A. S.; Sonawane, U. B.; Jani, V.; Joshi, R. R.; Ramteke, S. N.; Kulkarni, P. P.; Joshi, B. Ruthenium (II) polypyridyl complex as inhibitor of acetylcholinesterase and Aβ aggregation. Eur. J. Med. Chem. 2014, 75, 375−381. (23) Vyas, N. A.; Ramteke, S. N.; Kumbhar, A. S.; Kulkarni, P. P.; Jani, V.; Sonawane, U. B.; Joshi, R. R.; Joshi, B.; Erxleben, A. Ruthenium (II) polypyridyl complexes with hydrophobic ancillary ligand as Aβ aggregation inhibitors. Eur. J. Med. Chem. 2016, 121, 793−802. (24) Silva, D. E.; Cali, M. P.; Pazin, W. M.; Carlos-Lima, E.; Salles Trevisan, M. T.; Venâncio, T.; Arcisio-Miranda, M.; Ito, A. S.; Carlos, R. M. Luminescent Ru (II) Phenanthroline Complexes as a Probe for Real-Time Imaging of Aβ Self-Aggregation and Therapeutic Applications in Alzheimer’s Disease. J. Med. Chem. 2016, 59, 9215− 9227. (25) Walke, G. R.; Rapole, S.; Kulkarni, P. P. Cisplatin inhibits the formation of a reactive intermediate during copper-catalyzed oxidation of amyloid β peptide. Inorg. Chem. 2014, 53, 10003−10005. (26) Hureau, C.; Faller, P. Platinoid complexes to target monomeric disordered peptides: a forthcoming solution against amyloid diseases? Dalton Trans. 2014, 43, 4233−4237. (27) Singh, S. B.; Kumbhar, A. S.; Khan, A. Honeycomb-like Ordered Assembly of DNA Induced by Flexible Binuclear Ruthenium (II)− Polypyridyl Complexes. Chem. - Eur. J. 2016, 22, 15760−15771. (28) Sun, L.; Berglund, H.; Davydov, R.; Norrby, T.; Hammarström, L.; Korall, P.; Börje, A.; Philouze, C.; Berg, K.; Tran, A. Binuclear ruthenium-manganese complexes as simple artificial models for photosystem II in green plants. J. Am. Chem. Soc. 1997, 119, 6996− 7004. (29) Williams, R. L.; Toft, H. N.; Winkel, B.; Brewer, K. J. Synthesis, characterization, and DNA binding properties of a series of Ru, Pt mixed-metal complexes. Inorg. Chem. 2003, 42, 4394−4400. (30) Price, J. H.; Williamson, A. N.; Schramm, R. F.; Wayland, B. B. Palladium (II) and platinum (II) alkyl sulfoxide complexes. Examples of sulfur-bonded, mixed sulfur-and oxygen-bonded, and totally oxygenbonded complexes. Inorg. Chem. 1972, 11, 1280−1284. (31) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimeraa visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, 1605−1612.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]. Phone No: +91 (020) 25601395 Extn. 534. Fax: +91 (020) 25691728. ORCID

Avinash S. Kumbhar: 0000-0002-2087-4827 Prasad P. Kulkarni: 0000-0002-3929-2990 Author Contributions ⊥

N.A.V. and S.B.S. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS NAV acknowledges Department of Science and Technology (DST), New Delhi, India, (SR/WOS-A/CS-63/2010) for funding. ASK and SBS acknowledge Department of Science and Technology (DST), New Delhi, India, for financial support under the scheme SR/S1/IC-25/2010. GRW thanks CSIR, Government of India for SRF. This work was supported by the funds from Department of Science and Technology, New Delhi, India (EMR/2014/001235). The authors thank Mr. Umesh Kasbe, CIF, SPPU for NMR experiments.

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REFERENCES

(1) Carreiras, M. C.; Mendes, E.; Perry, M. J.; Francisco, A. P.; Marco-Contelles, J. The multifactorial nature of Alzheimer’s disease for developing potential therapeutics. Curr. Top. Med. Chem. 2013, 13, 1745−1770. (2) Mucke, L. Neuroscience: Alzheimer’s disease. Nature 2009, 461, 895−897. (3) Roberson, E. D.; Mucke, L. 100 years and counting: prospects for defeating Alzheimer’s disease. Science 2006, 314, 781−784. (4) Rosini, M.; Simoni, E.; Caporaso, R.; Minarini, A. Multitarget strategies in Alzheimer’s disease: benefits and challenges on the road to therapeutics. Future Med. Chem. 2016, 8, 697−711. (5) Anand, P.; Singh, B. A review on cholinesterase inhibitors for Alzheimer’s disease. Arch. Pharmacal Res. 2013, 36, 375−399. (6) Musial, A.; Bajda, M.; Malawska, B. Recent developments in cholinesterases inhibitors for Alzheimer’s disease treatment. Curr. Med. Chem. 2007, 14, 2654−2679. (7) Colombres, M.; Sagal, J. P.; Inestrosa, N. C. An overview of the current and novel drugs for Alzheimer’s disease with particular reference to anti-cholinesterase compounds. Curr. Pharm. Des. 2004, 10, 3121−3130. (8) Quinn, D. M. Acetylcholinesterase: enzyme structure, reaction dynamics, and virtual transition states. Chem. Rev. 1987, 87, 955−979. (9) Alvarez, A.; Alarcón, R.; Opazo, C.; Campos, E. O.; Muñoz, F. J.; Calderón, F. H.; Dajas, F.; Gentry, M. K.; Doctor, B. P.; De Mello, F. G. Stable complexes involving acetylcholinesterase and amyloid-β peptide change the biochemical properties of the enzyme and increase the neurotoxicity of Alzheimer’s fibrils. J. Neurosci. 1998, 18, 3213− 3223. (10) Bush, A. I. The metallobiology of Alzheimer’s disease. Trends Neurosci. 2003, 26, 207−214. (11) Watt, N. T.; Whitehouse, I. J.; Hooper, N. M. The role of zinc in Alzheimer’s disease. Int. J. Alzheimer's Dis. 2011, 2011, 1 (12) McCord, M. C.; Aizenman, E. The role of intracellular zinc release in aging, oxidative stress, and Alzheimer’s disease. Front. Aging Neurosci. 2014, 6, 77. (13) Smith, D. G.; Cappai, R.; Barnham, K. J. The redox chemistry of the Alzheimer’s disease amyloid β peptide. Biochim. Biophys. Acta, Biomembr. 2007, 1768, 1976−1990. K


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Inorganic Chemistry (32) Frisch, M.; Trucks, G.; Schlegel, H.; Scuseria, G.; Robb, M.; Cheeseman, J.; Montgomery, J.; Vreven, T.; Kudin, K.; Burant, J. Gaussian 03, revision C.02; 2008. (33) Moustakas, D. T.; Lang, P. T.; Pegg, S.; Pettersen, E.; Kuntz, I. D.; Brooijmans, N.; Rizzo, R. C. Development and validation of a modular, extensible docking program: DOCK 5. J. Comput.-Aided Mol. Des. 2006, 20, 601−619. (34) Levine, H. Thioflavine T interaction with synthetic Alzheimer’s disease β-amyloid peptides: Detection of amyloid aggregation in solution. Protein Sci. 1993, 2, 404−410. (35) Khurana, R.; Coleman, C.; Ionescu-Zanetti, C.; Carter, S. A.; Krishna, V.; Grover, R. K.; Roy, R.; Singh, S. Mechanism of thioflavin T binding to amyloid fibrils. J. Struct. Biol. 2005, 151, 229−238. (36) Nichols, M. R.; Colvin, B. A.; Hood, E. A.; Paranjape, G. S.; Osborn, D. C.; Terrill-Usery, S. E. Biophysical comparison of soluble amyloid-β (1−42) protofibrils, oligomers, and protofilaments. Biochemistry 2015, 54, 2193−2204. (37) Dickens, M. G.; Franz, K. J. A Prochelator Activated by Hydrogen Peroxide Prevents Metal-Induced Amyloid β Aggregation. ChemBioChem 2010, 11, 59−62. (38) Liu, J.-G.; Zhang, Q.-L.; Shi, X.-F.; Ji, L.-N. Interaction of [Ru (dmp)2 (dppz)]2+ and [Ru (dmb)2 (dppz)]2+ with DNA: effects of the ancillary ligands on the DNA-binding behaviors. Inorg. Chem. 2001, 40, 5045−5050. (39) Frisch, M. J.; Trucks, G. W., Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, revision C.02; Gaussian, Inc.: Pittsburgh, PA, 2003. (40) Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 1961, 7, 88IN191−9095. (41) Mulcahy, S. P.; Li, S.; Korn, R.; Xie, X.; Meggers, E. Solid-phase synthesis of tris-heteroleptic ruthenium (II) complexes and application to acetylcholinesterase inhibition. Inorg. Chem. 2008, 47, 5030−5032. (42) Carvajal, F. J.; Inestrosa, N. C. Interactions of AChE with Aβ aggregates in Alzheimer’s brain: therapeutic relevance of IDN 5706. Front. Mol. Neurosci. 2011, 4, 4. (43) Butini, S.; Brindisi, M.; Brogi, S.; Maramai, S.; Guarino, E.; Panico, A.; Saxena, A.; Chauhan, V.; Colombo, R.; Verga, L. Multifunctional cholinesterase and amyloid beta fibrillization modulators. Synthesis and biological investigation. ACS Med. Chem. Lett. 2013, 4, 1178−1182. (44) Dvir, H.; Silman, I.; Harel, M.; Rosenberry, T. L.; Sussman, J. L. Acetylcholinesterase: from 3D structure to function. Chem.-Biol. Interact. 2010, 187, 10−22. (45) Viayna, E.; Sabate, R.; Muñoz-Torrero, D. Dual inhibitors of βamyloid aggregation and acetylcholinesterase as multi-target antiAlzheimer drug candidates. Curr. Top. Med. Chem. 2013, 13, 1820− 1842. (46) Aljafari, A. A. Kinetics for the inhibition of acetylcholinesterase from human erythrocyte by cisplatin. Int. J. Biochem. Cell Biol. 1995, 27, 965−970. (47) Marco-Contelles, J.; León, R.; de los Ríos, C.; Guglietta, A.; Terencio, J.; López, M. G.; García, A. G.; Villarroya, M. Novel Multipotent Tacrine− Dihydropyridine Hybrids with Improved

Acetylcholinesterase Inhibitory and Neuroprotective Activities as Potential Drugs for the Treatment of Alzheimer’s Disease. J. Med. Chem. 2006, 49, 7607−7610. (48) Ranade, D. S.; Bapat, A. M.; Ramteke, S. N.; Joshi, B. N.; Roussel, P.; Tomas, A.; Deschamps, P.; Kulkarni, P. P. Thiosemicarbazone modification of 3-acetyl coumarin inhibits Aβ peptide aggregation and protect against Aβ-induced cytotoxicity. Eur. J. Med. Chem. 2016, 121, 803−809. (49) Valensin, D.; Anzini, P.; Gaggelli, E.; Gaggelli, N.; Tamasi, G.; Cini, R.; Gabbiani, C.; Michelucci, E.; Messori, L.; Kozlowski, H. fac{Ru (CO) 3} 2+ selectively targets the histidine residues of the βamyloid peptide 1−28. implications for new alzheimer’s disease treatments based on ruthenium complexes. Inorg. Chem. 2010, 49, 4720−4722. (50) Messori, L.; Camarri, M.; Ferraro, T.; Gabbiani, C.; Franceschini, D. Promising in vitro anti-Alzheimer properties for a ruthenium (III) complex. ACS Med. Chem. Lett. 2013, 4, 329−332. (51) Man, B. Y.-W.; Chan, H.-M.; Leung, C.-H.; Chan, D. S.-H.; Bai, L.-P.; Jiang, Z.-H.; Li, H.-W.; Ma, D.-L. Group 9 metal-based inhibitors of β-amyloid (1−40) fibrillation as potential therapeutic agents for Alzheimer’s disease. Chem.Sci. 2011, 2, 917−921. (52) Wang, X.; Wang, X.; Zhang, C.; Jiao, Y.; Guo, Z. Inhibitory action of macrocyclic platiniferous chelators on metal-induced A β aggregation. Chem. Sci. 2012, 3, 1304−1312. (53) Kumar, A.; Moody, L.; Olaivar, J. F.; Lewis, N. A.; Khade, R. L.; Holder, A. A.; Zhang, Y.; Rangachari, V. Inhibition of Aβ42 peptide aggregation by a binuclear ruthenium (II)− platinum (II) complex: potential for multimetal organometallics as anti-amyloid agents. ACS Chem. Neurosci. 2010, 1, 691−701. (54) Minicozzi, V.; Stellato, F.; Comai, M.; Dalla Serra, M.; Potrich, C.; Meyer-Klaucke, W.; Morante, S. Identifying the minimal copperand zinc-binding site sequence in amyloid-β peptides. J. Biol. Chem. 2008, 283, 10784−10792. (55) Alies, B.; Conte-Daban, A.; Sayen, S.; Collin, F.; Kieffer, I.; Guillon, E.; Faller, P.; Hureau, C. Zinc (II) binding site to the amyloidβ peptide: insights from spectroscopic studies with a wide series of modified peptides. Inorg. Chem. 2016, 55, 10499−10509. (56) Sasaki, I.; Bijani, C.; Ladeira, S.; Bourdon, V.; Faller, P.; Hureau, C. Interference of a new cyclometallated Pt compound with Cu binding to amyloid-β peptide. Dalton Trans. 2012, 41, 6404−6407. (57) Ginotra, Y. P.; Ramteke, S. N.; Walke, G. R.; Rapole, S.; Kulkarni, P. P. Histidine availability is decisive in ROS-mediated cytotoxicity of copper complexes of Aβ1−16 peptide. Free Radical Res. 2016, 50, 405−413. (58) Nicolini, G.; Miloso, M.; Zoia, C.; Di Silvestro, A.; Cavaletti, G.; Tredici, G. Retinoic acid differentiated SH-SY5Y human neuroblastoma cells: an in vitro model to assess drug neurotoxicity. Anticancer Res. 1997, 18, 2477−2481.

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