Luminescent Ru(II) Phenanthroline Complexes as a Probe for Real


Sep 26, 2016 - ... for Real-Time Imaging of Aβ Self-Aggregation and Therapeutic Applications in ... Citation data is made available by participants i...
0 downloads 4 Views 3MB Size


Article pubs.acs.org/jmc

Luminescent Ru(II) Phenanthroline Complexes as a Probe for Real-Time Imaging of Aβ Self-Aggregation and Therapeutic Applications in Alzheimer’s Disease Debora E. S. Silva,† Mariana P. Cali,† Wallance M. Pazin,‡ Estevaõ Carlos-Lima,§ Maria Teresa Salles Trevisan,∥ Tiago Venâncio,† Manoel Arcisio-Miranda,§ Amando S. Ito,‡ and Rose M. Carlos*,† †

Departamento de Química, Universidade Federal de São Carlos, São Carlos, São Paulo 13565-905, Brazil Departamento de Física, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, USP, Ribeirão Preto, São Paulo 14040-901, Brazil § Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, São Paulo 04023-062, Brazil ∥ Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, Ceará Fortaleza, 60451-970, Brazil ‡

S Supporting Information *

ABSTRACT: The complexes cis-[Ru(phen)2(Apy)2]2+, Apy = 4-aminopyridine and 3,4-aminopyridine, are stable in aqueous solution with strong visible absorption. They present emission in the visible region with long lifetime that accumulates in the cytoplasm of Neuro2A cell line without appreciable cytotoxicity. The complexes also serve as mixed-type reversible inhibitors of human AChE and BuChE with high active site contact. cis-[Ru(phen)2(3,4Apy)2]2+ competes efficiently with DMPO by the OH• radical. Luminescence using fluorescence lifetime imaging (FLIM) enables real-time imaging of the conformational changes of the self-aggregation of Aβ with incubation of complexes (0−24 h) in phosphate buffer at micromolar concentrations. By this technique, we identified protofibrills in the selfassembly of Aβ1−40 and globular structures in the short fragment Aβ15−21 in aqueous solution.



INTRODUCTION There has been increasing interest in the biochemical stages of β-amyloid peptide (Aβ) aggregation owing to its implications on the development and progression of Alzheimer’s disease (AD).1−3 The role of this peptide in AD is dictated by its accumulation over time as soluble monomers, oligomers, protofibrills, and subsequent formation of mature fibrils which deposit as insoluble plaques in specific regions of the brain.1−3 These amyloid plaques can disturb the normal function of the brain by changing the synaptic plasticity1−3 or promoting local inflammatory process, oxidative damage,4 and deregulation of Ca2+ homeostasis5 that lead to gradual but progressive synaptic loss, neuronal death, and cognitive decline.6 The net balance between production and clearance of Aβ deposits determines the degree of neuronal damage in the brain and the AD stage.7 For this reason, there is considerable interest in strategies to detect amyloid deposits8,9 because a definite diagnosis of Alzheimer’s disease can be made only through biopsy or autopsy.10 However, compounds able to recognize the early stages of aggregation, in particular the neurotoxic oligomeric form of Aβ,11,12 not only greatly contribute to fundamental studies on elucidation of Aβ self-assembly dynamics but also broaden the scope of potential application in AD therapy in patients at early stages of this disease. © 2016 American Chemical Society

The aggregation of Aβ is commonly studied in vitro, using a diversity of compounds13−17 and techniques.18−22 Among them, thioflavin-T (ThT)23−25 and Congo Red (CR)26−28 have been widely used to follow the Aβ self-assembly, to recognize senile plaques, and as probe for the studies of self-aggregation of Aβ by nonluminescent inhibitors. Both ThT and CR bind specifically to amyloid fibrils, but there are studies reporting that CR also binds to Aβ monomers reducing toxicity.27,28 Although molecular dynamics simulation indicates the hydrophobic region of Aβ12−28 as the major binding mode of CR as observed for ThT, the hydrophilic region is also appointed.29,30 Curcumin and chrysamine G also interact with Aβ and binds to the cross-β sheet structure of Aβ mostly by hydrophobic interactions.31,32 Common to all these organic dyes are the two aromatic and planar rings separated by a flexible linker region with different lengths. In general, these results show that the structural characteristics of a compound define their binding mode to Aβ. Indeed, results using the NMR spectroscopic technique show that 1,10-phenanthroline with methyl and phenyl substituents exhibit interactions, though very weak, with Aβ through π−π interactions with the aromatic amino acid Received: August 1, 2016 Published: September 26, 2016 9215

DOI: 10.1021/acs.jmedchem.6b01130 J. Med. Chem. 2016, 59, 9215−9227

Journal of Medicinal Chemistry

Article

Scheme 1. Molecular Structure of Complexes cis-[Ru(phen)2(4Apy)2]2+ (1) and cis-[Ru(phen)2(3,4Apy)2]2+ (2)

rearrangement on the structure of Aβ1−28 with the Ru(II) binding, suggesting a reduction of the aggregation process and neurotoxicity of the Aβ peptide.47 Another strategy is the use of tris-chelate ligands in Ru(II) complexes to target the hydrophobic domain of Aβ by noncovalent interactions.48−53 Among such complexes the Ru(II) polypyridine complexes are particularly interesting because of their unique spectroscopic properties, which can provide valuable opportunities for probing and bioimaging applications.54,55 A recent study explored this strategy in detail for the Ru(α-diimine)dppz complex, α-diimine = 2,2′-bipyridine, 1,10-phenanthroline, and dppz = dipyrido[3,2-a:2′,3′-c]phenazine, which presents light switching properties as observed for ThT under conditions that induce Aβ aggregation.49,50 On the basis of theoretical results, the strong emissive response of Ru(αdiimine)dppz in the presence of Aβ fibrils was attributed to the binding of the dppz ring of Ru(II) complex to the hydrophobic cleft formed on the surface of Aβ between the residues Val18 and Phe20 as observed for ThT.50 Overall, these studies show that the binding sites of a metal complex with Aβ and the resulting implications on the aggregation and inhibition of Aβ depend on the metal center, its oxidation state, the ligands in the coordination sphere, and the geometry of complex. Moreover, both the hydrophobic and the hydrophilic regions of Aβ are valuable targets for the development of markers and/or inhibitors to amyloid fibrils. These findings encouraged us to prepare the complexes cis[Ru(phen)L2]2+, phen = 1,10-phenanthroline, L = 4-aminopyridine (1), and 3,4-diaminopyridine (2), Scheme 1. These complexes were planned to be useful both as sensitive luminescent image sensors for Aβ aggregation into fibril plaques and as therapeutic agents. Our strategy was to design a luminescent Ru(II) complex containing a hydrophobic and a hydrophilic region around the metal center to disturb the backbone hydrogen bonds of the peptide. Thus, we expect that both these factors work in a synergic way to recognize, by the luminescence of complexes, specific sites of Aβ that play a role on the aggregation process and, consequently, interfere in the conformational changes of Aβ into fibrillar aggregates. Monodentate aminopyridine ligands are known by their cognitive and cholinergic properties;56,57 consequently, therapeutic properties are also expected for these complexes. In addition, the involvement of reactive oxygen species in the aggregation process of Aβ is well reported.58−60 For this reason,

residues Phe4 and Tyr10 located on the N terminal hydrophilic domain of Aβ.33 Coordination of the peptide to the metal centers by covalent bind has also been currently used to improve and enhance the scope of biologic applications of compounds to target and reduce the neurotoxicity of Aβ. For instance, the histidine residues His6, His13, and His14 located in the same hydrophilic domain of Phe4 and Tyr10 are good σ-donors, and therefore, metal−histidine interactions are expected and indeed observed for many transition metal complexes.14,34−41 Barnhams’s group exploited this idea intensively with Pt(II)phen and Pt(II)-NH3 complexes containing labile chloride ligands.34,38,41 The cis-[Pt(phen)Cl2] complex interacts with Aβ, inhibits aggregation, reduces the level of reactive species of oxygen, and restores the synaptic plasticity in mouse hippocampus slices that were submitted to long-term potentiation.38 ESI mass spectrometry together with NMR, EPR, HPLC, and EXAFS experiments demonstrated that the cis-[Pt(phen)2Cl2] complex binds to Aβ through both π−π stacking interactions and covalent bonds by replacement of chloride ligands with the N atom of histidine. The synergistic interaction resulted in conformational changes in the structure of Aβ to produce nontoxic structures.39−41 Binuclear complexes of Pt(II)−Ru(II) and mononuclear complexes of Ir(III), Rh(III), and Ru(II/III) have also been investigated.42−51 In particular, Ru complexes are of special interest due to their attractive biologic profiles compared with Pt(II).52 In this respect, the Ru(III) complexes NAMI-A ([Him][trans-RuCl4(DMSO)(im)], im = imidazole) and KP1019 ([Hind][trans-[RuCl4(ind)2], ind = indazole) and its Na+ analogue (KP1339) have been investigated using ThT fluorescence to follow the formation of Aβ fibril aggregates.44−46 Although NAMI-A and KP1339 are currently under clinical trials owing to their anticancer53 activity and low toxicity properties, they were inactive or exhibited poor activity against Aβ binding and toxicity.45,46 Nevertheless, a recent study using EPR, gel electrophoresis, and TEM images suggested a concentration dependent binding of KP1019 to Aβ1−42, and as observed for Pt(II), KP1019 binds to the N-terminus of Aβ through Hist6, Hist13, and/or Hist14.46 The Ru(II) complex fac-[Ru(CO)3Cl2thz], where thz = 1,3thiazole, is another interesting example of the potential of Ru complexes for investigation of Aβ aggregation process.47 This complex interacts strongly with the N-terminal Aβ1−28 fragment of Aβ producing a stable Ru-Aβ1−28 adduct by the release of chloride and thz ligands with histidine residues Hist13 and Hist14.47 NMR and ESI mass investigations indicated a local 9216

DOI: 10.1021/acs.jmedchem.6b01130 J. Med. Chem. 2016, 59, 9215−9227

Journal of Medicinal Chemistry

Article

Figure 1. (A) Absorption spectrum of complex 2 in aqueous solution at pH 7.4 (1 × 10−5 mol L−1). (B) Corrected excitation spectrum (blue) and uncorrected emission spectrum (λexc = 480 nm) at room temperature in aqueous solution at pH 7.4 (1 × 10−5 mol L−1) (black). (C, upper) Emission decays of complex 2 in aqueous solution at 22 °C (black line) by excitation at λexc = 480 nm and emission at λem = 650 nm. Also shown are the instrument response function (blue line) and best fit curve (red line) from a biexponential decay. (C, bottom) Plot of the weighted residues lifetime.

1,10-phenathroline, L = 4-aminopyridine (1) and 3,4-aminopyridine (2), were synthesized by reacting cis-Cl2Ru(phen)2 with aminopyridine in an H2O/EtOH (1:1) ratio using an experimental procedure similar to those found in the literature.61−64 The complex 1 has been previously characterized by our research group.61 The composition of the complex 2 was evidenced by CHNO elemental analysis. Structural insight came from NMR 1D (1H, 13C) and 2D (1H−1H COSY) measurements shown in Figures S1, S2, S3 in Supporting Information. The 1H NMR spectrum of 2 in DMSO-d6 shows duplication of the proton signals of 1,10-phenanthroline and 3,4Apy consistent with two phen and two 3,4Apy in the coordination sphere of Ru(II) in a cis- octahedral structure. The data corroborate the integration of proton signals of the coordinated ligand, Table S1. UV−Visible Spectroscopy. The complexes present suitable optic characteristics for fluorescent imaging in physiological medium, such as broad and intense absorption in the visible region attributed to metal-to-ligand charge transfer (MLCT) electronic transitions (ε480nm = 9500 mol−1 L cm−1) and strong luminescence at room temperature in aqueous solution with maximum intensity at 650 nm and a tail that extends to 800 nm, Figure 1A,B. This long wavelength emission can be assigned to phosphorescence from the triplet 3MLCT excited state, as seen for other ruthenium polypyridine complexes.54,61 Another notable feature of the emission behavior of these complexes relevant to biological applications is the large Stokes shift at approximately 5000 cm−1, which eliminates selfquenching, Figure 1B. The emission decay profiles measured in buffer solution (pH 7.4) required a biexponential fit,

the effect of complexes on the cholinesterase enzyme activity and their antioxidant activity in vitro will also be investigated. The cis-[Ru(phen)2(Apy)2]2+ complexes are easy to prepare in high yield and soluble in diluted aqueous solution with strong absorption. They also show emission in the visible region with reasonably long lifetime that does not interfere with biomolecule emission lifetime and large Stokes shift that precludes self-quenching of emission light. Our results (1) demonstrate the cell uptake of complexes in neuronal cells with no measurable cytotoxicity; (2) provide, in real time, images of the conformational changes of Aβ, during aggregation, after incubation with the luminescent complexes (0−24 h) in aqueous solution, using the time-correlated single photon counting fluorescence lifetime imaging microscopy (TCSPC-FLIM) technique; (3) exhibit important images of the early stages of aggregation, since the beginning of nucleation step to elongation phase; (4) identify wormlike structures that appear to be precursor of the protofibrills; (5) reveal important images of the short peptides Aβ15−21 self-aggregation into U-shaped and globular structures; (6) demonstrate the inhibitory activity of the complexes to human acetylcholinesterase and butyrylcholinesterase enzymes, using the methodology proposed by Ellman; and (7) show the antioxidant activity of the complexes to the hydroxyl radical generated by Fenton reaction and monitored by DMPO-OH adduct, using the electron paramagnetic resonance (EPR) technique.



RESULTS AND DISCUSSION Synthesis and Characterization of Complexes. The monodentate complexes [Ru(phen)2L2](PF6)2, where phen = 9217

DOI: 10.1021/acs.jmedchem.6b01130 J. Med. Chem. 2016, 59, 9215−9227

Journal of Medicinal Chemistry

Article

Table 1. Inhibition of Acetylcholinesterase from Electrophorus electricus (EeAChE), Human Acetylcholinesterase (hAChE), and Human Butyrylcholinesterase (hBuChE) by Complexes and a Series of Commercial Compounds: IC50 Values and Inhibition Constant Ki of Complex−Enzyme IC50 (μmol L−1) compd 2a 1a propidium iodidea [Ru(phen)3]2+ b [Ru(phen)2(bxbg)]2+ b donepezilc tacrined rivastigminee a

eeAChEhAChE 0.73 1.58 15.75 9.23 0.3 0.18

8.72 9.64

Ki (μmol L−1)

hBuChE

eeAChE

hAChE

hBuChE

39.75 50.34

1.37 1.4

2.52 2.64

22.08

4.1 0.04 0.0116 0.147 3.03

7.273 0.036 0.30

This work. bData from ref 77. cData from ref 78. dData from ref 79. eData from ref 89.

Electrophorus electricus (eeAChE), human recombinant (hAChE), and butyrylcholinesterase from serum enzyme (hBuChE) to inhibition by the complexes, using the spectrophotometric method proposed by Ellman.66 Assays were also conducted for free ligands and the precursor complex cis[RuCl2(phen)2]. The experimental plots of substrate absorbance at different concentrations of complex present typical substrate-saturation curves, Figure S6. The inhibition constant Ki between complex−enzyme and the IC50 values are presented in Table 1. ee-AChE. The inhibitory activity of complexes was significantly greater compared with that of the propidium ion (peripheral active site inhibitor)67 and of similar potency compared with that of tacrine (catalytic active site inhibitor).68 The inhibitory activity of the series 2 ∼ 1 > [Ru(phen)3]2+ ≫ Apy demonstrated that the coordination of Apy and phen to the Ru(II) center favors the complex/enzyme interactions through a synergistic effect. Coordination of aminopyridine to the Ru(II) center hinders the hydration effects of Apy that compete with cation−π interactions with AChE.69 Moreover, the planar and aromatic rings of the 1,10-phenanthroline ligand favor π−π-stacking interactions with this enzyme. hAChE/hBuChE. The RuApy complexes are good inhibitors of both hAChE and hBuChE, with no significant difference between them, Table 1. This finding is of special interest considering that dual inhibitors of cholinesterase have been extensively investigated for AD treatment because they can simultaneously improve cognition through interaction with the catalytic active site and indirectly inhibit Aβ aggregation through binding to the peripheral active site.70,71 The spectroscopic data were complemented with kinetic parameters by Lineweaver−Burk analysis,72 Figure S7. The results indicated a mixed-type reversible inhibition for both complexes, which is also a characteristic of many commercial pharmaceuticals, Table 1. The α > 1 for both complexes is indicative of an inhibitor with higher competitive character and higher affinity for the active site of enzyme. The RuApy complexes are 4-fold more potent to hAChE than to hBuChE. Donepezyl73 and huperzine A74 are also mixed-type reversible and selective inhibitors of AChE. As previously described, the binding of donepezil to enzyme is strongly dependent on π−π interactions formed between the aromatic residue Phe295 of the active site and the residue Trp286 of peripheral anionic site of the enzyme; therefore the high selectivity to AChE is justified by the absence of these residues in hBuChE.75 For the RuApy complexes, the planar and aromatic rings of the phenanthroline ligand could establish π−π interactions with

characterized by long 129.3 ns (99.7% contribution to total emission) and a short 1.23 ns (0.3% contribution to total emission) lifetime components, Figure 1C, Table S2. The long emission lifetime of complexes does not interfere with the fluorescent properties of biomolecules.65 Biological Properties. Stability and Solubility of Complexes. The complexes are stable in both solid state and aqueous solution. The chemical stability of the complex 2 in the dark in aqueous solution (phosphate buffer) as a function of the pH of solution was demonstrated by the absence of changes in the 1H NMR at pH values from 5 to 12 over a period of 24 h, Figure S4. Only at pH 2 a chemical shift and formation of new species is observed, which indicates the hydrolysis of the complex. The solubility of the complexes was determined by the shake-flask method and is listed in Table S3. At physiological pH (aqueous buffer phosphate solution, pH 7.4) the complexes are relatively soluble with values of 0.8436 mg mL−1 for 2 and 0.5544 mg mL−1 for 1. In Vitro Uptake of Neuro2A Cells and Cell Proliferation Assay. The cytotoxicity of complexes was evaluated for differentiated Neuro2A cells using MTT reduction assay. Neuro2A cells were incubated for 24 h with different concentrations of complexes, and the absorbance of formazan formation was obtained at 570 nm. Figure S5A shows that, over the course of this experiment, no adverse effects seemed to occur, with cell viability greater than 90% for concentrations up to 50 μmol L−1. Cellular uptake and distribution of the complexes into differentiated Neuro2A cells were probed by the luminescence of the complexes using confocal laser scanning microscopy technique. Figure S5B shows the overlaid differential interference contrast (DIC) and luminescence images for each compound. From these images, it is evident that both complexes accumulate in the cytoplasm region and apparently do not penetrate into the nucleus of the cells. Importantly, the retention of the luminescence of complexes into Neuro2A cells indicates the maintenance of the morphology and integrity of the cells and complexes considering that the aquo and/or hydroxo Ru(II) or Ru(III) complexes that could be formed during the uptake experiments are not emissive. In Vitro Inhibition of Cholinesterase Enzymes. Considering that aminopyridines have important implications on the cognitive and cholinergic system,56,57 it is expected that coordination of Apy to {Ru(phen)2}2+ fragment potentialize the effects on cholinesterase enzymes by avoiding free Apy hydration. Investigations were conducted to compare the in vitro sensitivities of 9218

DOI: 10.1021/acs.jmedchem.6b01130 J. Med. Chem. 2016, 59, 9215−9227

Journal of Medicinal Chemistry

Article

Phe295, and the quaternary N atom of Apy would contact with Trp286 by electrostatic cation−π interactions. In contrast, tacrine only contacts with the active site of AChE through π−π stacking interactions with the indole ring of Trp86 and Phe330, while the N atom of the tacrine ring forms a hydrogen bond with the backbone oxygen of His447.76 Similar interactions may also be expected for RuApy complexes. Hence, both the planar phenanthroline ring and the protonated ammonium substituent of the pyridine ring are critical for the binding of the complexes to cholinesterase enzyme. Further insight into complex−enzyme interactions came from saturation transfer difference (STD) NMR experiments, Figure 2. The signals in the STD-NMR originated from the

The EPR assay shown in Figure 3A was based on the competition between the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and the complexes in scavenging OH radicals generated by the Fenton reaction.82,83 The plot displayed in Figure 3B shows that the complex 2 competes with DMPO for the OH• radical in a concentration-dependent manner: 52.3% (33 μmol L−1), 46.7% (66 μmol L−1), and 31.24% (132 μmol L−1). Surprisingly, the complex 1 and the free ligands 3,4Apy and 4Apy were unable to compete with DMPO for the hydroxyl radical, indicating that the amino group meta-oriented of the pyridine ring of complex plays a role on the antioxidant activity. Moreover, the EPR spectrum of RuApy complexes was performed at X-band (13.81−1.05 g) and remained silent for Ru(III) in the conditions of Fenton reaction in the absence (f ≈ 9.51 GHz, 77 K) and presence of DMPO (f ≈ 9.75 GHz, 295 K). The antioxidant capacity of the complexes and free ligands was also assessed using the stable radicals 2,2-diphenil-1picrylhydrazyl (DPPH•) and 2,2-azinobis-3ethylbenzothiazoline 6-sulfonate (ABTS•+).84 The time-decay of EPR signal intensity of the DPPH• in the presence of the complex 2 and free ligand 3,4Apy is shown in Figure S8A and Figure S8B. It is interesting to note that the reaction of the free ligand 3,4Apy with the DPPH• radical was faster than that found for the complex 2. This result may be explained by steric hindrance, which accounts for the high activity of the 3,4Apy ligand compared with that of the complex 2. For evaluation of large molecules some studies show the limitations of DPPH• as an antioxidant activity methodology.85 Similar results were also observed for the reaction between the complexes and free ligands with ABTS•+ monitored via the decay of visible absorbance of radical, Figure S8C and Figure S8D. Trolox and ascorbic acid were used as positive control. The IC50 values are shown in Table 2. Aside from 1 and free 4Apy, which had no antioxidant activity in the latter two assays, the complex 2 and free ligand 3,4Apy showed a modest antioxidant potential in comparison with ascorbic acid and trolox. These data support the conclusion derived from the results obtained with DMPO spin trap; i.e., complex 2 exhibited a great capacity to scavenge all the radicals tested. In accordance, Zhanyong Guo86 reported the antioxidant activity of a series of inulin derivatives with amino-pyridines as substituents. In this study, the derivative compounds presented better antioxidant activity compared with inulin, and those

Figure 2. Off resonance and STD spectra of complex 2 in the presence of hAChE.

interaction of the protons of the complex with the enzyme. This finding allows us to design an epitope map, which shows that the phenanthroline protons are closer to hAChE; the aminopyridine proton also contributes to the contact between the complex and the enzyme because a single signal can be observed in the STD spectrum. Antioxidant Activity. Considering the implication of ROS overproduction on the pathology of AD,58−60 we evaluated the efficacy of the complexes on oxidative stress. Among the ROS, the hydroxyl radical is the most aggressive species to the organism.81

Figure 3. (A) EPR spectrum of DMPO-OH adduct generated in situ by the Fenton reaction Fe(II) + H2O2 → Fe(III) + OH− + OH• (black) and immediately after addition of 132 μmol L−1 of complex 2 (red). Spectrometer settings were the following: band X, receiver gain 5 × 104, modulation amplitude 1 G, modulation frequency 100 kHz, 2 mW, time constant 20.48 ms. (B) Inhibition of DMPO-OH adduct as a function of complex 2 concentration. 9219

DOI: 10.1021/acs.jmedchem.6b01130 J. Med. Chem. 2016, 59, 9215−9227

Journal of Medicinal Chemistry

Article

Table 2. IC50 for Scavenging of ABTS+• compd

IC50 (μmol L−1)

2 3,4Apy 1 4Apy trolox ascorbic acid

64.46 31.51 ≫100 ≫100 17.92 18.24

To further probe the complex/peptide interaction, we also obtained images of Aβ1−40 incubated with 2 in thin films produced at 1, 3, and 6 h from the respective aqueous solution samples. The thin films taken at 1 h incubation indicate that the bright luminescent spots generate a mixture of “spherical” type aggregates and short chains of peptides that resemble the beginning of the nucleation phase,88 Figure 5. The images taken after a 3 h incubation clearly show that the wormlike structures of Aβ1−40 come closer to each other to form an elongated structure, similar to protofibrills, that resembles the elongation phase of the formation of amyloid fibrils,88 Figure 5. The enlarged figure clearly shows that the protofibrill is formed by the continuous stacking of the curvilinear units, which are superimposed following an organized orientation. After 6 h of incubation, the enlarged images reveal a large number of wormlike, U-shaped structures and several arrangements of Aβ of different sizes, all of them randomly distributed. Note that all the images show that the luminescence of the complex 2 follows an ordered distribution which accompanies the fibril length, indicating that the complex can recognize and align specific sites of the Aβ peptide. On the basis of these results, it seems that the complexes interact with the Aβ by binding to the prefibrillar oligomeric species present at or near the nucleation phase. The most probable target would be the hydrophobic regions of Aβ. This hypothesis is supported by the lack of changes on the luminescent imaging of complexes when Aβ1−40 was replaced by Aβ22−35, whose apolar residues Val18 and Phe20 are absent, Figure 6. Therefore, it is likely that the site formed by the amino acids Phe19, Phe20, and/or Val18 are involved in the binding of the complex to Aβ1−40. These amino acids are located at the central hydrophobic core region of Aβ1−40 between residues 17 and 21.89 This region is critical for the growth of full-length Aβ fibril and for stabilizing aggregates; consequently, it is more

containing two amino groups had a better scavenging effect than that of derivatives with one amino group. Real-Time Following and Imaging Aβ Self-Aggregation by FLIM Technique. The luminescence of the complexes provided a valuable opportunity to monitor, in real time, the selfaggregation of Aβ by imaging of conformational changes of Aβ during aggregation after incubation with the complexes. The experiment was performed in aqueous solution and in thin films with a time correlated single photon counting fluorescence lifetime imaging microscopy (TCSPC-FLIM) equipment. It is important to note that the Aβ1−40 itself presents an intrinsic fluorescence that is visible as the background of the image shown in Figure 4. This behavior is in agreement with previous works regarding fluorescent images of the poly(ValGlyGlyLeuGly) amyloid-like fibril, under neutral aqueous solution conditions, by confocal microscopy.87 Figure 4 shows that the intensity of luminescence of the complexes is strong enough to be distinguished from the background fluorescence of Aβ, demonstrating that the complexes can be employed to monitor, in real time, the self-aggregation of Aβ using the FLIM technique. Also, the images show that the spread luminescent spots of complexes at incubation time zero change over time to form many bright spots inserted into the small clusters (assemblies) that correlate with the progress of aggregation. After 24 h, only a diffuse and dense cluster of aggregates is observed.

Figure 4. Luminescent confocal images at 470 nm light excitation and 488 nm long-pass emission corresponding to 0, 1, 3, 6, and 24 h of Aβ1−40 incubated with complexes 2, 1, and ThT in phosphate buffer (pH 7.4 and 100 mmol L−1 of NaCl). 9220

DOI: 10.1021/acs.jmedchem.6b01130 J. Med. Chem. 2016, 59, 9215−9227

Journal of Medicinal Chemistry

Article

Figure 5. Fluorescent lifetime images at 470 nm excitation and 488 nm long-pass emission, corresponding to 1 and 3 h of Aβ1−40 incubated with complex 2 in thin films produced from the respective phosphate buffer samples (pH 7.4 and 100 mmol L−1 of NaCl) and an expanded image of the highlighted region taken at a higher pixel resolution showing protofibrils formation.

Figure 6. Fluorescent lifetime images at 470 nm light excitation and 488 nm long-pass emission corresponding to (A) 0 and 24 h of Aβ22−35 incubated with 2, in phosphate buffer (pH 7.4 and 100 mmol L−1 of NaCl). (B) Light scattering of the image taken at 24 h incubation.

mainly by hydrophobic interactions through π−π stacking.90 Moreover, the C-terminus is also critical for self-assembly of Aβ.91−94 For example, mutations of the Phe19 and Phe20 produced spherical aggregates on the C-terminus and restrict

susceptible to neurotoxicity87−89 and became a good starting point for the design of Aβ markers as well as inhibitors of Aβ. Previous studies have indicated that the two phenyalanine residues (Phe19 and Phe20) favor nucleation aggregation 9221

DOI: 10.1021/acs.jmedchem.6b01130 J. Med. Chem. 2016, 59, 9215−9227

Journal of Medicinal Chemistry

Article

Figure 7. Luminescent confocal images at 470 nm light excitation and 488 nm long-pass emission, corresponding to 1 and 24 h of Aβ15−21 incubated with complex 2, in phosphate buffer (pH 7.4 and 100 mmol L−1 of NaCl).

of bright spots which follow a pattern of alignment but do not form the protofibrills. In a more comprehensive analysis, the enlarged images show various sequences of Aβ/Aβ interactions which suggest that Aβ15−21 polymerization is a dynamic process. The sequence of images shown in Figure 7 indicates that the globular structure is built by the contact of the U-shaped units surrounding a central axis in an ordered arrangement. Globular structures of Aβ are an alternative pathway to fibril formation proposed by theoretical and experimental techniques.95−97 However, to our knowledge, this is the first study to display images of the structural evolution of self-aggregation of the short fragment Aβ15−21 in aqueous solution in real time by FLIM. Also important is the imaging of the ordered arrangement of the protofibrill at the Aβ1−40 peptide. In summary, these results provide experimental evidence that the noncovalent interaction of complex with the Aβ induces site-specific conformational alterations in the peptide. The hydrophobic interactions induce protofibrill formation in the Aβ1−40. Moreover, the hydrogen donor interactions between the NH3+ group of complex and Ala21 (C-terminus) residues of the short fragment Aβ15−21 interfere with the capability of

the conformational freedom of Aβ to form an extended conformation. Both modifications were demonstrated to prevent fibrillization.1 For this reason, we selected the fragment Aβ15−21 to study the complex/Aβ interaction. We chose this fragment for two reasons: first, because it contains the hydrophobic amino acids of interest which would strengthen the π−π interaction between the phen moiety of complex and Phe20; second, at pH 7.4 and due to the proximity of Ala21 to Phe20, hydrogen donor bonds and electrostatic interactions between the protonated NH3+ groups of the Apy moiety of complex and the negative charged carboxylate CO2− groups of Ala21(C terminus) would be highly favored. If this pathway is feasible, then it certainly would restrict the conformational freedom of peptide, would be competing with hydrogen bonds either among protofibrills or β-sheets, and would serve as a limitation of the elongation phase to form the fibrils. As a consequence, an alternative pathway for Aβ self-assembly would be expected. The luminescent images of Aβ15−21/complex show U-shaped conformers and smaller aggregates at 1 h of incubation, Figure 7. After 24 h of incubation, it is possible to observe a large number 9222

DOI: 10.1021/acs.jmedchem.6b01130 J. Med. Chem. 2016, 59, 9215−9227

Journal of Medicinal Chemistry

Article

resonance experiments, which were run by irradiation at −400 and 20 000 Hz, respectively. A train of 2000 Gaussian pulses of 1 ms each with a 4 μs delay between pulses was used, with a total saturation time of 5s. Cell Culture and Differentiation. Mouse Neuro-2a cells (ATCC: CCL-131, kindly provided by Dr. Isaias Glesser, Unifesp) were routinely grown under 5% CO2 at 37 °C in DMEM (Sigma-Aldrich, USA) supplemented with 10% FBS (Invitrogen, USA) and 1% penicillin/streptomycin (Invitrogen, USA). Before each assay, the cellular differentiation was followed by neurites outgrowth and was achieved by replacing the culture medium with differentiation medium (DMEM supplemented with 2% FBS and 20 μM of retinoic acid) for 72 h. The Neuro2A cells were independently treated with 1 or 2 (0.5 μmol L−1) in differentiation medium for 24 h in a humidified incubator at 37 °C and 5% CO2. Confocal Laser Scanning Microscopy. The cell localization was obtained by following the intrinsic luminescence of the ruthenium complexes. The cells were grown in differentiation medium in 40 mm culture dishes. After 48 h, the cells were incubated with 1 or 2 (0.5 μM) for 24 h. The cellular nucleus was labeled by incubating the cells with Hoechst-33374 (Molecular Probes, USA) for 15 min. After that, the dishes were washed 3 times and kept in phosphate buffer solution (PBS) during the experiments. The images were obtained using an inverted Zeiss LSM 780 confocal laser scanning microscope (INFAR Confocal and Flow Cytometry Facility, Unifesp) and light excitation at 450 nm and emission at 590 nm. Hoechst-33374 spectrum was excited at 346 nm and emitted at 460 nm wavelengths. The images were analyzed using the ImageJ software.98 MTT Viability Assay. The cell toxicity of 1 or 2 was assessed by the MTT reduction inhibition assay.99,100 The cells were plated at a density of 5000 cells/well on 96-well plate in culture medium without penicillin/streptomycin. After 48 h, the cells were then incubated for 24 h with the desired concentration of 1 or 2. Next, an amount of 10 μL of MTT (10 mg mL−1) was added to each well. After 2 h, the solution of each well was carefully aspirated and an amount of 100 μL of DMSO was added to each sample that was incubated at room temperature until complete cell lysis. The absorbance of the formazan was measured at 570 nm in Ultra microplate reader EL 808 (Bio-Tek Instruments). The percentage of viable cells was calculated as the ratio of the absorbance of treated cells to the absorbance of control groups. Experiments were carried out in triplicate. Cholinesterases Inhibition. The inhibition of acetylcholinesterase was evaluated for enzymes from Electrophorus electricus (ee-AChE) and human recombinant (h-AChE) and the butyrylcholinesterase enzyme was human serum enzyme (h-BuChE), using the spectrophotometric method proposed by Ellman.66 Stock solutions of complexes and free ligands were prepared in methanol. A mixture containing 100 μL of the complex, 2.875 mL of a solution containing Tris-HCl buffer (50 mmol L−1, pH 8.0), DTNB (333 μmol L−1), NaCl (0.1 mol L−1), MgCl2 (0.02 mol L−1), and 15 μL of enzyme solution (prepared in 15 μmol L−1 solution of BSA in Tris-HCl buffer, 50 mmol L−1, pH 8.0) was incubated for 15 min at room temperature. After this period, the reaction was initiated with the addition of 10 μL of substrate, acetylthiocholine iodide, or butyrylthiocholine iodide. The hydrolysis of the substrate could be observed by the formation of a yellow compound (5-thionitrobenzoate). Absorbance was measured at 412 nm. Measurements were made after 5 min of the hydrolysis reaction. The inhibition type was justified by the linear Lineweaver− Burk regression. The value of Ki was obtained from the nonlinear regression using the GraphPad Prism software. EPR. Electron paramagnetic resonance (EPR) experiments were performed with a Bruker EMXplus spectrometer at the X-band frequency (9.4 GHz). Measurements were performed at 298 K. The samples were loaded into the cavity using a peristaltic pump (WatsonMarlow, Pump-Pro MPL-579). At the center of the resonant cavity, a quartz capillary tube (internal diameter = 1.0 mm, volume = 80 μL) was used as a sample holder. Hydroxyl Radical Scavenging. Hydroxyl radical was generated using the Fenton reaction. Solutions of 3.3 × 10−4 mol L−1 FeSO4 (final concentration) and 2.2 × 10−3 mol L−1 H2O2 (final concentration) were

hydrogen bonding of the C-terminal face of peptide and consequently induce globular structures in solution instead of fibril, as expected for Aβ17−21. These results open new frontiers for the application of cis[Ru(phen)2(Apy)2]2+ complex in diagnostic imaging, especially to gain further insight into the mechanism of self-aggregation of amyloid proteins.



CONCLUSION The RuApy complexes expand the potential applications of luminescent complexes for investigation of amyloid proteins. The feasible synthesis, dark stability, and water solubility in a large range of pH, combined with their broad and intense visible absorption and emission with a tail in the near IR region, large Stokes shift, and long emission lifetime indicate the potential of the complexes as theranostic agents. The therapeutic properties investigated in vitro suggested that the complexes are nontoxic to the Neuro2A cells and exhibit a protective effect against ROS and inhibitory effect to cholinesterase enzymes activity. The high sensitivity of the luminescence of complexes that allows in vitro image of neuronal cells and in real time the direct observation of structural evolution of Aβ monomers to protofibrills (Aβ1−40) and globular oligomers (Aβ15−21), with no apparent loss of luminescence, is feasible and provides a tool for visualization and opens new frontiers for the elucidation of the biochemical stages of amyloid proteins.



EXPERIMENTAL SECTION

General. The reagents were purchased from Aldrich and used without further purification. Elemental analysis shows that the complexes are greater than 95% pure. 1H NMR analyses were used to confirm the purity of all compounds. Materials. All chemicals used for inhibition experiments were obtained from Sigma-Aldrich: acetylcholinesterase from Electrophorus electricus (ee-AChE) and human recombinant (h-AChE), butyrylcholinesterase from human serum (h-BuChE), acetylthiocholine iodide, butyrylthiocholine iodide, and 5,5′-dithiobis(2-nitrobenzoic acid) (3,3′-6) (DTNB). The peptides Aβ1−40, Aβ22−35, and Aβ15−21 were purchased from GenScript. cis-[Ru(phen)2(3,4APy)2](PF6)2 (2). cis-RuCl2(phen)2 (0.1 mmol) was dissolved in a 1:1 EtOH/H2O mixture (10 mL), then 3,4APy (0.2 mmol) ligand was added. The solution was stirred under nitrogen atmosphere for 8 h under reflux. A stoichiometric amount of NH4PF6 was added to precipitate the complex. The dark red precipitate was filtered, washed with water and ethanol, and then dried under vacuum. Anal. Calcd for RuC34H30N10F12P2: C, 42.11; H, 3.12; N, 14.44. Found: C, 42.29; H, 3.19; N, 14.21%. Solubility. Solubility of the complexes in aqueous buffer phosphate pH 7.4 (100 mmol L−1) was determined by the shake-flask method at 37.0 ± 0.5 °C. These experiments were conducted by adding an appropriate amount of complex until saturation in 2 mL of buffer solution. Suspensions were shaken for 24 h at 50 rpm until equilibrium was attained. Samples were centrifuged at 220 rpm. The concentration of the complex in the filtrate was determined by UV−vis spectrophotometry. NMR Experiments. All NMR experiments were conducted in a Bruker Avance III 600 MHz spectrometer, by employing a 1H {13C,15N} TCI triple resonance cryogenic cooled probehead, equipped with a z-gradient coil producing a nominal maximum gradient of 53.5 G cm−1. To simulate the biological medium, all the experiments were run at 37 °C. STD-NMR. STD-NMR experiments in aqueous sodium phosphate buffer, pD 7.4 (75 mmol L−1; NaCl 150 mmol L−1), were conducted with hAChE enzyme (4 × 10−6 mol L−1) and the complex 2 (400 × 10−6 mol L−1). A recovery time of 4 s of acquisition time by collecting 64 k points was used. 256 scans were averaged for both on- and off 9223

DOI: 10.1021/acs.jmedchem.6b01130 J. Med. Chem. 2016, 59, 9215−9227

Journal of Medicinal Chemistry

Article

mixed. DMPO was used to probe the hydroxil radical (OH•) consumption using electron paramagnetic resonance (EPR) spectroscopy. The concentration of DMPO (5,5-dimethylpyrroline N-oxide, SigmaAldrich) was monitored using UV−vis spectroscopy (λmax = 236 nm, ε = 7200 L mol−1 cm−1). DPPH• and ABTS•+ Scavenging. A methanolic solution of DPPH• at a final concentration of 2.5 × 10−4 mol L−1 was mixed with complexes or free ligands solutions. The time-decay of EPR signal intensity of the DPPH• was evaluated. The ABTS•+ radical cation (ABTS•+) was produced by reacting ABTS (7 × 10−3 mol L−1) solution with 2.5 × 10−3 mol L−1 potassium persulfate (final concentration). The mixture was kept in the dark at room temperature for 16 h. The ABTS•+ stock solution was mixed with different concentrations of complexes or free ligands, and the reaction was monitored via the decay of visible absorbance at 734 nm. Preparation of Aβ. The Aβ peptide was dissolved in 200 μL of aqueous NaOH solution, sonicated in a bath sonicator, and then filtered through 0.2 μm centrifuge filters. The filtered solution was diluted in PBS (100 mmol L−1 sodium phosphate, 150 mmol L−1 NaCl, pH 7.4) to 50 μmol L−1. An aliquot of the concentrated complex solution was added to Aβ solution to obtain a concentration of 1 μmol L−1 of complex. The samples were incubated at 37 °C in an orbital shaker at 900 rpm for 24 h. The time evolution of Aβ1−40 aggregation was investigated by removing one sample from centrifuge at regular time intervals and examining it by FLIM. For comparison purposes, ThT was also assayed under the same experimental conditions. The thin films were obtained by a careful evaporation of the solution with a very slow and continuous N2 flux of the same sample prepared in phosphate buffer in a glass coverslip without using a stopper after evaporation. Time Resolved Fluorescence Decay. The fluorescence intensity decay was obtained using the time-correlated single photon counting (TCSPC) technique. The excitation source was a titanium sapphire laser (Tsunami 3950 Spectra Physics), pumped by the solid-state laser Millenia X (Spectra Physics). The pulse repetition rate was set to 2 MHz by using a 3980 Spectra Physics pulse picker. The laser was tuned so that a second-harmonic generator (GWN-23PL Spectra Physics) gave an emergent beam at 480 nm that was directed to an Edinburgh FL900 spectrometer. A refrigerated Hamamatsu R3809U microchannel plate photomultiplier detected the emitted photons; and software provided by Edinburgh Instruments was used to analyze the decays, by fitting to multiexponential curves. Fluorescence Lifetime Imaging (FLIM). The MicroTime 200 (PicoQuant) system was used to collect fluorescence lifetime images. A picosecond laser pulse with wavelength 470 nm (from LDH-D-C470 diode laser) was reflected with a dichroic beam splitter centered at about 490 nm (490dcxr) into an inverted Olympus IX 71 microscope base. The samples were placed in a special 20 mm × 20 mm coverslip (Knittel glass, Germany) and excited when the light was focused by a 60× water immersion objective, numerical aperture 1.2. Fluorescence emission was collected with the same objective, passing through dichroic filter to remove scattered light before reaching a pinhole for confocal detection. A 488 nm long-pass filter (BLP01-488R) was added in the optical path to guarantee only fluorescence emission, detected with a single avalanche photodiode (SPAD) with timecorrelated single photon counting (TCSPC) method. All images were obtained in a 10−15 μL volume, and the films were scanned in the XY plane by a piezoscan through the excitation focus, in a fixed Z. Data were processed and images were obtained by an operating software of the system (SymPhoTime, PicoQuant), and their resolution was maintained at either 256 × 256 or 512 × 512 pixels.





EPR spectrum of DPPH, IC50 for scavenging of ABTS+•, Lineweaver−Burk regression and plots of absorbance of substrate by complex 2 (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] Phone: +55 1633518780. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The authors acknowledge FAPESP (Grants 2014/12538-8, 2014/07935-8, 2012/02065-0, 2014/24643-0, and 2014/ 26895-7), CNPq (Grant 304981/2012-5), and CAPES for the grants and fellowships received. The authors also acknowledge Dr. Antonio Carlos Roveda Júnior and Thiago ́ Abrahão Silva from the Instituto de Quimica de São Carlos at USP for the EPR experiments.



ABBREVIATIONS USED 3,4Apy, 3,4-aminopyridine; Apy, aminopyridine; CHNO, carbon, hydrogen, nitrogen, and oxygen; CR, Congo red dye; DIC, differential interference contrast; DMPO, 5,5-dimethyl-1pyrroline N-oxide; DPPH, 2,2-diphenyl-1-picrylhydrazyl; eeAChE, acetylcholinesterase from Electrophorus electricus; EXAFS, extended X-ray absoption fine structure; FLIM, fluorescence lifetime imaging; hAChE, human recombinant acetylcholinesterase; hBuChE, butyrylcholinesterase from serum enzyme; MLCT, metal to ligand charge transfer; PBS, phosphate buffer saline; Phen, 1,10-phenanthroline; STDNMR, saturation-transfer difference nuclear magnetic resonance; TCSPC-FLIM, time correlated single photon counting fluorescence lifetime imaging; TCSPC, time correlated single photon counting



REFERENCES

(1) Saura, C. A.; Parra-Damas, A.; Enriquez-Barreto, L. Gene expression parallels synaptic excitability and plasticity changes in Alzheimer’s disease. Front. Cell. Neurosci. 2015, 9, 318. (2) Rauk, A. The chemistry of Alzheimer’s disease. Chem. Soc. Rev. 2009, 38, 2698−2715. (3) Serrano-Pozo, A.; Frosch, M. P.; Masliah, E.; Hyman, B. T. Neuropathological alterations in Alzheimer disease. Cold Spring Harbor Perspect. Med. 2011, 1, 1−23. (4) Nunomura, N.; Perry, G.; Aliev, A.; Hirai, K.; Takeda, A.; Balraj, E. K.; Jones, P. K.; Ghanbari, H.; Wataya, T.; ShimohamA, S.; Chiba, S.; Atwood, C. S.; Petersen, R. B.; Smith, M. A. Oxidative damage is the earliest event in Alzheimer disease. J. Neuropathol. Exp. Neurol. 2001, 60, 759−760. (5) Zündorf, G.; Reiser, G. Calcium dysregulation and homeostasis of neural calcium in the molecular mechanisms of neurodegenerative diseases provide multiple targets for neuroprotection. Antioxid. Redox Signaling 2011, 14, 1275−1288. (6) Kumar, A.; Singh, A.; Ekavali. A review on Alzheimer’s disease pathophysiology and its management: an update. Pharmacol. Rep. 2015, 67, 195−203. (7) Liu, C.-C.; Kanekiyo, T.; Xu, H.; Bu, G. Apolipoprotein E and Alzheimer disease: risk, mechanisms, and therapy. Nat. Rev. Neurol. 2013, 9, 106−118. (8) Masters, C. L.; Selkoe, D. J. Biochemistry of amyloid β-protein and amyloid deposits in Alzheimer disease. Cold Spring Harbor Perspect. Med. 2012, 2, a006262.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01130. 1 H NMR spectra in DMSO-d6 and in different pH for Ru3,4Apy, solubility, excited-state lifetimes, in vitro uptake of Neuro2A cells and cell proliferation assay, 9224

DOI: 10.1021/acs.jmedchem.6b01130 J. Med. Chem. 2016, 59, 9215−9227

Journal of Medicinal Chemistry

Article

(9) Karran, E.; Mercken, M.; De Strooper, B. The amyloid cascade hypothesis for Alzheimer’s disease: an appraisal for the development of therapeutics. Nat. Rev. Drug Discovery 2011, 10, 698−712. (10) Engelborghs, S.; De Vreese, K.; Van de Casteele, T.; Vanderstichele, H.; Van Everbroeck, B.; Cras, P.; Martin, J.-J.; Vanmechelen, E.; De Deyn, P. P. Diagnostic performance of a CSFbiomarker panelin autopsy-confirmed dementia. Neurobiol. Aging 2008, 29, 1143−1159. (11) Williams, S. C. Alzheimer’s imaging agents struggle to find a market outside trials. Nat. Med. 2013, 19, 1551. (12) Liang, H. C.; Russell, C.; Mitra, V.; Chung, R.; Hye, A.; Bazenet, C.; Lovestone, S.; Pike, I.; Ward, M. Glycosylation of human plasma clusterin yields a novel candidate biomarker of Alzheimer’s disease. J. Proteome Res. 2015, 14, 5063−5076. (13) Stefani, M.; Rigacci, S. Protein folding and aggregation into amyloid: the interference by natural phenolic compounds. Int. J. Mol. Sci. 2013, 14, 12411−12457. (14) Valensin, D.; Gabbiani, C.; Messori, L. Metal compounds as inhibitors of β-amyloid aggregation. Perspectives for an innovative metallotherapeutics on Alzheimer’s disease. Coord. Chem. Rev. 2012, 256, 2357−2366. (15) Gibson, T. J.; Murphy, R. M. Design of peptidyl compounds that affect β-amyloid aggregation: importance of surface tension and context. Biochemistry 2005, 44, 8898−8907. (16) Funke, S. A.; Willbold, D. Peptides for therapy and diagnosis of Alzheimer’s disease. Curr. Pharm. Des. 2012, 18, 755−767. (17) Nilsson, K. P. R. Small organic probes as amyloid specific ligands − Past and recent molecular scaffolds. FEBS Lett. 2009, 583, 2593−2599. (18) Li, H.; Rahimi, F.; Sinha, S.; Maiti, P.; Bitan, G.; Murakami, K. Amyloids and protein aggregation-analytical methods. In Encyclopedia of Analytical Chemistry; Meyers, R. A., Ed.; Wiley: New York, 2009; pp 1−23. (19) Munishkina, L. A.; Fink, A. L. Fluorescence as a method to reveal structures and membrane-interactions of amyloidogenic proteins. Biochim. Biophys. Acta, Biomembr. 2007, 1768, 1862−1885. (20) Anderson, V. L.; Webb, W. W. Transmission electron microscopy characterization of fluorescently labelled amyloid β 1-40 and α-synuclein aggregates. BMC Biotechnol. 2011, 11, 1−10. (21) Sepkhanova, I.; Drescher, M.; Meeuwenoord, N. J.; Limpens, R. W. A. L.; Koning, R. I.; Filippov, D. V.; Huber, M. Monitoring Alzheimer amyloid peptide aggregation by EPR. Appl. Magn. Reson. 2009, 36, 209−222. (22) Sarroukh, R.; Goormaghtigh, E.; Ruysschaert, J.-M.; Raussens, V. ATR-FTIR: A “rejuvenated” tool to investigate amyloid proteins. Biochim. Biophys. Acta, Biomembr. 2013, 1828, 2328−2338. (23) LeVine, H., III Quantification of β-sheet amyloid fibril structures with thioflavin T. Methods Enzymol. 1999, 309, 274−284. (24) Leuma-Yona, R.; Mazères, S.; Faller, P.; Gras, E. Thioflavin derivatives as markers for amyloid-beta fibrils: insights into structural features important for high-affinity binding. ChemMedChem 2008, 3, 63−66. (25) Groenning, M. Binding mode of thioflavin T and other molecular probes in the context of amyloid fibrilscurrent status. J. Chem. Biol. 2010, 3, 1−18. (26) Bose, P. P.; Chatterjee, U.; Xie, L.; Johansson, J.; Göthelid, E.; Arvidsson, P. I. Effects of Congo red on Aβ1−40 fibril formation process and morphology. ACS Chem. Neurosci. 2010, 1, 315−324. (27) Wu, C.; Scott, J.; Shea, J. E. Binding of congo red to amyloid protofibrils of the Alzheimer Aβ9−40 peptide probed by molecular dynamics simulations. Biophys. J. 2012, 103, 550−557. (28) Wu, C.; Wang, Z.; Lei, H.; Zhang, W.; Duan, Y. Dual binding modes of congo red to amyloid protofibril surface observed in molecular dynamics simulations. J. Am. Chem. Soc. 2007, 129, 1225− 1232. (29) Tao, K.; Wang, J.; Zhou, P.; Wang, C.; Xu, H.; Zhao, X.; Lu, J. R. Self-assembly of short Aβ(16−22) peptides: Effect of terminal capping and the role of electrostatic interaction. Langmuir 2011, 27, 2723−2730.

(30) Walsh, D. M.; Tseng, B. P.; Podlisny, M. B.; Selkoe, D. J. The oligomerization of amyloid beta-protein begins intracellularly in cells derived from human brain. Biochemistry 2000, 39, 10831−10839. (31) Ishii, K.; KlunK, W. E.; Arawaka, S.; Debnath, M. L.; Furiya, Y.; Sahara, N.; Shoji, S.; Tamaoka, A.; Pettegrew, J. W.; Mori, H. Chrysamine G and its derivative reduce amyloid-B induced neurotoxicity in mice. Neurosci. Lett. 2002, 333, 5−8. (32) Reinke, A. A.; Gestwicki, J. E. Structure−activity relationships of amyloid beta-aggregation inhibitors based on curcumin: influence of linker length and flexibility. Chem. Biol. Drug Des. 2007, 70, 206−215. (33) Yao, S.; Cherny, R. A.; Bush, A. I.; Masters, C. L.; Barnham, K. J. Characterizing bathocuproine self-association and subsequent binding to Alzheimer’s disease amyloid beta-peptide by NMR. J. Pept. Sci. 2004, 10, 210−217. (34) Kenche, V. B.; Barnham, K. J. Alzheimer’s disease & metals: therapeutic opportunities. Br. J. Pharmacol. 2011, 163, 211−219. (35) Hayne, D. J.; Lim, S.; Donnelly, P. S. Metal complexes designed to bind to amyloid-β for the diagnosis and treatment of Alzheimer’s disease. Chem. Soc. Rev. 2014, 43, 6701−6715. (36) Valensin, D.; Gabbiani, C.; Messori, L. Metal compounds as inhibitors of β-amyloid aggregation. Perspectives for an innovative metallotherapeutics on Alzheimer’s disease. Coord. Chem. Rev. 2012, 256, 2357−2366. (37) Spinello, A.; Bonsignore, R.; Barone, G.; Keppler, B. K.; Terenzi, A. L. Metal ions and metal complexes in Alzheimer’s disease. Curr. Pharm. Des. 2011, 163, 211−219. (38) Barnham, K. J.; Kenche, V. B.; Ciccotosto, G. D.; Smith, D. P.; Tew, D. J.; Liu, X.; Perez, K.; Cranston, G. A.; Johanssen, T. J.; Volitakis, I.; Bush, A. I.; Masters, C. L.; White, A. R.; Smith, P. J.; Cherny, R. A.; Cappai, R. Platinum-based inhibitors of amyloid-β as therapeutic agents for Alzheimer’s disease. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 6813−6818. (39) Ma, G.; Wang, E.; Wei, H.; Wei, K.; Zhu, P.; Liu, Y. PtCl2(phen) disrupts the metal ions binding to amyloid-β peptide. Metallomics 2013, 5, 879−887. (40) Pinato, O.; Musetti, C.; Sissi, C. Pt-based drugs: the spotlight will be on proteins. Metallomics 2014, 6, 380−395. (41) Streltsov, V. A.; Epa, V. C.; James, S. A.; Churches, Q. I.; Caine, J. I.; 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. (42) 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 agent. ACS Chem. Neurosci. 2010, 1, 691−701. (43) 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. (44) Kratz, F.; Hartmann, M.; Keppler, B.; Messori, L. The binding properties of two antitumor ruthenium(III) complexes to apotransferrin. J. Biol. Chem. 1994, 269, 2581−2588. (45) 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. (46) Jones, M. R.; Mu, C.; Wang, M. C. P.; Webb, M. I.; Walsby, C. J.; Storr, C. Modulation of the Aβ peptide aggregation pathway by KP1019 limits Aβ-associated neurotoxicity. Metallomics 2015, 7, 129− 135. (47) Valensin, D.; Anzini, P.; Gaggelli, E.; Gaggelli, N.; Tamasi, G.; Cini, R.; Gabbiani, C.; Michelucci, E.; Messori, L.; Kozlowski, H.; Valensin, G. fac-{Ru(CO)3}2+ selectively targets the histidine residues of the β-amyloid peptide 1−28. Implications for new Alzheimer’s disease treatments based on ruthenium complexes. Inorg. Chem. 2010, 49, 4720−4722. (48) 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 9225

DOI: 10.1021/acs.jmedchem.6b01130 J. Med. Chem. 2016, 59, 9215−9227

Journal of Medicinal Chemistry

Article

binding promiscuity to selective inhibition. Chem. Biol. 2003, 10, 341− 349. (69) Kearney, P. C.; Mizoue, L. S.; Kumpf, R. A.; Forman, J. E.; McCurdy, A.; Dougherty, D. A. Molecular recognition in aqueous media. New binding studies provide further insights into the cation-pi. interaction and related phenomena. J. Am. Chem. Soc. 1993, 115, 9907−9919. (70) Lane, R. M.; Potkin, S. G.; Enz, A. Targeting acetylcholinesterase and butyrylcholinesterase in dementia. Int. J. Neuropsychopharmacol. 2006, 9, 101−124. (71) Copeland, R. A. Evaluation of Enzyme Inhibitors in Drug Discovery; Wiley: New York, 2005; p 57. (72) Segel, I. H. Enzyme Kinetics: Behaviour and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems; Wiley: New York, 1975; pp 107−111, 132−134. (73) Sugimoto, H.; Ogura, H.; Arai, Y.; Iimura, Y.; Yamanishi, Y. Research and development of donepezil hydrochloride, a new type of acetylcholinesterase inhibitor. Jpn. J. Pharmacol. 2002, 89, 7−20. (74) Bai, D. L.; Tang, X. C.; He, X. C. Huperzine A, a potential therapeutic agent for treatment of Alzheimer’s disease. Curr. Med. Chem. 2000, 7, 355−374. (75) Chatonnet, A.; Lockridge, O. Comparison of butyrylcholinesterase and acetylcholinesterase. Biochem. J. 1989, 260, 625−634. (76) Nachon, F.; Carletti, E.; Ronco, C.; Trovaslet, M.; Nicolet, Y.; Jean, L.; Renard, P.-Y. Crystal structures of human cholinesterases in complex with huprine W and tacrine: elements of specificity for antiAlzheimer’s drugs targeting acetyl- and butyryl-cholinesterase. Biochem. J. 2013, 453, 393−399. (77) 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. (78) Camps, P.; Formosa, X.; Galdeano, C.; Gómez, T.; MuñozTorrero, D.; Scarpellini, M.; Viayna, M.; Badia, A.; Clos, M. V.; Camins, A.; Pallàs, M.; Bartolini, M.; Mancini, F.; Andrisano, V.; Estelrich, J.; Lizondo, M.; Bidon-Chanal, A.; Luque, F. A. Novel donepezil-based inhibitors of acetyl- and butyrylcholinesterase and acetylcholinesterase-induced β-amyloid aggregation. J. Med. Chem. 2008, 51, 3588−3598. (79) Marco-Contelles, J.; León, R.; delosRíos, C.; Samadi, A.; Bartolini, M.; Andrisano, V.; Huertas, O.; Barril, X.; Luque, F. J.; Rodríguez-Franco, M. I.; López, B.; López, M. G.; García, A. G.; do Carmo Carreiras, M.; Villarroya, M. Tacripyrines, the first tacrine dihydropyridine hybrids, as multitarget-directed ligands for the treatment of Alzheimer’s disease. J. Med. Chem. 2009, 52, 2724−2732. (80) Rizzo, S.; Rivière, C.; Piazzi, L.; Bisi, A.; Gobbi, S.; Bartolini, M.; Andrisano, V.; Morroni, F.; Tarozzi, A.; Monti, J.-P.; Rampa, A. Benzofuran-based hybrid compounds for the inhibition of cholinesterase activity, beta amyloid aggregation, and abeta neurotoxicity. J. Med. Chem. 2008, 51, 2883−2886. (81) Lloyd, R. V.; Hanna, P. M.; Mason, R. P. The origin of the hydroxyl radical oxygen in the Fenton reaction. Free Radical Biol. Med. 1997, 22, 885−888. (82) Metzker, G.; De Aguiar, I.; Souza, M. L.; Cardoso, D. R.; Franco, D. W. Reaction of ruthenium(II) complexes with 2,2diphenyl-1-picrylhydrazyl (DPPH•) and hydroxyl radicals. Can. J. Chem. 2014, 92, 788−793. (83) Boligon, A. A.; Machado, M. M.; Athayde, M. L. Technical evaluation of antioxidant activity. Med. Chem. 2014, 4, 517−522. (84) Floegel, A.; Kim, D.-O; Chung, S.-J.; Chun, O. K. Comparison of ABTS/DPPH assays for the detection of antioxidant capacity in foods. FASEB J. 2010, 24, 535−539. (85) Dawidowicz, A. L.; Wianowska, D.; Olszowy, M. On practical problems in estimation of antioxidant activity of compounds by DPPH method (Problems in estimation of antioxidant activity). Food Chem. 2012, 131, 1037−1043. (86) Hu, Y.; Zhang, J.; Yu, C.; Li, Q.; Dong, F.; Wang, G.; Guo, Z. Synthesis, characterization, and antioxidant properties of novel inulin

(II) polypyridyl complex as inhibitor of acetylcholinesterase and Aβ aggregation. Eur. J. Med. Chem. 2014, 75, 375−381. (49) Cook, N. P.; Torres, V.; Jain, D.; Martí, A. A. Sensing amyloid-β aggregation using luminescent dipyridophenazine ruthenium(II) complexes. J. Am. Chem. Soc. 2011, 133, 11121−11123. (50) Cook, N. C.; Ozbil, M.; Katsampes, C.; Prabhakar, R.; Martí, A. A. Unraveling the photoluminescence response of light-switching ruthenium(II) complexes bound to amyloid-β. J. Am. Chem. Soc. 2013, 135, 10810−10816. (51) 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. (52) Mari, C.; Pierroz, V.; Ferrari, S.; Gasser, G. Combination of Ru(II) complexes and light: new frontiers in cancer therapy. Chem. Sci. 2015, 6, 2660−2686. (53) Rademaker-Lakhai, J. M.; van den Bongard, D.; Pluim, D.; Beijnen, J. H.; Schellens, J. H. A phase I and pharmacological study with imidazolium-trans-DMSO-imidazole-tetrachlororuthenate, a novel ruthenium anticancer agent. Clin. Cancer Res. 2004, 10, 3717− 3727. (54) Juris, A.; Balzani, V.; Barigelletti, F.; Campagna, S.; Belser, P.; von Zelewsky, A. Ru(II) polypyridine complexes: photophysics, photochemistry, eletrochemistry, and chemiluminescence. Coord. Chem. Rev. 1988, 84, 85−277. (55) Gill, M. R.; Thomas, J. A. Ruthenium (II) polypyridil complexes and DNAfrom structural probes to cellular imaging and therapeutics. Chem. Soc. Rev. 2012, 41, 3179−3192. (56) Sinha, S. K.; Shrivastava, S. K. Synthesis and evaluation of some new 4-aminopyridine derivatives as a potent antiamnesic and cognition enhancing drugs. Med. Chem. Res. 2012, 21, 4395−4402. (57) Wang, Y.; Mattson, M. P. L-type Ca(2+) currents at CA1 synapses, but not CA3 or dentate granule neuron synapses, are increased in 3xTgAD mice in an age-dependent manner. Neurobiol. Aging 2014, 35, 88−95. (58) Rosini, M.; Simoni, E.; Milelli, A.; Minarini, A.; Melchiorre, C. Oxidative stress in Alzheimer’s disease: are we connecting the dots? J. Med. Chem. 2014, 57, 2821−2831. (59) Huang, X.; Moir, R. D.; Tanzi, R. E.; Bush, A. I.; Rogers, J. T. Redox-active metals, oxidative stress, and Alzheimer’s disease pathology. Ann. N. Y. Acad. Sci. 2004, 1012, 153−163. (60) Sayre, L. M.; Perry, G.; Smith, M. A. Oxidative stress and neurotoxicity. Chem. Res. Toxicol. 2008, 21, 172−188. (61) Camilo, M. R.; Cardoso, C. R.; Carlos, R. M.; Lever, A. B. P. Photosolvolysis of cis-[Ru(α-diimine)2(4-Aminopyridine)2]2+ complexes; photophysical, spectroscopic, and density functional analysis. Inorg. Chem. 2014, 53, 3694−3708. (62) Chun-Ying, D.; Zhong-Lin, L.; Xiao-Zeng, Y.; Mak, T. C. W. Crystal structure and photochemistry of bis(pyridine)-bis(4-aminopyridine) ruthenium(II). J. Coord. Chem. 1999, 46, 301−312. (63) Johnson, E. S.; Sullivan, B. P.; Salmon, D. J.; Adeyemi, A.; Meyer, T. Synthesis and properties of the chloro-bridged dimer [(bpy)2RuCl2]2+ and its transient 3+ mixed-valence ion. Inorg. Chem. 1978, 17, 2211−2215. (64) Durham, W.; Walsh, J. L.; Carter, C. L.; Meyer, T. J. Synthetic aplications of photosubstitution reactions of poly(pyridil) complexes of ruthenium (II). Inorg. Chem. 1980, 19, 860−865. (65) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer U.S.: New York, 2006. (66) Ellman, G. L.; Courtney, K. D.; Andres, V., Jr; Featherstone, R. M. A new and rapid colorimetric determination of acetycholinesterase activity. Biochem. Pharmacol. 1961, 7, 88−95. (67) Taylor, P.; Lappi, S. Interaction of fluorescent probes with acetylcholinesterase. The site and specificity of propidium binding. Biochemistry 1975, 14, 1989−1997. (68) Bencharit, S.; Morton, C. L.; Hyatt, J. L.; Kuhn, P.; Danks, M. K.; Potter, M. P.; Redinbo, M. R. Crystal Structure of human carboxylesterase 1 complexes with the Alzheimer’s drug tacrine: from 9226

DOI: 10.1021/acs.jmedchem.6b01130 J. Med. Chem. 2016, 59, 9215−9227

Journal of Medicinal Chemistry

Article

derivatives with amino-pyridine group. Int. J. Biol. Macromol. 2014, 70, 44−49. (87) Del Mercato, L. L.; Pompa, P. P.; Maruccio, G.; Della Torre, A.; Sabella, S.; Tamburro, A. M.; Cingolani, R.; Rinaldi, R. Charge transport and intrinsic fluorescence in amyloid-like fibrils. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 18019−18024. (88) Miti, T.; Mulaj, M.; Schmit, J. D.; Muschol, M. Stable, metastable, and kinetically trapped amyloid aggregate phases. Biomacromolecules 2015, 16, 326−335. (89) Li, J.; Liu, R.; Lam, K. S.; Jin, L.-W.; Duan, Y. Alzheimer’s diseases drug candidates stabilizes A-β protein native structure by interacting with the hydrophobic core. Biophys. J. 2011, 100, 1076− 1082. (90) Singh, V.; Rai, R. K.; Arora, A.; Sinha, N.; Thakur, A. K. Therapeutic implication of L-phenylalanine aggregation mechanism and its modulation by D-phenylalanine in phenylketonuria. Sci. Rep. 2013, 4, 3875. (91) Gazit, E. A possible role for pi-stacking in the self-assembly of amyloid fibrils. FASEB J. 2002, 16, 77−83. (92) Liu, L.; Niu, L.; Xu, M.; Han, M.; Han, Q.; Duan, H.; Dong, M.; Besenbacher, F.; Wang, C.; Yang, Y. Molecular tethering effect of Cterminus of amyloid peptide Aβ42. ACS Nano 2014, 8, 9503−9510. (93) Bett, C. K.; Serem, W. K.; Fontenot, K. R.; Hammer, R. P.; Garno, J. C. Effects of peptides derived from terminal modifications of the Aβ central hydrophobic core on Aβ fibrillization. ACS Chem. Neurosci. 2010, 1, 661−678. (94) Formaggio, F.; Bettio, A.; Moretto, V.; Crisma, M.; Toniolo, C.; Broxterman, Q. B. Disruption of the beta-sheet structure of a protected pentapeptide, related to the beta-amyloid sequence 17−21, induced by a single, helicogenic C-alpha tetrasubstituted alpha amino acid. J. Pept. Sci. 2003, 9, 461−466. (95) Kotler, S. A.; Brender, J. R.; Vivekanandan, S.; Suzuki, Y.; Yamamoto, K.; Monette, M.; Krishnamoorthy, J.; Walsh, P.; Cauble, M.; Holl, M. M. B.; Marsh, E. N. G.; Ramamoorthy, A. Highresolution NMR characterization of low abundance oligomers of amyloid-beta without purification. Sci. Rep. 2015, 5, 11811. (96) Luo, J.; Wärmländer, S. K.; Gräslund, A.; Abrahams, J. P. Alzheimer peptides aggregate into transient nanoglobules that nucleate fibrils. Biochemistry 2014, 53, 6302−6308. (97) Ehrnhoefer, D. E.; Bieschke, J.; Boeddrich, A.; Herbst, M.; Masino, L.; Lurz, R.; Engemann, S.; Pastore, A.; Wanker, E. E. EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers. Nat. Struct. Mol. Biol. 2008, 15, 558−566. (98) Schneider, C. A.; Rasband, W. S.; Eliceiri, K. W. NIH Image to Image J: 25 years of image analysis. Nat. Methods 2012, 9, 671−675. (99) Riboni, L.; Prinetti, A.; Bassi, R.; Caminiti, A.; Tettamanti, G. A mediator role of ceramide in the regulation of neuroblastoma neuro2a cell differentiation. J. Biol. Chem. 1995, 270, 26868−26875. (100) Freimoser, F. M.; Jakob, C. A.; Aebi, M.; Tuor, U. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities. Appl. Environ. Microbiol. 1999, 65, 3727−3729.

9227

DOI: 10.1021/acs.jmedchem.6b01130 J. Med. Chem. 2016, 59, 9215−9227