Photo-inhibition of Aβ fibrillation mediated by a newly designed fluorinated oxadiazole

Abstract

Uncontrolled aggregation of amyloid beta peptide (Aβ) is the main cause of Alzheimer's disease. Therapeutic approaches to intervention in amyloid diseases include the use of small molecules able to stabilize the soluble Aβ conformation, or to redirect the amyloidogenic pathway towards non-toxic and non-fibrillar states. Fluorometric measurements revealed that 3-(4′-trifluoromethylphenyl)-5-(4′-methoxyphenyl)-1,2,4-oxadiazole, when irradiated, is able to interact with the monomeric Aβ peptide readdressing the aggregation pathway toward the formation of amorphous aggregates as evidenced by CD, AFM, and SAXS measurements. We hypothesize that this compound, under radiation, forms a reactive intermediate that sticks on the Aβ peptide by interfering with its fibrillation process. Cytotoxicity assays performed on LAN5 neuroblastoma cells suggest that the presence of oxadiazole reduces the toxicity of Aβ. This finding might be the start of innovative therapies against Alzheimer's disease.

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Introduction

Alzheimer's Disease (AD) is a neurodegenerative pathology representing the most common form of dementia in the elderly. The affected individuals present progressive loss of memory, mood changes, communication and reasoning problems.^1–3 The pathology is characterized by the formation of extracellular amyloid plaques and intracellular neurofibrillary tangles in the brain.⁴ All these deposits are composed by well-ordered, β-sheet rich, fibers of the Amyloid β peptides (Aβ) produced by proteolysis of Amyloid Precursor Protein (APP) in residues of 39–43 amino acids. The evidence of the simultaneous presence of accumulated fibers and the cerebral tissue loss brought to the initial hypothesis that the proteinaceous aggregates are responsible for the neurodegenerative damage.^5–7 However, it has been shown that the species formed in the initial state of the aggregation kinetics could determine severe damage in the cerebral cells.^8–10 Much experimental evidence, in fact, highlighted the role of these species in the alteration of membranes, particularly of their solubility and permeability.^11,12 Moreover, the pathological presynaptic alteration was observed in transgenic mice brain before the neuronal degeneration associated with fibers accumulation. These observations suggest that the severity of the pathology is uncorrelated with the concentration of the mature fibers.^13–17 Despite much effort and the numerous studies conducted, the mechanism and the pathogenesis of AD are still a matter of debate. The problem is also compounded by the instability of the peptide both in the monomeric and early aggregated forms and by the presence of numerous polymorphic aggregates and fibers. In fact, Aβ aggregation is a complex process that seems to involve more than a simple conversion from an unstable monomer to mature fibrils.^18,19

Some therapeutic approaches trigger the disaggregation of the amyloid deposits and aggregates. Graphene-based materials or gold nanoparticles, with high affinity for deposits of Aβ fibers, were used to redissolve them, by using the local heat.^20–22

Over the last years, increasing interest has been focused on the study of natural or synthetic molecules capable of interfering with the aggregation and fibrillation of Aβ peptide.^23–25 The search of small inhibitors of the toxic effects of Aβ has produced a wide number of approaches and promising drugs.^26–32

Particularly, growing interest has been addressed to molecules able to drive the unstable monomers towards the formation of harmless and stable aggregates.³³ Indeed, the deviation of the aggregation mechanism toward off-pathway products seems to be a new promising strategy for the treatment of amyloid diseases.

For example, the inhibitor effects of curcumin on the fibrillation have been investigated by different authors.^25,27,34,35 Curcumin is a natural molecule characterized by anti-inflammatory and antioxidant activity. However, it crosses the blood–brain barrier (BBB) only when injected, and its effectiveness is reduced when added to the diet.^36,37 These limitations have prompted the synthesis of new derivatives having a major efficacy on the inhibition of aggregation.^38,39

The ferulic acid is another example of natural molecule with in vitro inhibitory activity. In fact, this molecule seems to interfere with the oligomer formation through the destabilization of Asp23–Lys28 salt bridge that is at “the basis of fibril stability”.^40,41

All these compounds share a common phenolic structure that seems to be responsible for the fibril destabilization.

A new branch of research in this field aims to the control of the amyloid aggregation through photochemical methods. For instance, it has been synthesized a “photo-clickable” Aβ_1–42 peptide with increased stability and solubility, whose primary structure is earned after photo-stimulation.³⁹ Very recently, the photochemical approach also allowed to control the peptide aggregation by means of riboflavin-mediated photooxygenation of Aβ_1–42, resulting into a decrease of Abeta toxicity and a reduction of the uncontrolled self-assembly.^42,43

The photo-stimulation is also used to trap, detect and characterize low molecular weight oligomers of Aβ-peptide using the PICUP technique (photo-induced protein cross-linking of unmodified proteins). This intriguing photo-chemical method seems to be well appropriate for structural studies of the transient and dynamic equilibrium between small oligomeric species of Aβ.⁴⁴ A characteristic of this technique is the short irradiation time required (few seconds) because of the involved metal complexes characterized by high photo-reactivity. However the short time of irradiation can make somehow difficult to redirect accurately the aggregation pattern toward the formation of small oligomers.⁴⁴ Moreover, the toxicity of involved metal complexes avoids the perspective of pharmaceutical development.

In this context, we decided to design a new not toxic photo-active molecule able to interact with Aβ in order to modulate its aggregation pattern by means of photo-stimulation.

Recent studies indicated 1,2,4-oxadiazoles as promising probes for detection of amyloid plaques in vivo due to their high affinity toward Aβ.⁴⁵ These interesting heterocycles, utilized in many pharmaceutical applications,^46–49 present a well known photochemical reactivity^50,51 characterized by the formation of intermediates able to induce by photostimulation an electron transfer (PET) with amines or an energy transfer (ET) with aromatic species.^52–54 In this study we designed, synthesized, characterized and tested a new fluorinated oxadiazolic compound which interfere with the Aβ self-assembly. The activation of the oxadiazolic compound at an appropriate wavelength is responsible for the formation of photo-active species exerting an inhibitory effect on Aβ fibrillation. Our experimental investigations were achieved by means of different techniques that are fluorescence, circular dichroism (CD), atomic force microscopy (AFM), and small angle X-ray scattering (SAXS). We aim to evidence with different structural resolutions the effects of the compound on Aβ aggregation pathway. Finally, preliminary investigation relative to the toxicity of the oxadiazolic compound were also examined by in vitro cellular experiments.

Materials and methods

Synthesis of 3-(4′-trifluoromethylphenyl)-5-(4′-methoxyphenyl)-1,2,4-oxadiazole

Pyridine (1.1 eq.) and 4-methoxybenzoyl chloride 2 (1.1 eq.) were added to a suspension of 4-trifluoromethylphenylamidoxime 1 (4.9 mmol, 1.0 g) in toluene (100 mL). The reaction mixture was refluxed for 8 hours. The solvent was removed under reduced pressure and the residue was chromatographed giving compound 3 (84%).

3-(4′-Trifluoromethylphenyl)-5-(4′-methoxyphenyl)-1,2,4-oxadiazole 3. Mp 145–147 °C. FT-IR (nujol) v: 1613 cm⁻¹.¹H NMR (300 MHz, CDCl₃) δ: 3.92 (s, 3H, OCH₃), 7.78 (d, J = 6.9 Hz, 2H, Ar), 7.83 (d, J = 8.1 Hz, 2H, Ar), 8.17 (d, 2H, J = 6.9 Hz, Ar), 8.26 (d, 2H, J = 8.1 Hz, Ar). GC-MS (m/z): 320 (M⁺, 100%). Elemental analysis for C₁₆H₁₁F₃N₂O₂ theor: C, 60.00; H, 3.46; N, 8.75. Exper.: C, 60.03; H, 3.48; N, 8.78.

Sample preparation. The oxadiazole was solubilized in ethanol at 1 mM concentration and then diluted to 10 μM in 10 mM phosphate buffer (PB) solution (NaH₂PO₄/Na₂HPO₄-RIEDEL DE HAEN in water MilliQ) at pH 7.4.

The lyophilized synthetic peptide Aβ_1–40 (Polypeptide) was solubilized with NaOH 5 mM (Sigma-Aldrich), pH 10, sonicated and already lyophilized according to Fezoui et al. treatment.⁵⁵ After this procedure the peptide was dissolved in PB 10 mM and then filtered with two filters in series having diameter of 0.20 μm (Whatman) and 0.02 μm (Millex-Lg) respectively, in order to eliminate whatever aggregates. The sample preparation was operated in a cool room at 4 °C. Aβ concentration (50 μM for all samples) was determined by tyrosine absorption at 276 nm using an extinction coefficient of 1390 cm⁻¹ M⁻¹.

The aggregation kinetics were followed at controlled temperature (37 °C) and under stirring (200 rpm) for 24 hours. Samples prepared for AFM images were aged at 37 °C for 4 days before the deposition on the mica surface. The same aged samples were concentrated at 150 μM using Millipore filters with a cut-off of 3 kDa before X-ray scattering measurements, in order to obtain the necessary scattering intensity. Samples for cytotoxicity assays were collected at 0, 5 and 24 hours of kinetic experiments in order to select aggregates at different states. All withdrawals were performed using a laminar flow hood to keep sterility.

Spectrofluorometric measurements. Emission spectra were recorded using a spectrofluorometer Jasco FP-6500. The oxadiazole emission spectra was recorded in ethanol and in PB–EtOH 99 : 1 using a quartz cell with 10 mm path length. The wavelength of excitation was 260 nm and the emission range was 250–500 nm.

Circular dichroism spectroscopy. The secondary structure of the Aβ_1–40 was studied by using a JASCO J-815 CD Spectrometer. Particularly, withdrawals of samples at appropriate time during the aggregation kinetic were observed. Spectra were recorded at 20 °C using a quartz cell with 0.2 mm path length. Each spectrum measurement was obtained by averaging over eight scans and subtracting the blank solvent contribution.

AFM analysis. Aliquots of 50 μM representative samples at the end of the aggregation kinetics were deposited onto freshly cleaved mica surfaces (Agar Scientific, Assing, Italy) and incubated for up to 2 minutes before rinsing with deionized water and drying with gentle nitrogen flow. AFM experiments were achieved with a Nanowizard II system (JPK Instruments, Germany), operating in tapping mode in air and at room temperature. Single beam silicon cantilevers (TESPA, NanoAndMore, USA) with a nominal spring constant of 42 N m⁻¹ and resonance frequency of 320 kHz, were used. Images with scan sizes of 2 × 2 μm² were acquired on different areas on each sample. Analysis of the aggregates size was performed by using the data processing software provided by JPK Instruments.

Small angle X-ray scattering. SAXS experiments were performed at ID2 beamline at the European Synchrotron Radiation Facility in Grenoble, France. The sample temperature was maintained at 20 °C. SAXS patterns were recorded using a fiber optically coupled two-dimension detector, FReLoN (Fast-Readout, Low-Noise). The distance from the sample and the detector was set to 1.5 m, in order to obtain a Q-range (Q = 4π sin θ/λ, where 2θ is the scattering angle and λ = 0.995 Å the X-ray wavelength) from 0.01 to 0.34 Å⁻¹. We recorded simultaneously the incident and transmitted intensities to the purpose of obtaining data in an absolute scale, hence the normalized SAXS patterns were azimuthally averaged to obtain the one-dimension profiles of scattered intensity. The buffer contribution was subtracted from protein solution data for each investigated condition, considering the correction for the protein volume fraction. To prevent radiation damage, all solutions were degassed before transferring into the capillary. Each measurement was performed for 100 ms, and followed by a dead time of 3 s in order to avoid radiation damage. This strategy was repeated for 50 times in order to obtain a satisfactory signal-to-noise ratio, despite the low protein concentration which was investigated.

Cell cultures, treatment and cell viability determination. Cells were cultured in 96 well plates at 1.5 × 10⁶ cells per well in RPMI 1640 medium (Celbio srl, Milan, Italy) supplemented with 10% fetal bovine serum (Gibco-Invitrogen, Milan, Italy) and 1% penicillin, 1% streptomycin (50 mg mL). Cells were maintained in a humidified 5% CO₂ atmosphere at 37 °C. LAN5 cells were treated for 72 h with samples of Aβ without or with compound 3 taken according to sample preparation. The final concentrations were 6.25 μM for Aβ and 1.25 μM for 3. Cell viability was measured by MTS assay (Promega Italia, S.r.l., Milan, Italy). MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium] was utilized according to the manufacturer's instructions. After cell treatments, 20 μL of the MTS solution was added to each well, and the incubation was continued at 37 °C for 4 h. The absorbance was read at 490 nm on the Microplate reader WallacVictor 2 1420 Multilabel Counter (PerkinElmer, Inc. Monza, Italy). Results were expressed as the percentage MTS reduction in the control cells. The control and treated cells were morphologically analyzed by microscopy inspection using Axio scope 2 microscope (Zeiss).

Results and discussion

Synthesis and photochemical characterization of 1,2,4-oxadiazole 3

The compound 3-(4′-trifluoromethylphenyl)-5-(4′-methoxyphenyl)-1,2,4-oxadiazole 3 was synthesized accordingly to the general scheme of the amidoxime route, by refluxing the trifluoromethylbenzamidoxime 1 with the appropriate benzoyl chloride in the presence of pyridine (Fig. 1).

Fig. 1 Synthesis of 1,2,4-oxadiazole 3.

The photochemical features and photostability of this compound were studied by time course measurement of fluorescence emission spectra in different solvents. The Fig. 2A shows the kinetic evolution of the emission spectrum of 1,2,4-oxadiazole 3 diluted in ethanol. The emission spectra present a Gaussian-like shape characterized by a time-dependent emission quenching.

Fig. 2 Emission spectra at different kinetic times of compound 3 (10 mM) in: (A) ethanol with continue photo-excitation; (B) in PB–ethanol (99 : 1) with continue photo-excitation; (C) in PB–ethanol (99 : 1) without continue photo-excitation.

Otherwise the oxadiazole dissolved in a solution of PB–ethanol at 99 : 1 ratio, has an emission spectra (Fig. 2B) with an initial intensity quench coupled with a change of the shape. In detail, the emission signal, after about 45 minutes, shows a fine structure with 3 characteristic bands: an hypsochromic band around 340–350 nm, an halfway band around 360 nm and a bathochromic band near 380 nm.

To investigate if the well-known photochemical reactivity of oxadiazoles^50–54 is due to the photo-excitation, a sample was incubated at 37 °C under stirring (200 rpm) in dark conditions. Interestingly, the emission spectra of sample registered at different times starting from the dissolution in phosphate buffer (Fig. 2C) display minor changes in intensity and shape. Overall, these results indicate that while the solvent is responsible for the emission maximum shift, the appearance of a fine structure in aqueous solution is a secondary phenomenon due to photo-stimulation of the compound.

A similar photochemical behavior has been already observed in cyanine dyes,^56,57 and correlated with the strong inter- and intra-molecular interactions of these molecules in aqueous solvent. In fact, it has been suggested that the kinetic evolution of the cyanine emission spectra is correlated to the formation of aggregates. Unlike cyanine dyes, spectral change of oxadiazole are instead induced by photo-excitation. We hypothesize that the oxadiazole ring may easily form a photoactive derivative under the high electronic stress induced by photo-stimulation. Furthermore, the three bands observed in the spectrum, could be related to the formation of aggregates composed by the same photo-excited derivatives but with different slippage angle.⁵⁷

Interaction between 1,2,4-oxadiazole 3 and amyloid peptide

The interaction between 1,2,4-oxadiazole 3 and Aβ_1–40 was investigated both with and without photo-stimulation with different experimental techniques. The formation of amyloid fibers was induced by following a method previously described⁵⁸ that assures reproducible results. Briefly, a solution of Aβ_1–40 monomers at 50 μM concentration in 10 mM PB was incubated at 37 °C under stirring at 200 rpm. In these conditions the fibrillation started up in about 5 hours, as suggested by ThT assay and CD spectra (see ESI†). The same incubation protocol was applied to a sample of monomeric Aβ_1–40 in presence of the oxadiazolic compound. The fluorescence emission spectrum evolution under continuous irradiation at 260 nm has been followed. Fig. 3 shows the spectra recorded at the start and at the end of the kinetic study. The fine structure developed over time is quite similar to that observed in the sample of 1,2,4-oxadiazole 3 alone (Fig. 2B). A higher intensity of emission was reached in 12 hours and after that the signal remained stable for a long time (1 month). We suggest that the increase of the signal might be related both to an increased solubility of the aromatic compound in presence of the peptide and to a direct interaction with Aβ. The latter phenomenon could be accounted for the high affinity of the 1,2,4-oxadiazole for the amyloid protein already observed in the case of similar compounds.⁴⁵ On the opposite, the initial and final spectra recorded for the same sample incubated without photo-excitation present minor spectral changes without any appearance of fine structure (Fig. 4), thus confirming implication of a photo-reaction.

Fig. 3 Aβ_1–40 (dotted line), Aβ_1–40 with the oxadiazole 3 at the initial step (black line) and at the end (pink line) of kinetic with continuous photo-stimulation. Reaction conditions: T = 37 °C, stirring at 200 rpm, λ_exc = 260 nm.

Fig. 4 Aβ_1–40 (dotted line), Aβ_1–40 with the oxadiazole 3 at the initial step (black line) and at the end (pink line) of kinetic without continuous photo-stimulation. Reaction conditions: T = 37 °C, stirring at 200 rpm, λ_exc = 260 nm.

To verify the oxadiazole effect on the aggregation pathway of Aβ_1–40, the peptide's secondary structure was inspected by CD measurements. Fig. 5 shows the dichroic spectra, for both irradiated and not irradiated samples, at the start and at the end of the aggregation kinetics.

Fig. 5 CD spectra for Aβ_1–40 at initial time (dotted line); final spectra of: Aβ_1–40 (black curve), Aβ_1–40 with 3, without excitation (green curve), Aβ_1–40 with 3, under irradiation, λ_exc = 260 nm (red curve).

Furthermore, CD spectra for a sample of Aβ alone at the starting and final time steps are also presented for comparison. No change in the typical signal of random coil structure of the monomeric peptide was observed for the sample kept under continuous photo-excitation (red line) while a signal of β-sheet structure formation was found for all other cases. Therefore, the 1,2,4-oxadiazole 3 seems to inhibit the formation of amyloid fibers only when activated by photo-excitation.

AFM and SAXS measurements performed on samples irradiated and opportunely treated (see sample information for details) confirm the previous results. As shown in Fig. 6, amyloid fibers are visible in a sample of Aβ_1–40 alone incubated at 37 °C (Fig. 6A), whereas only small aggregates, with a disordered secondary structure, are present when Aβ was incubated a 37 °C in presence of compound 3 under continuous photo-excitation (Fig. 6B). Structural differences between the two samples are also evidenced by SAXS measurements, as shown in Fig. 7.

Fig. 6 AFM images at the final stage for Aβ_1–40 (A) and for and for Aβ_1–40 with 3 prepared under photo-stimulation (B).

Fig. 7 SAXS experimental data and theoretical fitting (continuous lines) obtained at the final stage for Aβ_1–40 (right) and for Aβ_1–40 with 3 prepared under photo-stimulation (left).

First of all, the macroscopical differential cross section at Q ≈ 0, which is proportional to the molecular weight of the macromolecules in solution,⁵⁹ clearly indicates that the photoexcited sample presents aggregates at lower aggregation numbers. The whole shape of SAXS curves of the two samples is significantly different, as it can be appreciated by their representation in the form of Kratky plot⁶⁰ (see Fig. S2 in the ESI†), too. In fact, while Kratky plot corresponding to Aβ sample alone presents the bell shape indicating the presence of compact objects in solution, the same plot related to Aβ sample with 3 under irradiation is typical of flexible, hence disordered molecules. Hence, Kratky plots determine that Aβ structural features in solution are in agreement with the information provided by AFM images (Fig. 6). In view of these findings, SAXS data corresponding to Aβ sample alone were also represented in the form of modified Guinier plot for elongated objects,⁶⁰ suggesting the presence of cylinders whose average cross section is about 37 Å (see Fig. S3† and related caption in the ESI†). In light of what we have shown up to this point, SAXS curve of Aβ sample alone was successfully fitted considering the presence of mature fibrils that can be approximated to cylindrical objects. The theoretical fitting, reported as a continuous line in the left panel of Fig. 7, corresponds to cylinders of length l = 900 ± 50 Å and average radius r_a = 38 ± 4 Å, which result to be poly-dispersed. We stress that the fitting procedure also takes into account Aβ nominal concentration, because scattering data were obtained in absolute scale, according to the same approach reported in literature.⁶¹ The average radius is in agreement with literature result which claim that Aβ_1–40 fibrils have constant apparent diameters of 70 ± 1 Å,⁶² and the resulting polydispersity is confirmed by AFM image shown in Fig. 6A.

On the other side, SAXS curve corresponding to the photo-excited sample cannot be fitted considering cylindrical or other compact objects in solution, as it was inferred by its representation in the form of Kratky plot (Fig. S2†).⁶⁰ The macroscopical differential scattering cross section was hence adequately fitted by considering the presence of flexible molecules, as illustrated in the sketch of Fig. 7. Particularly, the theoretical curve reported in the right panel of Fig. 7 was obtained by considering the Pedersen–Schurtenberger worm-like chain form factor, P_PS(Q), multiplied by a two-electron density level cross section for the protein chain,^61,63

where N_A is Avogadro's number and r_e is the electron scattering length. According to this model, the cross-sectional radius of the chain r_c, the contour length of the chain c_l, the statistical segment (Kuhn) length representing the separation between two adjacent rigid scattering domains b, considered in the expression for P_PS(Q), and the thickness δ and electron density ρ_s of the surrounding shell, are the fitting parameters. Solvent electron density ρ₀, Aβ_1–40 concentration c, its molecular weight M_Aβ and electron density ρ_Aβ are fixed according to experimental conditions (c) and literature values. The aggregation number N_agg represents the number of Aβ monomers that form the flexible chain and can be simply determined by the ratio πr_c²c_l/V_Aβ, where V_Aβ is Aβ_1–40 monomer volume. The theoretical fitting on the experimental SAXS curve reported in the right panel of Fig. 7 was performed by considering the simultaneous presence of two species of worm-like chains, as it could be suggested by observing the change of slope of the SAXS curve in the region of Q ≈ 0.03 Å⁻¹, according to:where x₁ and x₂ account for the fractions of protein molecules occurring as worm-like species 1 and 2, respectively. However, in order to limit the number of the fitting parameters, only the aggregation number N_agg, was left as a free parameter, while the structural parameters r_c and b were considered to be common to both the worm-like species. All the theoretical fitting of SAXS curves were performed by GENFIT software package.⁶⁴ The resulting parameters are reported in Table 1, including the weight fraction of each worm-like population.

Table 1 Fitting parameters of the SAXS curve corresponding to the photoexcited sample, reported in Fig. 7, right panel

	Aggregation number Nagg	Chain radius rc (Å)	Kuhn length b (Å)	Weight fraction x (%)
Worm-like 1	2.0 ± 0.4	4.3 ± 0.3	10 ± 2	30 ± 5
Worm-like 2	11.2 ± 0.5			70 ± 5

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The aggregation numbers and the low value of the cross section radius confirm that the photo-excited sample does not present big aggregates, as evidenced by AFM images. Moreover, the shortness of the statistical segment length is consistent with the lack of secondary structure revealed by CD results.

The mechanism of inhibition of the oxadiazolic compound on Aβ fibrillogenesis could be traced to its photo-reactivity. We hypothesize that the photo-stimulation induces the formation of a reactive intermediate. The latter may react with the Aβ_1–40 amino acid backbone through an electron and/or energy transfer mechanism and induce a structural modification responsible for fibrillogenesis inhibition, as pictorially represented in Fig. 8.

Fig. 8 Pictorial representation of photo-induced Aβ_1–40 modification.

We suggest a mechanism analogous to the PICUP method used for stabilizing metastable amyloid oligomers previously described.⁴⁴ More in details, the method is based on the photo-stimulation of ruthenium or palladium complexes which form radicals capable of extracting an electron from nearby amyloid peptides. The formed reactive species of Aβ may carry out an alternative cross-linking with other neighboring Aβ peptides.⁴⁴

Particularly, we suggest that the electron-transfer from Tyr residues could initially generate the coupling of two peptide chain, forming a cross-linked dimers.

We would like to stress that a PICUP-like mechanism is strongly suggested by finding that the oxadiazolic compound exerts an inhibitory effect only under photo-stimulation. Obviously, further experiments will clarify the specific molecular mechanism behind the interaction between amyloid peptide and the oxadiazolic compound.

In vitro cytotoxicity assay

We further investigate the cytotoxicity of compound 3 and its possible effect on the formation of neurotoxic Aβ_1–40 aggregates, MTS assay was realized on LAN5 neuroblastoma cell line.

LAN5 cells were treated using sample withdrawals taken at 0, 5 and 24 hours of aggregation kinetic of Aβ_1–40 alone or in presence of the oxadiazole 3, and their viability was estimated. As shown in Fig. 9A, the main toxicity was caused by the sample of Aβ_1–40 alone incubated for 5 hours, which, accordingly to our previous studies⁵⁸ should contain only oligomeric species. The Aβ_1–40 sample at the initial time and the fibers obtained after 24 hours do not result as toxic as the 3 compound. To verify the effect of oxadiazole 3 on the toxicity ofAβ_1–40 photo-stimulated samples for 5 and 24 hours in presence of the compound 3 were dosed on LAN5 cells. No toxic effect was observed for cells treated with samples incubated 5 hours. On the contrary, the samples photo-stimulated for 24 hours cause a reduction on the cell viability if compared with Aβ fibers. However the species formed after this irradiation time result less toxic if compared with Aβ aggregates obtained after 5 hours. This evidence suggests a different structural organization obtained in presence of photo-stimulated compound 3. Microscopic images acquired on the same samples, before MTS assay, showed different cell morphology (Fig. 9B), consistently with cell viability results. In fact, LAN5 cells treated with Aβ_1–40 alone incubated for 5 hours, presented a reduction of the cellular body and a decrease of a neuritis and neuronal cell number with respect to the control (Fig. 9B). On the opposite a regular neuronal morphology is evident for the other samples, included the cells treated with Aβ-3 incubated for 24 hours.

Fig. 9 (A) Viability for LAN5 cells after 72 h of incubation with sample at different kinetic time of Aβ_1–40 (t = 0, 5, and 24 h) and Aβ_1–40 with oxadiazole 3 (t = 5, and 24 h) compared with control and oxadiazole 3. (B) Microscopic images for the same samples.

On the whole, these results support the hypothesis that the oxadiazole 3 is able to modulate the aggregation pathway of Aβ peptide and to inhibit the formation of toxic oligomer if properly photo-stimulated.

Conclusion

Uncontrolled aggregation of amyloid beta peptide is the main cause of Alzheimer's disease.^1,2 In the last years the attention has been focused on the potential use of small molecules for stabilizing or redirecting the amyloidogenic pathway towards the non-toxic and non-fibrillar states. In this work, we present the newly designed compound 3, namely (3-(4′-trifluoromethylphenyl)-5-(4′-methoxyphenyl)-1,2,4-oxadiazole) able to interfere, upon photo-stimulation, with Aβ fibrillation, and produce non-toxic oligomers, if treated for appropriate irradiation time. Although this photo-stimulated compound cannot be used, at this stage, as the direct drug for AD treatment, we consider our results a crucial step to elucidate the molecular specific mechanism of action of small stabilizing molecules. Furthermore, we suggest that the compound 3 could be used to build new technological and experimental strategies in this direction. In fact, very interesting is the possibility, offered by our study, to use the compound 3 for the production of non-toxic oligomers. In fact, it is known, that the study of amyloid oligomers structure requires their easy production. In this respect, the production of stable oligomers induced by the Aβ_1–40 peptide interaction with the photo-stimulated compound 3, has been tested and it could be an attractive possibility to finely tuning in a photo-catalytic way this production. The atoxic profile of compound 3 suggests the further development of 1,2,4-oxadiazole derivatives that could be photo-stimulated with NIR light for the practical perspective development of a photo-therapy for AD.

Funding

This work has been supported by Italian grant FIRB “Future in research” RBFR12SIPT MIND: “Multidisciplinary Investigations for the development of Neuro-protective Drugs”, and Progetto Bandiera N-CHEM.

Acknowledgements

We thank Dr R. Carrotta and Dr F. Librizzi for useful discussions; Ing F. D'Anca for technical help in AFM measurements; Dr A. Provenzano and Mr M. Lapis for their technical support; Dr T. Narayanan and Dr F. Spinozzi for help in SAXS experiments.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra13556c

‡ The two authors have contributed equally to the present work.

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