Roles of DMSO-type ruthenium complexes in disaggregation of prion neuropeptide PrP106–126

Abstract

Ruthenium complexes are potential anticancer metallodrugs and inhibitors of various proteins, such as enzymes and even amyloid peptides. Studies on Aβ protein, human islet amyloid polypeptide, and prion neuropeptide have indicated that Ru complexes can inhibit amyloidosis. However, the interaction mechanism of peptides with Ru complexes remains unclear. In this study, we selected four dimethyl sulfoxide (DMSO)-type Ru complexes containing large aromatic ligands to explore and compare the interactions of Ru complexes with the prion neuropeptide PrP106–126. Results showed that, unlike new anti-tumor metastasis inhibitor-A-like compounds, these complexes can bind to PrP106–126 mainly through metal coordination and hydrophobic interaction. The Ru complexes disaggregated the PrP106–126 fibrils into scattered fragments or amorphous forms, thereby reducing the toxicity of PrP106–126. Among the four Ru complexes, complex 1, which consists of bipyridyl and DMSO ligands, exhibited the highest disaggregation ability and relatively high cell viability, which may be attributed to its molecular configuration and low cytotoxicity. These results suggested that Ru complexes are promising metallodrugs against amyloidosis-related diseases.

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Introduction

The discovery of cisplatin as an anticancer agent aroused the interest on metallodrugs since the 1970s.¹ Research on different metal complexes (such as Au, Ru, Ir, and Bi) has advanced considerably owing to their significance in treating tumors, ulcers, and other diseases.^2–5 Recent studies have reported the use of complexes of metals with various metal centers and chelating ligands against amyloid disorders.^6–15 The low cytotoxicity of these compounds, as well as their permeability to the blood–brain barrier, resulted in their rapid development for medical applications.

Ru complexes, which are non-platinum-based metallodrugs that demonstrate high activity and low toxicity, exhibit a great potential as anticancer drugs.^16–20 A promising group of Ru complexes includes ammonia and imines ([HL][trans-RuL₂Cl₄]), pyridines, EDTA classes, dimethyl sulfoxide (DMSO) types, and NAMI-A-like compounds.^21–27 NAMI stands for new anti-tumor metastasis inhibitor, which are related to tetrachlororuthenium(III) DMSO complexes (X) [trans-RuCl₄(dmso-S)L]; X is either HL or Na, which corresponds to NAMI-A or NAMI, respectively, and L is imidazole.^26,27 Ru complexes can induce tumor-cell apoptosis and regulate some related proteins. In addition, Ru complexes can effectively bind to proteins, such as serum albumin, apotransferrin, and apolactoferrin, resulting in an altered protein structure and binding ability of these molecules.^28–31 Versatile Ru complexes also demonstrate inhibitory effect on fibril formation of Aβ protein, prion neuropeptide, and human islet amyloid polypeptide.^{10,11,32–34} Moreover, Ru complexes act as luminescent probes for fibrillar protein detection.³⁵

Prion diseases, including scrapie in sheep, BSE, and CJD in humans, are certified genetic, infectious, and sporadic neurodegenerative diseases.^36,37 A suggested pathogenic mechanism of these diseases involves the transition of the normal cellular form of a prion protein (PrP^c), which is rich in α-helix, into a pathogenic scrapie isoform (PrP^Sc), which is rich in β-sheet. The major conformational change that occurs during conversion of PrP^c into PrP^Sc is localized in residues 90–112; moreover, residues 113–126 constitute the conserved hydrophobic region that also displays structural plasticity.³⁸ The N-terminal fragment of PrP corresponding to the sequence of PrP106–126 exhibits physicochemical and biophysical properties similar to those of similar PrP^Sc, such as induction of neuronal apoptosis, anti-proteinase K digestion, fiber formation, and mediation of conversion of PrP^C into PrP^Sc.^39–41 Despite the reports on the roles of short peptides, such as PrP118–135 and PrP octapeptide, PrP106–126 is a common research model used to investigate neural degeneration of prion disease.^42–44 Among the prion sequences, PrP106–126 contains a core structure and a typical hydrophobic region.^45–48 Some transition metal ions, such as Cu²⁺, Zn²⁺, and Ni²⁺, bind with PrP106–126 and alter the properties of peptide aggregation.^32,49–52 The effects of some metal complexes, such as Au, Pt, and Ru complexes, on PrP106–126 aggregation were investigated.^8,10,13 Ru compounds exert low cytotoxicity and significantly inhibit PrP106–126 aggregation. Different molecular configurations result in distinct binding modes and inhibitory effects of some Ru compounds, and NAMI-A-like Ru complexes mainly bind to peptides through electrostatic interaction.^10,53 However, the disaggregation ability of Ru complexes is unclear. The present study used a series of DMSO-type Ru complexes consisting of large aromatic configuration to compare the interaction and disaggregation ability of Ru complexes on PrP106–126 (Scheme 1). The results demonstrated that these Ru complexes can remarkably disaggregate PrP106–126 fibrils and bind to the peptide not only through metal coordination but also through peptide–ligand interaction. Ru complexes also effectively rescued the cytotoxicity of PrP106–126-induced SH-SY5Y nerve cells.

Scheme 1 Molecular structures of mer-[RuCl₃(dmso)(bpy)] (1), mer-[RuCl₃(dmso)(phen)] (2), mer-[RuCl₃(dmso)(dpq)] (3), and mer-[RuCl₃(dmso)(dppz)] (4).

Experimental section

Materials

The human prion protein fragment PrP106–126 and its mutant peptide M109FPrP106–126 were chemically synthesized by SBS Co., Ltd. (Beijing, China). The synthesized PrP106–126 and M109FPrP106–126 were purified and identified using HPLC, MS, and NMR. The samples were more than 95% pure. The metal complexes were synthesized as previously described.^54,55 All other reagents were of analytical grade.

Thioflavin T (ThT) assay

Formation of β-sheet-rich amyloid fibrils was detected over time through ThT dye-binding assay. Fluorescence was monitored using an F-4500 fluorescence spectrometer (Hitachi, Japan) equipped with a programmable temperature controller (PolyScience, USA). After incubating PrP106–126 for 24 h at 310 K, the metal compound was added to PrP106–126 in 10 mM phosphate buffer at pH 7.5 and 310 K for 1 day. The sample was then mixed with 100 μM of ThT for further detection at 298 K. The final concentration of the peptide was 100 μM. The ThT signal was quantified by averaging the fluorescence emission at 484 nm over 10 s when the sample was excited at 432 nm. The final spectrum was obtained from the mean of three spectra obtained from repeated experiments. For the determination of concentration dependence, the concentrations of the Ru complexes were 0, 20, 40, 60, 80, 100, and 120 μM.

To determine the effect of Ru complexes on ThT fluorescence, samples were prepared in 10 mM phosphate buffer at pH 7.5. The ThT concentration was 100 μM, and the concentrations of the Ru complexes were 20, 50, 80, and 100 μM. The samples were excited at 432 nm, and the fluorescence emission at 484 nm over 10 s was measured.

UV spectra

A Cary 50 UV spectrometer (Varian, USA) was used to obtain UV spectra at room temperature. The Ru complex was dissolved in 10 mM of phosphate buffer at pH 7.5, and the sample concentration was 50 μM. To prepare the mixture of ThT and Ru complex, we used 100 μM of both ThT and metal complex. The spectrum was obtained after subtracting the absorption of metal complex. The RuCl₃ sample was also used for comparison. The scan wavelength used for each sample was 200–800 nm.

Transmission electron microscopy (TEM)

The PrP106–126 samples were incubated for 24 h in the absence of metal complex followed by continuous cultivation for 24 h at 310 K in the presence of different concentrations of metal complex. The final peptide concentration of all the samples was 10 μM, and the molar ratio of metal complex was 0.2, 1.0, and 3.0. TEM observations were performed on Hitachi-800 TEM at a magnification of 50 000× and an acceleration voltage of 220 kV at room temperature. All of the results were obtained from multiple parallel experiments.

Atomic force microscopy (AFM)

The samples were prepared by incubating 1 mM of peptide solution for 24 h and adding equivalent amounts of Ru complexes at 310 K for another 24 h. The final peptide concentration used in the AFM experiment was 10 μM with 1% DMSO. By using Veeco D 3100 instrument (Veeco Instruments 151, Inc., USA), we obtained images in tapping mode with a silicon tip under ambient condition and under a scanning rate of 1 Hz and a scanning line of 512.

Spectrofluorometric measurements

Steady-state fluorescence measurements of intrinsic phenylalanine residue were performed at 298 K to investigate the binding affinity of Ru complexes with PrP106–126. Given that PrP106–126 contains no luminescent aromatic residue, the single PrP106–126 mutant, M109FPrP106–126, was selected and used as a model considering its similarity with other species in this residue position. Moreover, an excitation wavelength of 260 nm was determined based on previous reports.^56–59 The dissociation constant (K_d) was calculated from the plot of the fluorescence intensity based on the Ru complex concentration.^13,53 The M109FPrP106–126 concentration was 100 μM. The results were obtained from three repeated experiments.

NMR

All of the NMR experiments were performed using Bruker Advance 400 or 600 MHz spectrometer at 298 K. The samples for NMR measurements were prepared in H₂O containing 10% d₆-DMSO at a final peptide concentration of 0.5 mM. The pH of the solution was carefully adjusted to 5.7 by adding either DCl or NaOD. The NMR spectrum was obtained and processed by Bruker Topspin 2.1 software using a gradient pulse to suppress the residual H₂O signal.

Cyclic voltammetry (CV)

Electrochemical measurements were conducted on an Epsilon Electrochemical Workstation (USA). A platinum wire and glassy carbon electrode were used as counter and working electrodes, respectively. All of the potential values were reported according to the Ag/AgCl reference electrode. The working electrode was polished using 0.3 mm alumina slurry and ultrasonic bath in ultrapure H₂O prior to the test. The glassy carbon electrode was replaced after every scan to eliminate possible electrodeposition of Ru complex. Phosphate buffer (10 mM) at pH 7.5 was used as supporting electrolyte. The final concentration of the Ru complex with or without PrP106–126 (50 μM) was 150 μM. In addition, the scan rate was set at 100 mV s⁻¹. Each spectrum represents an average of three accumulated scans.

Electrospray ionization-mass spectrometry (ESI-MS)

The ESI-MS spectra were recorded in positive mode by directly introducing the samples at a flow rate of 3 mL min⁻¹ on an APEX IV FT-ICR high-resolution MS (Bruker, USA) equipped with a conventional ESI source. The working conditions were as follows: end-plate electrode voltage, 3500 V; capillary entrance voltage, 4000 V; skimmer voltages, 1 and 30 V; and dry gas temperature, 473 K. The flow rates of the drying and nebulizer gases were set to 12 and 6 L min⁻¹, respectively. Data analysis 4.0 software (Bruker) was used to acquire data. Deconvoluted masses were obtained using an integrated deconvolution tool. The concentration of the peptide sample was 50 μM, and quintuple amounts of Ru complex were added into the peptide solution for detection.

3-(4,5-Dimethyl-2-thiazolyl-2,5-diphenyl-terazolium bromide) (MTT) assay

Human SH-SY5Y neuroblastoma cells were cultured in a 1 : 1 mixture of Dulbecco's modified Eagle's medium and F12 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U mL⁻¹ penicillin, and 100 U mL⁻¹ streptomycin. The cells were grown at 310 K in a humidified incubator with 95% air and 5% CO₂. Cell survival was assessed by measuring reduction of MTT. PrP106–126 was incubated for 24 h in the absence of Ru complexes, and then continuous cultivation was performed for another 24 h in the presence of 10 μM of Ru complex. The mixture was then added into the cells and allowed to react for 4 days. Subsequently, the cells were incubated with 10 mL of MTT at 310 K for 4 h. The absorbance at 570 nm was measured using a UV-vis spectrophotometer. Each experiment was repeated four times. Data were calculated as percentage of the value for untreated control.

Results

Synthesis of Ru complexes

Four DMSO-type Ru complexes, namely, mer-[RuCl₃(dmso)(bpy)] (1), mer-[RuCl₃(dmso)(phen)] (2), mer-[RuCl₃(dmso)(dpq)] (3), and mer-[RuCl₃(dmso)(dppz)] (4) were synthesized and identified according to reported methods.^54,55 These complexes were synthesized considering their potential ligand effects on PrP106–126 disaggregation. For complex 1, equal amounts of trans-[Ru(dmso)₄Cl₂] and 2,2-dipyridine were dissolved in the mixture of CH₃OH and CHCl₃ (1 : 1, v/v) and then reacted at 373 K for 2 h. The resulting mixture was filtered to obtain an orange-red clear solution. After natural evaporation in air for several days, orange-red crystals were generated with a final yield of 49%.

For complex 2, a bright yellow mixture containing cis-[RuCl₂(dmso)₄] and 1,10-phenanthroline monohydrate in 6 M HCl (10 mL) and CH₃CH₂OH (10 mL) were heated at 353 K for 2 h; the resulting clear red solution was subsequently concentrated in a rotary evaporator and then dried in vacuum. The crude product was purified by column chromatography on a neutral alumina with dried CH₂Cl₂ and CH₃OH (10 : 1, v/v) as eluent, resulting in a dark red solid complex 2 with a final yield of 46%. Complex 3 was synthesized by adding dipyrido[3,2-f:2′,3′-h]quinoxaline (dpq) instead of 1,10-phenanthroline using the method mentioned above, and the final yield was 37%. Complex 4 was synthesized by adding dipyrido[3,2-a:2′,3′-c]phenazine (dppz) instead of 1,10-phenanthroline using the same method mentioned above with a final yield of 41%. Fig. S1† shows the NMR spectra, which correspond to the structures of the four complexes. Fig. S2† shows the UV spectra of these complexes, which are consistent with a reported finding.⁵⁵

ThT assay

Previous studies used the fluorescent dye ThT as a marker in PrP106–126 aggregation.^53,60 An excitation at 432 nm and a strong emission at approximately 484 nm were observed when ThT bound to an aggregated peptide. Fibril formation of PrP106–126 resulted in intense fluorescence intensity (Fig. 1). ThT fluorescence intensity dramatically dropped when Ru complexes were incubated with the aggregated peptide. However, one cannot conclude that the decreased ThT fluorescence is associated to peptide disaggregation. UV experiments were performed to clarify the interaction of metal complex with ThT. Addition of the compound into ThT led to a slight hyperchromic effect, indicating the binding of metal complex with ThT (Fig. S3†). The fluorescence experiments on metal complexes with ThT showed that complexes 1, 2, and 3 can quench ThT fluorescence up to a certain extent, whereas complex 4 and Ru cation demonstrated a relatively strong quenching effect (Fig. S4A–E†). Considering the interaction of metal complexes with ThT, the deceased fluorescence in ThT assay cannot directly reflect the interaction of Ru complexes with the aggregated peptide. Hence, the Ru complexes and aggregated PrP106–126 were morphologically characterized.

Fig. 1 Ability of the four complexes to disaggregate the PrP106–126 fibrils as measured by ThT assay. PrP106–126 in the absence and presence of complexes 1 (A), 2 (B), 3 (C), and 4 (D). The final concentration for the peptide was 100 μM and that of the metal complex was 0, 20, 40, 60, 80 100, and 120 μM (from the top to bottom, respectively).

Morphology of aggregated PrP106–126

TEM and AFM were performed to reveal the effects of Ru complexes on peptide disaggregation. Studies have effectively applied these techniques to determine the different states of amyloidogenic peptides. Images of fine fibrils distributed on the surface of carbon membrane support suggested the strong aggregation of PrP106–126 (Fig. 2A). However, after incubation of the peptide with equivalent amounts of Ru complex, such an aggregation was reversed in the presence of Ru complex as revealed by the TEM micrographs of PrP106–126. Complexes 1 and 2 (Fig. 2B and C) inhibited PrP106–126 aggregation better than 3 and 4 (Fig. 2D and E). Moreover, morphological characterization conducted at low (0.2) and high (3.0) molar ratios illustrated different effects of the compounds on peptide aggregation, thereby clarifying the ThT assay results. The images showed thin fibrils at low molar ratio (Fig. S5†). However, after incubating the aggregated PrP106–126 with triple amounts of Ru complexes, all of the samples exhibited amorphous forms, implying an intense disaggregation caused by Ru complexes at high molar ratio (Fig. S6†). The AFM experiments also proved the ability of Ru complexes to disaggregate PrP106–126 (Fig. 3). These data are consistent with the TEM results, thus providing strong support for the roles of Ru complexes in PrP106–126 fibril disaggregation.

Fig. 2 TEM images of PrP106–126 in the absence (A) and presence of equivalent amounts of complexes 1 (B), 2 (C), 3 (D), and 4 (E). The scale bar is 500 nm.

Fig. 3 AFM images of PrP106–126 in the absence (A) and presence of equivalent amounts of complexes 1 (B), 2 (C), 3 (D), and 4 (E). The scan size is 5 μm.

K_d determination of PrP106–126 with Ru complexes

Numerous studies have used intrinsic fluorescence quenching to calculate K_d.^13,53 We assume that peptide fluorescence will change when a Ru complex is added, reflecting the amounts of binary complex produced. The mutant peptide M109FPrP106–126 was used to produce an intrinsic fluorescence, which was different from that in ThT assay. In addition, quenching of phenylalanine fluorescence caused by the Ru complexes can represent structural change in the peptide. Fluorescence intensities at 287 nm in the presence of Ru complexes were therefore used to estimate K_d through a nonlinear least-squares regression. The obtained apparent K_d values were 2.7 ± 0.5 × 10⁻⁶, 7.6 ± 1.8 × 10⁻⁷, 7.4 ± 2.1 × 10⁻⁸, and 3.2 ± 0.4 × 10⁻⁶ M for complexes 1, 2, 3, and 4, respectively (Fig. 4). These results showed that complex 3 exhibited the strongest binding affinity but showed weaker disaggregation ability among the four Ru complexes. The disaggregation ability of complex 3 was not remarkable despite its great aromaticity that contributes to its better binding affinity.

Fig. 4 Intrinsic fluorescence spectra of PrP106–126 plotted at 287 nm by titration of complexes 1 (black), 2 (red), 3 (blue), and 4 (magenta). The error bar was obtained by three repeated experiments.

¹H NMR studies

¹H NMR spectra were acquired at pH 5.7 and 298 K to investigate the interaction of Ru complex with PrP106–126. The residues His111 and Met109/112 are the most possible metal binding sites, and their characteristic side chain protons are clearly assigned.^49,53 The ¹H NMR spectrum of PrP106–126 is consistent with that of a previous finding.^40,53 After incubating the Ru complex with the peptide, the characteristic peak of His111 side chain C_δHs at 7.08 ppm was upshifted for the four complexes, showing a slight decrease in intensity (Fig. 5 and S7–S9†). However, the featured peak C_εHs of Met109/112 at 2.08 ppm remained unchanged, suggesting that methionine residues were not involved in the binding. In addition, His111 may have participated in the interaction of metal complex with peptide, which indicated a plausible metal coordination. Furthermore, changes in relaxation properties of some resonance peaks of complexes 1 and 4 were observed, implying the hydrophobic interaction of the ligand with the peptide, whereas no obvious effect was found for complexes 2 and 3, indicating a different binding affinity to some extent.

Fig. 5 ¹H NMR spectra of PrP106–126 in H₂O/DMSO at pH 5.7 and 298 K. PrP106–126 alone (A), PrP106–126 in the presence of triple amounts of complexes 1 (B), and complex 1 alone (C). The peaks assigned to C_δHs of His111 (dot) and from the metal complex (star) were remarkably affected by the interaction.

CV

Investigating the interaction between a macromolecule and a small molecule is one of the applications of electroanalytical technique.^61–65 The present study used CV to obtain information regarding the binding mode between Ru complexes and PrP106–126. The CV curves of the Ru complexes in the absence and presence of PrP106–126 were obtained (Fig. 6). All of the four Ru complexes showed a reductive peak current at approximately 1.5 V. Adding PrP106–126 apparently reduced the reductive peak with a slight potential shift for complexes 2, 3, and 4 (Table 1), indicating the formation of non-electric active substances. In addition, changes in the peak currents suggested hydrophobic interaction between PrP106–126 and the four complexes.^11,12

Fig. 6 Cyclic voltammograms of ruthenium complexes in the absence (black line) and presence (red line) of PrP106–126 obtained at the glassy carbon electrode for complex 1 (A), 2 (B), 3 (C), and 4 (D), respectively.

Table 1 Cyclic voltammograms of Ru complexes in the absence and presence of PrP106–126 obtained at glassy carbon electrode^a

	Ru complex alone				Ru complex in the presence of PrP106–126
	Epa/V	Epc/V	Ipa/μA	Ipc/μA	Epa/V	Epc/V	Ipa/μA	Ipc/μA
a Errors were estimated by three repeated scans.
1	1.45 ± 0.13	−1.59 ± 0.08	−56.93 ± 1.76	81.07 ± 5.90	1.49 ± 0.14	−1.53 ± 0.05	−43.61 ± 5.66	38.19 ± 2.41
2	1.44 ± 0.01	−1.56 ± 0.01	−65.34 ± 10.24	80.03 ± 3.87	1.61 ± 0.05	−1.49 ± 0.03	−46.72 ± 6.18	40.66 ± 3.55
3	1.53 ± 0.04	−1.47 ± 0.03	−86.8 ± 3.56	58.23 ± 4.16	1.57 ± 0.02	−1.43 ± 0.02	−52.73 ± 5.73	41.49 ± 5.54
4	1.41 ± 0.02	−1.54 ± 0.02	−69.58 ± 8.5	64.74 ± 0.3	1.47 ± 0.01	−1.53 ± 0.02	−46.69 ± 3.87	39.61 ± 2.66

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ESI-MS spectra of PrP106–126 with Ru complexes

Quintuple amounts of Ru complexes were incubated with the peptide to determine whether the Ru complexes directly bind to PrP106–126 and to identify the probable binding pattern between them. The final solution was analyzed using ESI-MS. The free PrP106–126 exhibited an intense peak at 1912 ± 1 corresponding to its expected mass. Adding complex 1 resulted in adduct [PrP106–126 + Ru(bpy) + Ru] formation as indicated by a value of 2273.85(1+) (Fig. 7A). Table 2 shows the adduct peaks for all of the metal complexes. The ESI-MS results demonstrated the binding of Ru complexes to the peptide in the form of either [PrP106–126 + Ru(ligand)] or [PrP106–126 + Ru] without ligands corresponding to the peaks at 2345.67(1+) [PrP106–126 + Ru(dmso)(phen)(H₂O)₃] (Fig. 7B), 2035.07(1+) [PrP106–126 + Ru(H₂O)] (Fig. 7C), and 1276.0(2+) [PrP106–126 + Ru(dppz) + 2Ru(H₂O)] (Fig. 7D) for complexes 2, 3, and 4, respectively. The combination of the peptide and a second Ru atom possibly indicates binding of PrP106–126 with Ru complex via metal coordination. The results for complex 4 were obtained by adding decuple amounts of the complex to the peptide. The MS results indicated a significant binding of the complexes with the peptide.

Fig. 7 ESI-MS spectra of PrP106–126 in the presence of quintuple amounts of complex 1 (A), 2 (B), 3 (C), and decuple amounts of 4 (D).

Table 2 ESI-MS values of the bound species of peptides with the Ru complexes and the corresponding calculated values

Complex	Calculated	Measured	Binding species
1	2271.20(1+)	2273.85(1+)	[PrP106–126 + Ru + Ru(bpy)]
2	2017.88(1+)	2013.07(1+)	[PrP106–126 + Ru]
	2153.77(1+)	2150.14(1+)	[PrP106–126 + 2Ru(H2O)]
	2343.32(1+)	2345.67(1+)	[PrP106–126 + Ru(dmso)(phen)(H2O)3]
3	2033.05(1+)	2035.07(1+)	[PrP106–126 + Ru(H2O)]
4	1274.50(2+)	1276.00(2+)	[PrP106–126 + Ru(dppz) + 2Ru(H2O)]

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Effect of Ru complexes on PrP106–126 neurotoxicity

Ru complexes can bind to the prion neuropeptide PrP106–126, resulting in the altered aggregation behavior of the latter. The ability of Ru complexes to reduce PrP106–126 neurotoxicity was assessed using human SH-SY5Y neuroblastoma cells to explore their potential as valid metallodrugs. Cell survival was evaluated after treating the SH-SY5Y cells with PrP106–126 alone or with the mixture of the peptide and Ru complexes. The aggregated PrP106–126 remarkably decreased the cell viability to 33% ± 3.0% compared with the control sample as measured by MTT assay with a *P value of <0.01. Adding complexes 1, 3, and 4 to PrP106–126 increased the cell viability to 60.1% ± 1.8%, 55.3% ± 2.3%, and 44.2% ± 1.7%, respectively, with a *P value of <0.01 (Fig. 8). By contrast, cell viability of SH-SY5Y reverted to 22.0% ± 2.3% after incubation with complex 2. The self-toxicity of the Ru complexes to SH-SY5Y cells was notably low except for complex 2, which cannot inhibit the cytotoxicity of PrP106–126 (Fig. S10†).

Fig. 8 Effects of ruthenium complexes on the neurotoxicity of PrP106–126, as determined by MTT assay. Human SH-SY5Y neuroblastoma cells were treated with PrP106–126 (100 μM) alone or with the mixture of the peptide and ruthenium complexes (10 μM). **P < 0.01 versus control group, and *P < 0.01 versus sample treated with PrP106–126. The data were from the average of four repeated experiments.

Discussion

Disaggregation of the four Ru complexes against PrP106–126 fibrils

The prion neuropeptide PrP106–126 is an essential fragment of PrP^Sc, which can self-aggregate. The formation of amyloid fibrils involves β-sheet, oligomer, and profibril transitions. This characteristic is exhibited by other amyloid peptides, such as Aβ protein, hIAPP, and α-synuclein.^66–74 After incubating Ru complexes with aggregated PrP106–126, the fluorescence intensity decreased dramatically in the presence of ThT, reflecting disaggregation of the peptide PrP106–126. However, all of the four complexes quenched ThT fluorescence to some extent (Fig. S3†), which interfered with the results of the ThT assay. Morphological analysis is crucial to the disaggregation ability of these complexes. The morphological observation of complexes 3 and 4 indicated that their actual effects on peptide fibril formation were weaker than those of complexes 1 and 2. Given the aggregation behavior of PrP106–126, in which hydrophobic residues surrounded the formed fibrils, the disaggregation ability of metal complex consisting of large ligand can possibly weaken because of notable steric barrier, which is similar to that found in Au complexes.¹³ In this situation, disaggregation resulting from the binding of metal complexes not only requires metal binding sites but also a suitable residue–ligand interaction, similar to the effect of heterocyclic aromatic ligands on Aβ.⁷⁵

Binding affinity between PrP106–126 and Ru complexes

Studies have mainly considered Ru complexes as potential anticancer metallodrugs owing to their ability to bind to the DNA of cancer cells.^76–78 In fact, these complexes also interact with proteins, enzymes, or polypeptide fragments.^2,79,80 The results of our ESI-MS experiments showed that metal coordination predominated the binding interaction; this finding is consistent with that of a previous report.¹⁰ Moreover, the decrease in CV peak current, proton relaxation in NMR, and K_d determination suggested that hydrophobic interaction existed between the peptide and Ru complexes. NMR data also showed that the Ru complex did not affect the methionine signal, and this phenomenon is different from that in Pt and Au complexes, illustrating the distinct role of center metal ions.^8,13,14 Furthermore, a previous study indicated that NAMI-A-like compounds predominantly interact with a peptide through electrostatic interaction, according to specific ESI-MS and CV analyses.⁵³ These results suggested that center metal ion not only contributed to the binding mode and binding affinity but also to ligand configuration. In addition, the disaggregation ability was not necessarily correlated with the binding affinity as reflected by results of K_d determination (Fig. 4). Actually, the property of the mutant peptide M109FPrP106–126 used in K_d determination is not completely similar to that of the peptide PrP106–126. Moreover, the intrinsic fluorescence quenching was distinct from the procedure of disaggregation, in which peptide incubation increased the peptide hydrophobicity. Disaggregation of PrP106–126 involved several factors, including the configuration of Ru complexes, the binding ability of Ru complexes to monomers, oligomers, and fibrils, as well as the physicochemical environment of the aggregated peptide, which might alter the disaggregation behaviors of Ru complexes on PrP106–126.

Influence of Ru complexes on PrP106–126 neurotoxicity

PrP106–126 plays an important role in the amyloidogenic and neurotoxic properties of the entire pathological PrP^Sc.^39–41 The viability of the SH-SY5Y cell induced by PrP106–126 is similar to that observed in a previous report.⁵³ Cytotoxicity mainly results from oligomer species of amyloid peptides.^81–83 Fig. S6† shows that addition of Ru complexes can disaggregate mature fibrils into monomers and oligomers, implying that the decreased cytotoxicity should be attributed to a greater number of monomer species. Unlike the complexes 1, 3, and 4, complex 2 reduces the cell viability compared with PrP106–126 treatment alone. Although complex 2 demonstrated better disaggregation ability than complexes 3 and 4, the reduced cell viability is possibly ascribed to the self-toxicity of the complex 2 to the cells. Ru complexes are widely used in biomedical field because of their relatively low cytotoxicity and excellent ability to cross the blood–brain barrier.^17–20 Cells are a complicated system, and their inherent mechanism is beyond what can be expounded in the present study. Analysis of cell viability and disaggregation data elucidated that among the four Ru compounds, complex 1 is possibly the best candidate as a potential metallodrug against peptide amyloid fibril formation.

Conclusion

This study investigated the ability of four DMSO-type Ru complexes with large aromatic ligand to induce disaggregation of the prion neuropeptide PrP106–126. Metal complexes possess higher plasticity in molecular structure than small organic compounds. In addition, metal complexes can be easily introduced into ligands for structural modification. This paper presented a series of Ru complexes demonstrating notable steric effects and which can mainly bind to PrP106–126 through metal coordination and hydrophobic interaction. The Ru complexes induced disaggregation of mature PrP106–126 fibrils into scattered fragments or amorphous forms, thereby reducing the cytotoxicity of PrP106–126. Complex 1, which consists of bipyridyl and DMSO ligands, exhibited the highest disaggregation ability and cell viability among the four Ru complexes. The results imply that adequate molecular configuration rather than a large steric effect is vital for the ability of a metal complex to disaggregate amyloid peptides. The results of this study suggested that Ru complexes are promising metallodrugs against amyloidosis-related diseases.

Acknowledgements

We are grateful for the support of the National Natural Science Foundation of China (No. 21271185 and 21473251).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra21523d

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