Platinum(II) O,S Complexes Inhibit the Aggregation of Amyloid Model Systems

Platinum(II) complexes with different cinnamic acid derivatives as ligands were investigated for their ability to inhibit the aggregation process of amyloid systems derived from Aβ, Yeast Prion Protein Sup35p and the C-terminal domain of nucleophosmin 1. Thioflavin T binding assays and circular dichroism data indicate that these compounds strongly inhibit the aggregation of investigated peptides exhibiting IC50 values in the micromolar range. MS analysis confirms the formation of adducts between peptides and Pt(II) complexes that are also able to reduce amyloid cytotoxicity in human SH-SY5Y neuroblastoma cells. Overall data suggests that bidentate ligands based on β-hydroxy dithiocinnamic esters can be used to develop platinum or platinoid compounds with anti-amyloid aggregation properties.


Pt(II) Complexes with Bidentate Ligands Inhibit the Aggregation of Amyloidogenic Peptides
The ability of the Pt(II) complexes reported in Figure 1 to inhibit the aggregation process of the investigated amyloid peptides was evaluated through fluorescence spectroscopy. Sequences and isoelectric points of the analyzed peptides are reported in Table 1. Thioflavin T (ThT) binding assay was employed; it is frequently used to analyze the kinetic of the self-recognition process associated with amyloid fibers formation [51].
The time course of the ThT fluorescence when NPM1264-277 is incubated with the complexes 1, 2, 3 and 4 is reported in Figure 2. Fluorescence values were registered for the peptide in the presence of the different concentrations of the Pt compounds, according to the peptide to Pt(II) compound molar ratios indicated in the legends.

Pt(II) Complexes with Bidentate Ligands Inhibit the Aggregation of Amyloidogenic Peptides
The ability of the Pt(II) complexes reported in Figure 1 to inhibit the aggregation process of the investigated amyloid peptides was evaluated through fluorescence spectroscopy. Sequences and isoelectric points of the analyzed peptides are reported in Table 1. Thioflavin T (ThT) binding assay was employed; it is frequently used to analyze the kinetic of the self-recognition process associated with amyloid fibers formation [51].
The time course of the ThT fluorescence when NPM1 264-277 is incubated with the complexes 1, 2, 3 and 4 is reported in Figure 2. Fluorescence values were registered for the peptide in the presence of the different concentrations of the Pt compounds, according to the peptide to Pt(II) compound molar ratios indicated in the legends.

Pt(II) Complexes with Bidentate Ligands Inhibit the Aggregation of Amyloidogenic Peptides
The ability of the Pt(II) complexes reported in Figure 1 to inhibit the aggregation process of the investigated amyloid peptides was evaluated through fluorescence spectroscopy. Sequences and isoelectric points of the analyzed peptides are reported in Table 1. Thioflavin T (ThT) binding assay was employed; it is frequently used to analyze the kinetic of the self-recognition process associated with amyloid fibers formation [51].
The time course of the ThT fluorescence when NPM1264-277 is incubated with the complexes 1, 2, 3 and 4 is reported in Figure 2. Fluorescence values were registered for the peptide in the presence of the different concentrations of the Pt compounds, according to the peptide to Pt(II) compound molar ratios indicated in the legends.  NPM1 264-277 alone, pre-treated with hexafluoroisopropanol (HFIP), exhibits a t 1/2 value for aggregation of 7 min, as reported in Table 2. When NPM1 264-277 is treated with the Pt compounds at a molar ratio of 1:1, a clear inhibitor effect is observed. The inhibition of the aggregation is faster for compounds 1 and 3 (Figure 2a,c) than for 2 and 4 (Figure 2b,d). In the case of 1 and 3, the ThT fluorescence value decreases in less than 5 min, whereas it reduces after 5 min and 20 min in the case of 2 and 4, respectively. The complete decrease of the fluorescence signal at 481 nm over time of the ThT/NPM1 264-277 systems in the presence of different Pt complexes suggests that the sample remains in the same monomeric state when treated with Pt compounds at 1:1 equivalents (Table 2). On the other hand, the anti-aggregation abilities of the investigated compounds are different at lower peptide to Pt complex molar ratios. When NPM1 264-277 is treated with 1 and 3 at 1:0.1, molar ratio its aggregation is completely inhibited after 50 min. On the contrary, when it is treated with 2 and 4, the inhibition of the amyloid aggregation is not completed in the investigated time scales. Furthermore, at 1:0.5 peptide to metal complex molar ratio, NPM1 264-277 provides ThT signals similar to those observed at 1:1 molar ratio in the presence of 1, 2 and 3, while it shows a ThT signal that suggests an incomplete inhibition of the aggregation process in the presence of 4. Potential variations of the fluorescence intensity of ThT caused by Pt(II) complexes are negligible and comparable to the blank signal registered in absence of the complexes.
This analysis indicates that 1 and 3 are the most effective inhibitors for amyloid aggregation of NPM1 264-277 ; therefore 1 was chosen for further analyses.
To verify if 1 could have a similar inhibitory effect on other amyloid systems, the ThT assay was also carried out using Sup35p 7-13 ( Table 1). The time courses of the ThT signals of Sup35p 7-13 , in the presence and in the absence of 1 are reported in Figure 3. The ThT fluorescence intensity of the peptide alone displays two distinct transitions, suggesting a seeding effect of first soluble aggregates to secondly achieve higher levels of oligomerization, as already suggested by other studies [52].
Comparing the ThT signal of the peptide alone with that observed in the presence of 1, it is clear that the Pt compound affects the aggregation process of Sup35p 7-13 , leading to species with a lower oligomeric state than those found in the case of the peptide alone.
We have also evaluated the ability of 1 to disaggregate soluble amyloid oligomers, monitoring the ThT signals versus time upon the addition of 1 to NPM1 264-277 and Sup35p 7-13 aggregates ( Figure 4). The two peptides have different aggregation kinetics, due to their differences in sequence and structure. For this reason, Sup35p 7-13 was pre-aggregated in the absence of the complexes for one night, whereas NPM1 264-277 was partially aggregated at t 0 as already reported [43]. Interestingly, we observed a decrease of ThT fluorescence intensity upon the addition of 1 for both peptides. These findings clearly indicate the disaggregating ability of the compound for both amyloid systems.
This analysis indicates that 1 and 3 are the most effective inhibitors for amyloid aggregation of NPM1264-277; therefore 1 was chosen for further analyses.
To verify if 1 could have a similar inhibitory effect on other amyloid systems, the ThT assay was also carried out using Sup35p7-13 ( Table 1). The time courses of the ThT signals of Sup35p7-13, in the presence and in the absence of 1 are reported in Figure 3. The ThT fluorescence intensity of the peptide alone displays two distinct transitions, suggesting a seeding effect of first soluble aggregates to secondly achieve higher levels of oligomerization, as already suggested by other studies [52].
Comparing the ThT signal of the peptide alone with that observed in the presence of 1, it is clear that the Pt compound affects the aggregation process of Sup35p7-13, leading to species with a lower oligomeric state than those found in the case of the peptide alone. We have also evaluated the ability of 1 to disaggregate soluble amyloid oligomers, monitoring the ThT signals versus time upon the addition of 1 to NPM1264-277 and Sup35p7-13 aggregates ( Figure  4). The two peptides have different aggregation kinetics, due to their differences in sequence and structure. For this reason, Sup35p7-13 was pre-aggregated in the absence of the complexes for one night, whereas NPM1264-277 was partially aggregated at t0 as already reported [43]. Interestingly, we observed a decrease of ThT fluorescence intensity upon the addition of 1 for both peptides. These findings clearly indicate the disaggregating ability of the compound for both amyloid systems.  A similar experiment was performed using Aβ21-40. For this peptide, the ThT fluorescence signal as function of time is reported in Figure 5. The addition of 1 to soluble aggregates of Aβ21-40, formed within 1000 min resulted in an instantaneous decrease of the fluorescence intensity, which indicates a disaggregating effect. The ability of 1 to dose-dependently inhibit the aggregation of NPM1264-277, Sup35p7-13 and Aβ21- 40 was quantitatively assessed through the comparison of experimental ThT fluorescence values [53], using different metal compound concentrations. The best fittings of experimental data, reported in Figure 6, provide IC50 values of 62.3 ± 1.3, 55.03 ± 1.12, 19.9 ± 1.6 µM for NPM1264-277, Sup35p7-13 and  A similar experiment was performed using Aβ21-40. For this peptide, the ThT fluorescence signal as function of time is reported in Figure 5. The addition of 1 to soluble aggregates of Aβ21-40, formed within 1000 min resulted in an instantaneous decrease of the fluorescence intensity, which indicates a disaggregating effect.  The ability of 0.9 to dose-dependently inhibit the aggregation of NPM1 264-277 , Sup35p 7-13 and Aβ [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] was quantitatively assessed through the comparison of experimental ThT fluorescence values [53], using different metal compound concentrations. The best fittings of experimental data, reported in Figure 6, provide IC 50 values of 62.3 ± 1.3, 55.03 ± 1.12, 19.9 ± 1.6 µM for NPM1 264-277 , Sup35p 7-13 and Aβ 21-40 , respectively.

Pt Complexes Inhibit Conformational β-Transition
The inhibitory effects of 1 and of the other Pt compounds here investigated could be associated with conformational variations of the analyzed peptides. To study these potential conformational variations, circular dichroism (CD) spectra of NPM1264-277 incubated with different equivalents of 1 for one night were compared. CD spectra are superimposed in Figure 7a.
A transition from a mixed α-helix+random coil structure towards a β-sheet structure was previously demonstrated for a variant of NPM1264-277 which includes helix 2 and the loop between the 1st and the 2nd helix of the bundle of the C-terminal domain of NPM1 [35].
The spectra of NPM1264-277 indicate that, upon overnight incubation, the peptide fibrillates and converts from α-helix to β-sheet (green vs. blue line in Figure 7a). Notably, the presence of the Pt compounds inhibits the α-helix to β-sheet conversion at all the investigated NPM1264-277:metal compound molar ratios. Indeed, spectra of NPM1264-277 in the presence of the Pt compounds show minima at wavelengths ≤ 210 nm (Figure 7a), which are diagnostic of the presence of a significant helical content and suggest the formation of ligand-specific secondary structures. A similar behavior has been previously observed when other Pt compounds interacted with Aβ peptides [42].
The same experiment was carried out using Aβ21-40. CD spectra of freshly prepared samples of this peptide are already indicative of the presence of a β-structure, as reported in Figure 7b (blue line), thus precluding the possibility to follow the α-helix to β-sheet transition. However, it is

Pt Complexes Inhibit Conformational β-Transition
The inhibitory effects of 1 and of the other Pt compounds here investigated could be associated with conformational variations of the analyzed peptides. To study these potential conformational variations, circular dichroism (CD) spectra of NPM1 264-277 incubated with different equivalents of 1 for one night were compared. CD spectra are superimposed in Figure 7a. one night of incubation, is more similar to that of the freshly prepared sample of Aβ21-40 than to that of Aβ21-40 incubated for one night in the absence of 1.
Attempts to carry out similar experiments using Sup35p7-13 failed, since during the aggregation process of this peptide only a significant decrease of the Cotton effect occurred [52] and no substantial differences were observed when the peptide was incubated in presence of 1.

Mass Spectrometry Analysis
The peptides and selected Pt compounds (1 and 3) at 1:10 ratio, were incubated for 24 h and analyzed by electrospray ionization mass spectrometry (ESI-MS) [54]. As an example, a portion of the spectra obtained for Aβ21-40 incubated with 1 is reported in Figure 8.
The presence of double charged signals at 1173.076 and 1212.586 m/z confirms the formation of adducts between Aβ21-40 and 1 at 1:1 molar ratio, which are formed upon the release of the chloride and DMSO or solely the chloride, respectively. Furthermore, the signal at 1421.596 m/z corresponds to the double charged peptide ion generated by Aβ21-40 bound to 2 Pt complexes, one with the release of the chloride and the other missing a chloride and DMSO. The theoretical and measured molecular masses are reported in Table 3. A transition from a mixed α-helix + random coil structure towards a β-sheet structure was previously demonstrated for a variant of NPM1 264-277 which includes helix 2 and the loop between the 1st and the 2nd helix of the bundle of the C-terminal domain of NPM1 [35].
The spectra of NPM1 264-277 indicate that, upon overnight incubation, the peptide fibrillates and converts from α-helix to β-sheet (green vs. blue line in Figure 7a). Notably, the presence of the Pt compounds inhibits the α-helix to β-sheet conversion at all the investigated NPM1 264-277 :metal compound molar ratios. Indeed, spectra of NPM1 264-277 in the presence of the Pt compounds show minima at wavelengths ≤210 nm (Figure 7a), which are diagnostic of the presence of a significant helical content and suggest the formation of ligand-specific secondary structures. A similar behavior has been previously observed when other Pt compounds interacted with Aβ peptides [42].
Attempts to carry out similar experiments using Sup35p 7-13 failed, since during the aggregation process of this peptide only a significant decrease of the Cotton effect occurred [52] and no substantial differences were observed when the peptide was incubated in presence of 1.

Mass Spectrometry Analysis
The peptides and selected Pt compounds (1 and 3) at 1:10 ratio, were incubated for 24 h and analyzed by electrospray ionization mass spectrometry (ESI-MS) [54]. As an example, a portion of the spectra obtained for Aβ 21-40 incubated with 1 is reported in Figure 8.

Mass Spectrometry Analysis
The peptides and selected Pt compounds (1 and 3) at 1:10 ratio, were incubated for 24 h and analyzed by electrospray ionization mass spectrometry (ESI-MS) [54]. As an example, a portion of the spectra obtained for Aβ21-40 incubated with 1 is reported in Figure 8.
The presence of double charged signals at 1173.076 and 1212.586 m/z confirms the formation of adducts between Aβ21-40 and 1 at 1:1 molar ratio, which are formed upon the release of the chloride and DMSO or solely the chloride, respectively. Furthermore, the signal at 1421.596 m/z corresponds to the double charged peptide ion generated by Aβ21-40 bound to 2 Pt complexes, one with the release of the chloride and the other missing a chloride and DMSO. The theoretical and measured molecular masses are reported in Table 3. The presence of double charged signals at 1173.076 and 1212.586 m/z confirms the formation of adducts between Aβ 21-40 and 1 at 1:1 molar ratio, which are formed upon the release of the chloride and DMSO or solely the chloride, respectively. Furthermore, the signal at 1421.596 m/z corresponds to the double charged peptide ion generated by Aβ 21-40 bound to 2 Pt complexes, one with the release of the chloride and the other missing a chloride and DMSO. The theoretical and measured molecular masses are reported in Table 3.

m/z Relative intensity
Noticeably the sequence NPM1 264-277 revealed the ability to form adducts with two, three or four Pt derivatives for both compounds 1 and 3. Results are reported in supplementary Tables S1 and S2.

Inhibition of Cytotoxic Effects of NPM1 264-277 Peptide in SH-SY5Y Cells
The ability of the Pt(II) complexes to reduce the neurotoxicity of the NPM1 264-277 peptide was assessed using human SH-SY5Y neuroblastoma cells. Cell survival was evaluated after treating SH-SY5Y cells with the peptide alone or with the mixture of the peptide and Pt(II) complexes.
In comparison with the control sample, the aggregated NPM1 264-277 peptide showed the highest toxicity at 2 h (cell viability <75%), becoming less effective after 24 h of incubation (cell viability <82%), probably because of the conversion of the early aggregates into larger and less toxic aggregates as already reported for similar amyloids [55] and for NPM1 264-277 peptide [43].
Compounds 1 and 3 incubated with NPM1 264-277 , at 1:10 molar ratio, seemed to have a protective function against the toxicity induced by the amyloid peptide. Indeed, their presence increased the cell viability values similar to untreated cells (Figure 9), as already reported for similar platinoid compounds [56].  Noticeably the sequence NPM1264-277 revealed the ability to form adducts with two, three or four Pt derivatives for both compounds 1 and 3. Results are reported in supplementary Tables S1 and S2.

Inhibition of Cytotoxic Effects of NPM1264-277 Peptide in SH-SY5Y Cells
The ability of the Pt(II) complexes to reduce the neurotoxicity of the NPM1264-277 peptide was assessed using human SH-SY5Y neuroblastoma cells. Cell survival was evaluated after treating SH-SY5Y cells with the peptide alone or with the mixture of the peptide and Pt(II) complexes.
In comparison with the control sample, the aggregated NPM1264-277 peptide showed the highest toxicity at 2 h (cell viability <75%), becoming less effective after 24 h of incubation (cell viability <82%), probably because of the conversion of the early aggregates into larger and less toxic aggregates as already reported for similar amyloids [55] and for NPM1264-277 peptide [43].
Compounds 1 and 3 incubated with NPM1264-277, at 1:10 molar ratio, seemed to have a protective function against the toxicity induced by the amyloid peptide. Indeed, their presence increased the cell viability values similar to untreated cells (Figure 9), as already reported for similar platinoid compounds [56].

Peptide Synthesis
Amyloid peptides analyzed in this study were synthesized as already reported [35,43]. Their sequences are reported in Table 1. Reagents for peptide synthesis were from Iris Biotech (Marktredwitz, Germany), solvents for peptide synthesis and HPLC analyses were from Romil (Dublin, Ireland); reversed phase columns for peptide analysis and the LC-MS system were from ThermoFisher (Waltham, MA, USA). Peptides' purity and identity were confirmed by LC-MS. Purified peptides were lyophilized and stored at −20 • C until use. Prior to be analyzed they were all treated for 30 min with HFIP to ensure a monomeric state (at 50% (v/v) in water), and then the organic solvent was removed by evaporation.

Synthesis of the Complexes
The Pt compounds were synthetized as previously described [11,12]. The stability of the compound under the investigated experimental conditions were tested using UV-Vis absorption spectroscopy, as previously done in ref [11].

Cell Culture
Human SH-SY5Y neuroblastoma cells (A.T.C.C., Manassas, VA, USA) were cultured in DMEM, supplemented with 10% FBS, 1.0 mM glutamine and antibiotics. Cell cultures were maintained in a 5.0% CO 2 humidified atmosphere at 37 • C and grown until they reached 80% confluence for a maximum of 20 passages.

Conclusions
In conclusion, we have studied the capability of Pt(II) complexes bearing O,S bidentate ligands to inhibit the aggregation process of three different amyloidogenic peptides. The results indicate that the O,S bidentate ligands of new Pt or Platinoid complexes are promising compounds able to inhibit aggregation of small model amyloid systems. Future studies on full-length proteins should confirm the anti-aggregation properties of these Pt(II) compounds and their potential application as drugs in neurodegenerative diseases.

Conflicts of Interest:
The authors declare no conflict of interest.