A Diruthenium Metallodrug as a Potent Inhibitor of Amyloid-β Aggregation: Synergism of Mechanisms of Action

The physical and chemical properties of paddlewheel diruthenium compounds are highly dependent on the nature of the ligands surrounding the bimetallic core. Herein, we compare the ability of two diruthenium compounds, [Ru2Cl(D-p-FPhF)(O2CCH3)3]·H2O (1) (D-p-FPhF– = N,N′-bis(4-fluorophenyl)formamidinate) and K3[Ru2(O2CO)4]·3H2O (2), to act as inhibitors of amyloid aggregation of the Aβ1–42 peptide and its peculiar fragments, Aβ1–16 and Aβ21–40. A wide range of biophysical techniques has been used to determine the inhibition capacity against aggregation and the possible mechanism of action of these compounds (Thioflavin T fluorescence and autofluorescence assays, UV–vis absorption spectroscopy, circular dichroism, nuclear magnetic resonance, mass spectrometry, and electron scanning microscopy). Data show that the most effective inhibitory effect is shown for compound 1. This compound inhibits fiber formation and completely abolishes the cytotoxicity of Aβ1–42. The antiaggregatory capacity of this complex can be explained by a binding mechanism of the dimetallic units to the peptide chain along with π–π interactions between the formamidinate ligand and the aromatic side chains. The results suggest the potential use of paddlewheel diruthenium complexes as neurodrugs and confirm the importance of the steric and charge effects on the properties of diruthenium compounds.


Figure S2 .
Figure S2.CD spectra of compound 1 (panel A) and compound 2 (panel B), at the indicated times of stirring.

Figure
Figure S3. 1 H NMR spectra of Aβ 21-40 in the absence (blue) and in the presence of compound 1 (green) or compound 2 (red).H N /aromatic and Hα/side chains regions of the spectra are shown on the left and on the right panels, respectively.

Figure S4 .
Figure S4.Absorption spectra of compound 1 (left panel) and 2 (right panel) upon the addition of increasing amount of (a and b) Aβ 1--42 , (c and d) Aβ 1--16 , and (e and f) Aβ 21--40 peptides.The arrows indicate the spectral variations.As insets, UV intensities at indicated wavelengths versus concentration of Aβ peptides are reported.

Figure S5 .
Figure S5.ESI-MS spectrum of Aβ 21-40 peptide at 0 h (panel A).ESI-MS spectra of Aβ 21-40 peptide incubated with compound 1 after 0 (panel B) and 24 h (panel C).The peaks marked with b n derive from spontaneous in source fragmentation of Aβ 21-40 peptide (b series elements); the asterisk highlighted the species present in the control (compound 1 alone).

Figure S6 .
Figure S6.ESI-MS spectrum of Aβ 1-42 peptide at 0 h (panel A).ESI-MS spectra of Aβ 1-42 peptide incubated with compound 1 after 0 (panel B) and 24 h (panel C).The peaks marked with b n derive from spontaneous in source fragmentation of Aβ 1-42 peptide (b series elements); the asterisk highlighted the species present in the control (compound 1 alone).

Figure S7 .
Figure S7.ESI-MS spectrum of Aβ 1-16 peptide alone at 0 h (panel A).ESI-MS spectra of Aβ 1-16 peptide incubated with compound 2 at 0 h (panel B) and at 24 h (panel C) conditions.The peaks marked with b n derive from spontaneous in source fragmentation of Aβ 1-16 peptide (b series elements).§ indicates K + adducts.

Figure S8 .
Figure S8.ESI-MS spectrum of Aβ 21-40 peptide alone at 0 h (panel A).ESI-MS spectra of Aβ 21-40 peptide incubated with compound 2 at 0 h (panel B) and at 24 h (panel C) conditions.The peaks marked with b n derive from spontaneous in source fragmentation of Aβ 21-40 peptide (b series elements).The § indicates K + adducts.

Figure S9 .
Figure S9.ESI-MS spectrum of Aβ 1-42 peptide alone at 0 h (panel A).ESI-MS spectra of Aβ 1-42 peptide incubated with compound 2 at 0 h (panel B) and at 24 h (panel C) conditions.The peaks marked with b n derive from spontaneous in source fragmentation of Aβ 1-42 peptide (b series elements); the asterisk highlighted the species present in the control (compound 2 alone).The § indicates K + adducts.

Figure S10 .
Figure S10.(a-c) Fluorescence emission spectra at different times of Aβ 1-16 in the absence and presence of compound 1. (d-e) Fluorescence emission spectra at different times of compounds 1 and 2 ( ex = 440 nm).

Table S1 :
Experimental m/z values detected in the spectra of Aβ 1-16 , Aβ 21-40 , Aβ 1-42 alone (Control) and with the addition of compound 1 at 0 and 24 h of incubation.The ion species corresponding to each experimental m/z, the expected m/z value (theoretical) and their charge states are also reported.

Table S2 :
Experimental m/z values detected in the spectra of Aβ 1-16 , Aβ 21-40 , Aβ 1-42 alone (Control) and with the addition of compound 2 at 0 and 24h of incubation.The ion species corresponding to each experimental m/z, the expected m/z value (theoretical) and their charge states are also reported.

Table S3 :
SEM analysis.Average diameter and length of fibers obtained for A peptides in the presence and in the absence of compound 1.