A Half-Sandwich Os(II) Glucoconjugated NHC Complex as a Modulator of Amyloid Aggregation

Abstract

Herein, the effects of a novel half-sandwich Os(II) complex on the aggregation of an amyloid model system, derived from the C-terminal domain of the nucleophosmin 1 protein (NPM1_264-277), were investigated. The thioflavin T (ThT) binding assay revealed that the complex [(η⁶-toluene)Os(NHCglu)Cl₂] (where NHCglu is the N-heterocyclic carbene ligand 1-methyl-3-{2,3,4,6-tetra-O-acetyl-1-glucosyl}imidazol-2-ylidene), hence named Os-Tolu, was able to repress amyloid aggregation in a dose-dependent way. Conformational studies through circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies clearly indicated that this inhibitory effect occurred through the stabilization of α-helical structures of monomeric NPM1_264-277, thus hampering self-recognition. Electrospray ionization mass spectrometry (ESI-MS) studies evidenced, through the formation of coordination adducts, direct interactions of the amyloid peptide with the Os-glucoconjugate complex that, in turn, promote chemical modifications of the sequence further disfavoring the self-assembly process. Noticeably, the presence of Os-Tolu completely repressed the formation of amyloid fibers in scanning electron microscopy (SEM) analysis and induced a slight rescue of cell viability, in contrast to its reduction caused by the amyloid model in human SH-SY5Y neuroblastoma cells. These data strongly support the hypothesis of expanding the use of osmium-based agents to neurodegenerative diseases, positioning them as potential neurodrugs.

Figure 1

(A) Structure of the Os(II) NHC complex Os-Tolu. (B) Primary structure of NPM1_264–277.

Scheme 1. Synthetic Route to Access Os-Tolu

Figure 2

¹H NMR spectrum of Os-Tolu in CDCl₃ at 263 K. In the inset, the highlighted signals are attributed to the NHC imidazole (*) and the anomeric glucosyl (#) protons.

Figure 3

Left panel: (A) ThT fluorescence assay of NPM1_264–277 alone (black) and NPM1_264–277 in the presence of Os-Tolu 1:1 (blue) and 1:5 (green). Os-Tolu alone is reported in red. Right panel: overlay of florescence emission spectra: λ_exc= 440 nm of (B) NPM1_264–277 alone and (C) NPM1_264–277 in the presence of Os-Tolu 1:5.

Figure 4

Upper panel: overlay of CD spectra over time of (A) NPM1_264–277 and (B) NPM1_264–277 + Os-Tolu 1:1 molar ratio. Lower panel: FTIR spectra of (C) NPM1_264–277 alone and in the presence of Os-Tolu 1:1 molar ratio and (D) deconvoluted and second-derivative FTIR spectra.

Figure 5

ESI-MS spectra of (A) NPM1_264–277 + Os-Tolu 1:1 molar ratio and of (B) NPM1_264–277 alone reported as reference. Asterisks (*) highlight the species present in the complex alone.

Figure 6

SEM micrographs of NPM1_264–277 (100 μM) alone (A, A′) and in the presence of Os-Tolu (B, B′) at 1:1 peptide/metal complex molar ratio. Overviews of the surface of the samples at 300 μm (A, B) and 30 μm (A′, B′).

Figure 7

Effects of Os-Tolu on the cytotoxicity of the NPM1 _264–277 peptide in SH-SY5Y neuroblastoma cells: the MTT assay of NPM1 _264–277 peptide in the absence and in the presence of Os-Tolu, incubated under stirring, at t = 0 and 2 h. Control refers to untreated cells as 100% of viable cells. Data are reported as mean ± standard deviation. Statistical comparisons were performed with a Student’s unpaired t-test.