Metal-Complexes Bearing Releasable CO Differently Modulate Amyloid Aggregation

Abstract

Neurodegenerative diseases are often associated with an uncontrolled amyloid aggregation. Hence, many studies are oriented to discover new compounds that are able to modulate self-recognition mechanisms of proteins involved in the development of these pathologies. Herein, three metal-complexes able to release carbon monoxide (CORMs) were analyzed for their ability to affect the self-aggregation of the amyloidogenic fragment of nucleophosmin 1, corresponding to the second helix of the three-helix bundle located in the C-terminal domain of the protein, i.e., NPM1_264-277, peptide. These complexes were two cymantrenes coordinated to the nucleobase adenine (Cym-Ade) and to the antibiotic ciprofloxacin (Cym-Cipro) and a Re(I)-compound containing 1,10-phenanthroline and 3-CCCH₂NHCOCH₂CH₂-6-bromo-chromone as ligands (Re-Flavo). Thioflavin T (ThT) assay, UV-vis absorption and fluorescence spectroscopies, scanning electron microscopy (SEM), and electrospray ionization mass spectrometry (ESI-MS) indicated that the three compounds have different effects on the peptide aggregation. Cym-Ade and Cym-Cipro act as aggregating agents. Cym-Ade induces the formation of NPM1_264-277 fibers longer and stiffer than that formed by NPM1_264-277 alone; irradiation of complexes speeds the formation of fibers that are more flexible and thicker than those found without irradiation. Cym-Cipro induces the formation of longer fibers, although slightly thinner in diameter. Conversely, Re-Flavo acts as an antiaggregating agent. Overall, these results indicate that metal-based CORMs with diverse structural features can have a different effect on the formation of amyloid fibers. A proper choice of ligands attached to metal can allow the development of metal-based drugs with potential application as antiamyloidogenic agents.

Figure 1

Chemical structures of the metal-based carbon monoxide-releasing molecules (CORMs) and sequence of amyloid model, investigated in this study.

Figure 2

UV–vis spectra overtime of Cym-Ade (panels A and B), Cym-Cipro (panels C and D) and Re-Flavo (panels E and F), not irradiated (left) and irradiated at 365 nm (right) in 10 mM borate buffer at pH 8.5 (1% DMSO, v/v). Complex concentration was 200 μM for Cym-Cipro and 50 μM for Cym-Ade and Re-Flavo.

Figure 3

Overlay of time-courses of ThT fluorescence emission intensity of NPM1_264–277 (100 μM) in the presence of Cym-Ade (panels A and B), Cym-Cipro (panels C and D), and Re-Flavo (panels E and F), at indicated peptide:metal complex molar ratios, without (left panel) and with irradiation at 365 nm (right panel) (10 mM borate buffer, 1% DMSO (v/v)). Results are representative of two independent experiments.

Figure 4

ESI-MS spectra of (A) not-irradiated, (B) irradiated Cym-Ade incubated with NPM1_264–277.

Figure 5

ESI-MS spectra of (A) not-irradiated, (B) irradiated Cym-Cipro incubated with NPM1_264–277. The asterisk highlights the species present also in the MS spectrum of the Cym-Cipro alone.

Figure 6

ESI-MS spectra of (A) not-irradiated, (B) irradiated Re-Flavo incubated with NPM1_264–277.

Figure 7

Overlay of fluorescence emission spectra, λ_exc= 440 nm, of NPM1_264–277 (100 μM) in (A) the absence and in (B) the presence of Cym-Ade at 1:5 NPM1_264–277: Cym-Ade molar ratio (10 mM borate buffer at pH 8.5, 1% DMSO, v/v), under stirring.

Figure 8

Cytotoxic effect of molecules in SHSY5Y cells: MTT assay of NPM1 _264–277 alone and in the presence of Cym-Ade and Cym-Cipro, incubated under stirring at times t = 0, 2 h. We refer to the control untreated cells (CTRL) as 100% of viable cells. Statistical analysis was calculated by GraphPad Prism 9 by two-way Anova with Šídák’s multiple comparison test (***p = 0.0001).

Figure 9

SEM micrographs of NPM1_264–277 (100 μM) incubated with Re-Flavo (A-A′′) or Cym-Cipro (B–B′′) at 1:5 peptide:metal complex molar ratio. Overviews of the surface of samples at 1 mm (A,B), 50 μm (A′,B′) and 5 μm (A′′,B′′). In red, measured diameters: (A′) 23.09 μm, 18.96 μm, 20.01 μm; (B′) 14.30 μm, 18.46 μm, 10.08 μm, 9.722 μm, 11.69 μm.

Figure 10

Morphology of assembled structures. SEM micrographs of NPM1_264–277 peptide (100 μM) incubated with Cym-Ade not-irradiated (A-A′′) and irradiated (B–B′′) at 1:5 peptide:complex molar ratio. Overviews of the surface of samples at 1 mm (A,B), 50 μm (A′,B′) and 5 μm (A′′,B′′). In red, measured diameters: (A′) 11.66 μm, 11.57 μm, 10.97 μm; (B′) 18.01 μm, 14.82 μm, 11.61 μm, 9.634 μm, 19.50 μm.