Mixed Ligand Cu²⁺ Complexes of a Model Therapeutic with Alzheimer’s Amyloid‑β Peptide and Monoamine Neurotransmitters

8-Hydroxyquinolines (8HQ) have found widespread application in chemistry and biology due to their ability to complex a range of transition metal ions. The family of 2-substituted 8HQs has been proposed for use in the treatment of Alzheimer’s disease (AD). Most notably, the therapeutic PBT2 (Prana Biotechnology Ltd.) has been shown to act as an efficient metal chaperone, disaggregate metal-enriched amyloid plaques comprised of the Aβ peptide, inhibit Cu/Aβ redox chemistry, and reverse the AD phenotype in transgenic animal models. Yet surprisingly little is known about the molecular interactions at play. In this study, we show that the homologous ligand 2-[(dimethylamino)­methyl]-8-hydroxyquinoline (HL) forms a CuL complex with a conditional (apparent) dissociation constant of 0.33 nM at pH 6.9 and is capable of forming ternary Cu²⁺ complexes with neurotransmitters including histamine (HA), glutamic acid (Glu), and glycine (Gly), with glutathione disulfide (GSSG), and with histidine (His) side chains of proteins and peptides including the Aβ peptide. Our findings suggest a molecular basis for the strong metal chaperone activity of PBT2, its ability to attenuate Cu²⁺/Aβ interactions, and its potential to promote neuroprotective and neuroregenerative effects

Oligomerisation of α-syn on treatment with DA.

<p><b>A</b>. Schematic of the different α-syn constructs used in this study. The black bars represent the imperfect repeats (residues 10–16, 21–27, 32–37, 43–49, 57–63, 80–86). NAC region (stippled bar, residues 60–95). <b>B</b>. Silver stain SDS-PAGE gel of truncated α-syn incubated in the presence or absence of DA. Lane 1: α-syn 1–140. Lane 2: α-syn 1–140 + DA. Lane 3: α-syn 1–95. Lane 4: α-syn 1–95 + DA. Lane 5: α-syn 43–140. Lane 6: α-syn 43–140 + DA. The α-syn to DA ratio was 1:10. <b>C</b>. Size exclusion chromatography of DA:α-syn 43–140 and DA:α-syn 1–95 oligomers. 200 μM α-syn was incubated with 2 mM DA for 7 days. The reaction was centrifuged at 100,000 rpm, 1 hour, 4°C and then analysed on a Superdex 200 10/300GL column using 10 mM sodium phosphate pH 7.5 buffer with a flow rate of 0.5 mL/min. Proteins were detected at A280nm.</p

Synthesis of 2‑Pyridyl-benzimidazole Iridium(III), Ruthenium(II), and Platinum(II) Complexes. Study of the Activity as Inhibitors of Amyloid‑β Aggregation and Neurotoxicity Evaluation

The design of small molecules that can target the aggregation of Aβ as potential therapeutic agents for Alzheimer’s disease is an area of study that has attracted a lot of attention recently. The novel ligand methyl 1-butyl-2-pyridyl-benzimidazole carboxylate was prepared for the synthesis of a series of new iridium­(III), ruthenium­(II), and platinum­(II) 2-pyridyl-benzimidazole complexes. The crystal structure of the half-sandwich iridium­(III) complex was established by X-ray diffraction. An arrangement of two cationic complexes in the unit cell is observed, and it seems to be organized by weak π···π interactions that are taking place between two symmetry-related benzimidazole ring systems. All new compounds inhibited aggregation of Aβ1–42 in vitro as shown by both thioflavin T fluorescence assay and transmission electron microscopy. Among them the Ir compound rescued the toxicity of Aβ1–42 in primary cortical neurons effectively

Stability of DA:α-syn 43–140 dimer exposed to denaturants.

<p>Silver stain SDS-PAGE gel of DA:α-syn 43–140 dimer incubated with different amounts of denaturants HFIP or TFE. The dimers did not dissociate under any of the conditions tested, suggesting a covalent cross-link. <b>Lane 1:</b> DA:α-syn 43–140 dimer + 1% HFIP. <b>Lane 2:</b> DA:α-syn 43–140 dimer + 2% HFIP. <b>Lane 3:</b> DA:α-syn 43–140 dimer + 4% HFIP. <b>Lane 4:</b> DA:α-syn 43–140 dimer + 5% HFIP. <b>Lane 5:</b> DA:α-syn 43–140 dimer + 4% TFE. <b>Lane 6:</b> DA:α-syn 43–140 dimer + 8% TFE. <b>Lane 7:</b> DA:α-syn 43–140 dimer + 10% TFE. <b>Lane 8:</b> DA:α-syn 43–140 dimer + 15% TFE. Arrow indicates the DA:α-syn 43–140 dimer band.</p

Comparison of ¹H, ¹⁵N-HSQC spectra acquired on DA:α-syn 43–140 monomer and α-DA:syn 43–140 dimer showing the characteristics expected for an intrinsically disorder protein.

<p>The proton-nitrogen correlation spectrum (HSQC) of 0.5 mM DA:α-syn 43–140 monomer or DA:α-syn 43–140 dimer in 10 mM sodium phosphate buffer, pH5.5. Spectra were acquired at 5°C and 800MHz. The HSQC spectrum of DA:α-syn 43–140 monomer is depicted in black and the DA:α-syn 43–140 dimer in pink. Amide resonance assignments are indicated for the DA:α-syn 43–140 monomer as sequence position and the poor chemical shift dispersion indicates little if any secondary structure is present. * indicate residues of interest, those colored orange indicate resonances with exchange broadening (see the text).</p

SDS-dependent conformational transition in DA:α-syn 43–140 monomer, but not DA:α-syn 43–140 dimer.

<p>Far UV CD spectrum of 15 μM DA:α-syn 43–140 monomer or DA:α-syn 43–140 dimer in 10 mM sodium phosphate buffer, pH 7.5. <b>A</b>. DA:α-syn 43–140 monomer minus (thin line) or plus 16 mM SDS (thick line). <b>B</b>. DA:α-syn 43–140 dimer minus (thin line) or plus 16 mM SDS (thick line). The CD spectrum shows the monomer and dimer in the absence of SDS have an intensity at λ = 200 nm is indicative of random coil structure. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116497#pone.0116497.ref058" target="_blank">58</a>] In the presence of SDS, the α-syn 43–140 monomer, but not the α-syn 43–140 dimer, showed a double minima at 208 and 220 nm with a maximum at 190nm are indicative of α-helical content. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116497#pone.0116497.ref059" target="_blank">59</a>]</p

Truncation mutants of α-syn form oligomers after treatment with DA.

<p>Truncated α-syn and mutants were treated with DA and the soluble fraction subjected to SDS-PAGE and visualised by silver staining <b>A</b>. Tricine SDS-PAGE. Lane 1: α-syn 43–140, Lane 2: α-syn 43–140 + DA, Lane 3: α-syn 43–60, Lane 4: α-syn 43–60 + DA. <b>B</b>. Lane 1: α-syn 43–140, Lane 2: α-syn 43–140 + DA (1:4), Lane 3: α-syn 43–140 + DA (1:10), Lane 4: α-syn 1–60, Lane 5: α-syn 1–60 + DA (1:4), Lane 6: α-syn 1–60 + DA (1:10). <b>C</b>. SEC of the α-syn 43–140 and α-syn 1–60 DA induced oligomers. 200 μM α-syn was incubated with 2 mM DA for 7 days. The reaction was centrifuged at 100,000 rpm, 1 hour, 4°C and then analysed on a Superdex 200 10/300GL column using 10 mM sodium phosphate pH 7.5 buffer with a flow rate of 0.5 mL/min. Proteins were detected at A280nm. The solid line represents α-syn 43–140 and broken line α-syn 1–60.</p

The NAC region is not required for DA mediated oligomerisation.

<p>The NAC fibrillsation mutant T75P did not alter DA mediated oligomerisation of α-syn 43–140. α-syn 43–140 T75P was incubate with different ratios of DA from 1:1 to 1:20 and analysed by SDS-PAGE and silver stain. Lane 1: α-syn 43–140 T75P, Lane 2: α-syn 43–140 T75P + DA (1:1), Lane 3: α-syn 43–140 T75P + DA (1:2), Lane 4: α-syn 43–140 T75P + DA (1:5), Lane 5: α-syn 43–140 T75P + DA (1:10), Lane 6: α-syn 43–140 T75P + DA (1:20), Lane 7: α-syn 43–140, Lane 8: α-syn 43–140 + DA (1:10)</p

Chemical shift deviations between DA:α-syn 43–140 monomer and DA:α-syn 43–140 dimer.

<p>The Cα resonances (Panels A and B) of the DA:α-syn 43–140 dimer do not differ significantly from those of the DA:α-syn 43–140 monomer. <b>A</b>. Cα chemical shift deviations from random coil values predict that the DA:α-syn 43–140 monomer is largely unstructured. <b>B</b>. Plot of chemical shift difference for Cα chemical shifts between DA:α-syn 43–140 monomer and DA:α-syn 43–140 dimer. The chemical shift deviation is plotted in ppm as a function of residue position in DA:α-syn 43–140 monomer. <b>C</b>. Plot of difference in Cα resonance position (ΔδCα (ppm)) from random coil values [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116497#pone.0116497.ref060" target="_blank">60</a>] for DA:α-syn 43–140 monomer. The largest differences arise from residues preceding a proline (A107, M116, D119, M127, E138). <b>D</b>. Plot of difference in Cα resonance positions between DA:α-syn 43–140 monomer and DA:α-syn 43–140 dimer (δ_dimer—δ_monomer). The absence of significant chemical shift differences between the DA:α-syn 43–140 dimer and DA:α-syn 43–140 monomer indicates their structural similarity. The largest differences are localised to residues near the methionine residues (M116, M127) that are likely to be oxidised under the conditions of DA-mediated α-syn 43–140 dimer formation.</p

α-syn point mutants form oligomers.

<p>Silver stain SDS-PAGE gel of soluble fraction of mutant α-syn reacted with DA. Lane 1: α-syn 43–140, Lane 2: α-syn 43–140 + DA (1:10), Lane 3: α-syn 43–140 E83A, Lane 4: α-syn 43–140 E83A + DA (1:10), Lane 5: α-syn 43–140 H50N, Lane 6: α-syn 43–140 H50N+ DA (1:10)</p