Reactivity of diphenylpropynone derivatives toward metal-associated amyloid-β species

Abstract

In Alzheimer's disease (AD), metal-associated amyloid-β (metal-Aβ) species have been suggested to be involved in neurotoxicity; however, their role in disease development is still unclear. To elucidate this aspect, chemical reagents have been developed as valuable tools for targeting metal-Aβ species, modulating the interaction between the metal and Aβ, and subsequently altering metal-Aβ reactivity. Herein, we report the design, preparation, characterization, and reactivity of two diphenylpropynone derivatives (DPP1 and DPP2) composed of structural moieties for metal chelation and Aβ interaction (bifunctionality). The interactions of these compounds with metal ions and Aβ species were confirmed by UV-vis, NMR, mass spectrometry, and docking studies. The effects of these bifunctional molecules on the control of in vitro metal-free and metal-induced Aβ aggregation were investigated and monitored by gel electrophoresis and transmission electron microscopy (TEM). Both DPP1 and DPP2 showed reactivity toward metal-Aβ species over metal-free Aβ species to different extents. In particular, DPP2, which contains a dimethylamino group, exhibited greater reactivity with metal-Aβ species than DPP1, suggesting a structure-reactivity relationship. Overall, our studies present a new bifunctional scaffold that could be utilized to develop chemical reagents for investigating metal-Aβ species in AD.

Figure 1

Incorporation approach (top) and structures of small molecules (bottom). Left to right: 3-(4-(dimethylamino)phenyl)-1-(4-iodophenyl)-2-propyn-1-one; DPP1 = 3-phenyl-1-(pyridin-2-yl)prop-2-yn-1-one; DPP2 = 3-(4-(dimethylamino)phenyl)-1-(pyridin-2-yl)-2-propyn-1-one.

Figure 2

Solution speciation studies of DPP1 and DPP2. Top: UV-Vis spectra of DPP1 (40 µM, left) and DPP2 (20 µM, right) in the range of pH 2–10. Middle: Solution speciation diagrams for DPP1 (left) and DPP2 (right) (F_L = fraction of compound with given protonation). Bottom: Acidity constants (pK_a) of L (L = DPP1 or DPP2). Charges are omitted for clarity. ^a Error in the parentheses is shown in the last digit. Conditions: I = 0.10 M NaCl; room temperature.

Figure 3

Cu²⁺ or Zn²⁺ binding of DPP1 and DPP2. (a) UV-Vis spectra of DPP1 (left) and DPP2 (right) with CuCl₂ (1–20 equiv) in EtOH at room temperature (incubation for 2.5 h (for DPP1) and 5 min (for DPP2)). (b) ¹H NMR spectra of DPP1 (left, black) or DPP2 (right, black) with ZnCl₂ (red) in CD₃CN at room temperature ([compound] = 4 mM; [ZnCl₂] = 4 mM).

Figure 4

Solution speciation investigation of the Cu²⁺–DPP2 complexes. Top left: UV-Vis spectra (pH 2–7) for the Cu²⁺–DPP2 complexes ([Cu²⁺]/[L] = 1:2; [Cu²⁺]_total = 10 µM; 7 h incubation with ligand (L) prior to pH titration, L = DPP2; room temperature). Top right: Solution speciation diagram of the Cu²⁺–DPP2 complexes (F_Cu = fraction of free Cu and Cu complexes). Bottom: Stability constants (logβ) of the Cu²⁺–DPP2 complexes. Charges are omitted for clarity. ^aError in the parentheses is shown in the last digit. ^b The species containing CuL₂ was introduced into the calculation model yielding a good fit to the data.

Figure 5

Aβ interactions of DPP1 and DPP2. (a) MS data for the complexes of Aβ_1–40 and DPP1 or DPP2 ([Aβ] =100 µM; [compound] = 600 µM; M = monomer, D = dimer, and T = trimer). Many binding stoichiometries were detected, including 1:1 (star), 2:1 (square), and 3:1 (triangle), (b) A histogram showing the total bound MS signal intensity, normalized for non-specific interactions and ESI-MS artifacts, for each binding stoichiometry observed in (a), (c) Docking studies of DPP1 (orange) and DPP2 (green) with Aβ_1–40 (PDB 2LFM) by AutoDock Vina. Poses for both compounds were overlapped in this conformation (other conformations, Figure S4). The helical region of Aβ (H13-D23) is highlighted in color (tan) in both the cartoon (left) and surface (right) representations.

Figure 6

Inhibition experiment (scheme, top). Analysis of various-sized Aβ species by (a) native gel electrophoresis and (b) SDS-PAGE (non-reducing conditions) with Western blot using an anti-Aβ antibody (6E10). (c) TEM images of the 24 h incubated samples. Conditions: [Aβ] = 25 µM; [CuCl₂ or ZnCl₂] = 25 µM; [compound] = 50 µM; pH 6.6 (for Cu²⁺ samples) or 7.4 (for metal-free and Zn²⁺ samples); 4, 8, or 24 h incubation; 37 °C; constant agitation.

Scheme 1

Synthesis of DPP2.