Rational design of a structural framework with potential use to develop chemical reagents that target and modulate multiple facets of Alzheimer's disease

Abstract

Alzheimer's disease (AD) is characterized by multiple, intertwined pathological features, including amyloid-β (Aβ) aggregation, metal ion dyshomeostasis, and oxidative stress. We report a novel compound (ML) prototype of a rationally designed molecule obtained by integrating structural elements for Aβ aggregation control, metal chelation, reactive oxygen species (ROS) regulation, and antioxidant activity within a single molecule. Chemical, biochemical, ion mobility mass spectrometric, and NMR studies indicate that the compound ML targets metal-free and metal-bound Aβ (metal-Aβ) species, suppresses Aβ aggregation in vitro, and diminishes toxicity induced by Aβ and metal-treated Aβ in living cells. Comparison of ML to its structural moieties (i.e., 4-(dimethylamino)phenol (DAP) and (8-aminoquinolin-2-yl)methanol (1)) for reactivity with Aβ and metal-Aβ suggests the synergy of incorporating structural components for both metal chelation and Aβ interaction. Moreover, ML is water-soluble and potentially brain permeable, as well as regulates the formation and presence of free radicals. Overall, we demonstrate that a rational structure-based design strategy can generate a small molecule that can target and modulate multiple factors, providing a new tool to uncover and address AD complexity.

Figure 1

Rational structure-based design principle (incorporation approach) of a multifuncitonal ligand (ML). Atoms responsible for metal binding are in bold. Chemical structures: ML = 4-(dimethylamino)-2-(((2-(hydroxymethyl)quinolin-8-yl)-amino)-methyl)phenol; p-I-stilbene = (E)-4-(4-iodostyryl)-N,N-dimethylaniline); L2-b = N¹,N¹-dimethyl-N⁴-(pyridin-2-ylmeth-yl)benzene-1,4-diamine; 1 = (8-aminoquinolin-2-yl)methanol.

Figure 2

Interactions of ML with soluble metal-free or Cu(ii)-treated Aβ species monitored by mass spectrometry or SOFAST-HMQC NMR. Mass spectra of (a) 1:5 mixture of Aβ42 and ML and (b) pure Aβ42 (z/n = charge/oligomer number). (c and d) 2D SOFAST-HMQC NMR spectra of ML-titrated metal-free monomeric Aβ40. Freshly dissolved Aβ40 (80 µM) in 50 mM Tris-DCl (pD 7.3) was titrated to (c) 40–160 µM or (d) 400 µM ML at 4 °C. The contour level of (d) has been adjusted to clearly show ligand-bound resonances. (e) Chemical shift perturbations within Aβ40 following addition of ML (Aβ:ML = 1:2). (f) Residues with largest changes in chemical shift at 1:1 Aβ40:ML mapped onto the NMR structure of the helical conformer of the Aβ40 structural ensemble (PDB 2LFM). Mass spectra of (g) Aβ42, Cu(ii), and ML (1:1:2) and of (h) Aβ42 and Cu(ii) (1:1). Peaks for pure Aβ42, Cu(ii)-bound Aβ42, ML-bound Aβ42, and Aβ42–Cu(ii)–ML complexes are noted with triangles, rectangles, circles, and stars, respectively.

Figure 3

(a–g) Interaction of ML with fibrillar Aβ40 species by saturation transfer difference (STD) NMR and (h–k) influence of ML on early Aβ42 oligomerization monitored by mass spectrometry and ion mobility studies. (a) Chemical shift changes in the ¹H spectra of ML upon the addition of 10 mol % metal-free Aβ40 fibers in 100% D₂O (20 mM deuterated Tris–DCl, pD 7.4). Large chemical shift changes can be seen in the aniline ring and dimethylamino groups (marked with an asterisk). (b) ¹H STD NMR spectra of ML with Aβ40 fibers (Aβ:ML = 1:10). Comparison of STD signal intensity (red) to the STD reference (black) reflects the relative proximity of the corresponding proton to the Aβ40 fiber. (c) ¹H STD NMR spectra of ML alone showing the absence of an STD signal in the absence of Aβ40 fibers. (d) Normalized STD intensities mapped to ML’s structure. Larger blue circles indicate a more intense STD effect; gray circles indicate the absence of an STD signal. (e) Lowest energy docked conformation of ML to Aβ40 fibers (PDB 2LMO). Other docked conformations and a cluster analysis can be found in Supporting Information Figures S4 and S5. (f) Comparison of the ¹H spectra of ML (200 µM) with Aβ40 fibers (20 µM) in 100% D₂O (20 mM deuterated Tris–DCl, pD 7.4) with (black) and without (red) 500 µM ZnCl₂. The large chemical shift changes are evidence of binding of Zn(ii) to ML. (g) ¹H STD NMR spectra of ML with Aβ40 fibers in a ratio of 10:1 in the presence of ZnCl₂ (500 µM). Arrival time distriubtions (ATDs) for the z/n = −5/2 peak of (h) pure Aβ42 and (i) 1:5 mixture of Aβ42 and ML sample, respectively. (j) ATD for the −5/2 peak of the Aβ42 sample prepared and placed on ice for ca. 4 h. (k) ATD for the −5/2 peak of the preincubated Aβ42 sample immediately following the addition of ML (ca. 5 min).

Figure 4

Influence of ML or 1 on the formation of metal-free and metal-induced Aβ40/42 aggregates. (a) Scheme of the inhibition experiment. (b) Aβ species were visualized by gel electrophoresis using immunoblotting with an anti-Aβ antibody (6E10). Experimental conditions: Aβ (25 µM); CuCl₂ or ZnCl₂ (25 µM); ML or 1 (50 µM); 4, 8, or 24 h; pH 6.6 (for metal-free and Cu(ii) experiments) or 7.4 (for metal-free and Zn(ii) experiments); 37 °C; constant agitation. Lanes: (1) Aβ; (2) Aβ + 1; (3) Aβ + ML; (4) Aβ + [CuCl₂ or ZnCl₂]; (5) Aβ + [CuCl₂ or ZnCl₂] + 1; (6) Aβ + [CuCl₂ or ZnCl₂] + ML. (c) TEM images of the 24 h incubated samples from (b).

Figure 5

Biological activities of ML. (a) Effect of ML on toxicity triggered by metal-free Aβ and metal–Aβ species in N2aAPPswe cells. Cells treated with Aβ40/42 (10 µM), a metal chloride salt (CuCl₂ or ZnCl₂; 10 µM), or ML (10 µM) were incubated for 24 h at 37 °C. Cell viability (%) was determined by the MTT assay compared to cells treated with DMSO only (0–1%, v/v) (MTT = 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide). Data are mean ± SEM, P < 0.05, n = 3. (b) Inhibitory activity toward ROS formation in the absence and presence of freshly prepared Aβ40 (normal condition) and Aβ40 aggregates (inhibition and disaggregation conditions), determined by the 2-deoxyribose assay. The absorbance values are normalized compared to ligand-free condition (Aβ/CuCl₂/ML = 25/10/125 µM). (c) Antioxidant activity of ML, DAP, 1, and a mixture of DAP and 1 (DAP + 1) identified by the TEAC assay. The TEAC values are relative to a vitamin E analogue, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid).

Scheme 1

Synthetic Route to ML