Small molecule modulators of copper-induced Abeta aggregation

Abstract

Our design of bifunctional metal chelators as chemical probes and potential therapeutics for Alzheimer's disease (AD) is based on the incorporation of a metal binding moiety into structural frameworks of Abeta aggregate-imaging agents. Using this strategy, two compounds 2-[4-(dimethylamino)phenyl]imidazo[1,2-a]pyridine-8-ol (1) and N(1),N(1)-dimethyl-N(4)-(pyridin-2-ylmethylene)benzene-1,4-diamine (2) were prepared and characterized. The bifunctionality for metal chelation and Abeta interaction of 1 and 2 was verified by spectroscopic methods. Furthermore, the reactivity of 1 and 2 with Cu(II)-associated Abeta aggregates was investigated. The modulation of Cu(II)-triggered Abeta aggregation by 1 and 2 was found to be more effective than that by the known metal chelating agents CQ, EDTA, and phen. These studies suggest a new class of multifunctional molecules for the development of chemical tools to unravel metal-associated events in AD and potential therapeutic agents for metal-ion chelation therapy.

Figure 1

Strategy of designing metal chelators. Chemical structures of ¹²⁵IMPY, p-¹²⁵I-stilbene, CQ, 1, and 2 are depicted.

Figure 2

NMR studies of Aβ with 2. (a) Overaly of 2D TROSY ¹H-¹⁵N HSQC spectra of Aβ upon addition of 2 (900 MHz, 200 mM SDS, 20 mM sodium phosphate, pH 7.3, 25 °C). Black and red chemical shifts were obtained from the Aβ sample (¹⁵N-labeled Aβ_1–40 was used, ca. 192 μM) containing ca. 2 μL of DMSO-d₆ or ca. 1.2 equivalent of 2 (ca. 1.4 μL DMSO-d₆), respectively. (b) Change in the combined ¹H and ¹⁵N chemical shifts as a function as the amino acid sequence to identify the major interaction sites of Aβ with 2. * Denotes absent or overlapped signals.

Figure 3

Inhibition experiments. TEM images of samples of Cu(II)-treated Aβ (a) incubated with the chelator [(b) 1, (c) 2, (d) CQ, (e) EDTA, or (f) phen] or with the control molecule [(g) MPY or (h) stilbene] ([Aβ] = 25 μM, [Cu^II] = 25 μM, [chelator] = 50 μM, 24 h, 37 °C, constant agitation). The scale bar indicates 500 nm.

Figure 4

Cytotoxicity of Cu-associated Aβ with the chelators in SK-N-BE(2)-M17 cells using a MTT assay. Cell viability (%) with [chelator] (green), [the chelator and Cu^II] (blue), or [Cu^II, Aβ, and chelator] (orange) after 24 h incubation ([Aβ] = 20 μM, [Cu^II] = 20 μM, [chelator] = 40 μM). Treatment of Aβ in the absence and presence of Cu^II for 24 h results in ~90 and ~70% survival of cells, respectively.

Scheme 1

Experiments of inhibition and disaggregation.