Diaryl hydrazones as multifunctional inhibitors of amyloid self-assembly

Abstract

The design and application of an effective, new class of multifunctional small molecule inhibitors of amyloid self-assembly are described. Several compounds based on the diaryl hydrazone scaffold were designed. Forty-four substituted derivatives of this core structure were synthesized using a variety of benzaldehydes and phenylhydrazines and characterized. The inhibitor candidates were evaluated in multiple assays, including the inhibition of amyloid β (Aβ) fibrillogenesis and oligomer formation and the reverse processes, the disassembly of preformed fibrils and oligomers. Because the structure of the hydrazone-based inhibitors mimics the redox features of the antioxidant resveratrol, the radical scavenging effect of the compounds was evaluated by colorimetric assays against 2,2-diphenyl-1-picrylhydrazyl and superoxide radicals. The hydrazone scaffold was active in all of the different assays. The structure-activity relationship revealed that the substituents on the aromatic rings had a considerable effect on the overall activity of the compounds. The inhibitors showed strong activity in fibrillogenesis inhibition and disassembly, and even greater potency in the inhibition of oligomer formation and oligomer disassembly. Supporting the quantitative fluorometric and colorimetric assays, size exclusion chromatographic studies indicated that the best compounds practically eliminated or substantially inhibited the formation of soluble, aggregated Aβ species, as well. Atomic force microscopy was also applied to monitor the morphology of Aβ deposits. The compounds also possessed the predicted antioxidant properties; approximately 30% of the synthesized compounds showed a radical scavenging effect equal to or better than that of resveratrol or ascorbic acid.

Figure 1

General structures of hydrazones and resveratrol

Figure 2

Synthesis of diaryl-hydrazones

Figure 3

Structure of diaryl-hydrazones used in this study

Figure 4

Atomic Force Microscopy images of Aβ_1–40 samples incubated without (control) and with the affector compounds for 6 days. The compounds numbers denote the inhibitor compounds as they are shown in Fig. 3.

Figure 5

The effect of incubation time on the amount of fibrils formed (expressed as fluorescence intensity, I_THT) in (A) Aβ_1–40 and (B) Aβ_1–42 samples incubated without (control) and with the affector compounds for 6 days. The compounds numbers denote the inhibitor compounds as they are shown in Fig. 3 (♦ - control; in the presence of compound: ■ – 3; ▲ – 16; X – 35; ● – 37) The values represent means±standard deviation of the data (n=3)

Figure 6

TSK G3000SWXL SEC-HPLC chromatograms of Aβ samples after centrifugation at 16, 100 xg (to remove fibrils) with/without selected hydrazone inhibitors. The compound numbers refer to structures shown in Fig. 3/Table 1. The molecular weight globular protein standards used to calibrate the column elution profile were β-galactosidase (132 kDa), bovine serum albumin (66 kDa), superoxide dismutase (32 kDa) and lysozyme (14.7 kDa). The intense peaks at ~24 min represent DMSO used as solvent during the preparation of the inhibitor solutions.

Figure 7

Radical scavenging activity of selected hydrazones (activity > 10%) illustrated as % reduction of the absorbance of DPPH at λ=519 nm after 60 min incubation. The compound numbers refer to structures shown in Fig. 3 and Table 1. Ascorbic acid (aa) and resveratrol were used as reference compounds. The values represent means ± standard deviation of the data (n=3–4).

Figure 8

Superoxide scavenging activity of selected hydrazones (activity > 2%) illustrated as % reduction of the absorbance of NBT at λ=560 nm after 10 min incubation. The compound numbers refer to structures shown in Fig. 3 and Table 1. Ascorbic acid (aa) and resveratrol were used as reference compounds. The values represent means ± standard deviation of the data (n=3–4).