Autoxidation Enhances Anti-Amyloid Potential of Flavone Derivatives

Abstract

The increasing prevalence of amyloid-related disorders, such as Alzheimer's or Parkinson's disease, raises the need for effective anti-amyloid drugs. It has been shown on numerous occasions that flavones, a group of naturally occurring anti-oxidants, can impact the aggregation process of several amyloidogenic proteins and peptides, including amyloid-beta. Due to flavone autoxidation at neutral pH, it is uncertain if the effective inhibitor is the initial molecule or a product of this reaction, as many anti-amyloid assays attempt to mimic physiological conditions. In this work, we examine the aggregation-inhibiting properties of flavones before and after they are oxidized. The oxidation of flavones was monitored by measuring the UV-vis absorbance spectrum change over time. The protein aggregation kinetics were followed by measuring the amyloidophilic dye thioflavin-T (ThT) fluorescence intensity change. Atomic force microscopy was employed to image the aggregates formed with the most prominent inhibitors. We demonstrate that flavones, which undergo autoxidation, have a far greater potency at inhibiting the aggregation of both the disease-related amyloid-beta, as well as a model amyloidogenic protein-insulin. Oxidized 6,2',3'-trihydroxyflavone was the most potent inhibitor affecting both insulin (7-fold inhibition) and amyloid-beta (2-fold inhibition). We also show that this tendency to autoxidize is related to the positions of the flavone hydroxyl groups.

Keywords: aggregation; amyloid-beta; autoxidation; flavones; inhibition; insulin.

Figure 1

UV-visible absorbance spectra of flavones, recorded at 0 h (black), 5 h (red), 40 h (blue), and 100 h (green). Spectra were baseline corrected at 800 nm. Most of the flavone spectra experienced a significant change in the 250–450 nm region. In contrast, 21, 30, 43, 45, 54, 56, 60, and 62 experienced only a slight transition of maxima or decrease in the magnitude of the initial absorbance spectrum.

Figure 2

Effects of non-oxidized and oxidized flavones on insulin aggregation kinetics (A) and relative ThT fluorescence intensity (B). Effect of oxidized flavones on Aβ₄₂ aggregation kinetics (C) and relative ThT fluorescence intensity (D). Error bars are for one standard deviation (n = 4). None of the non-oxidized flavones, except 56, inhibited insulin aggregation; after the oxidation, more than half of the flavones showed an inhibitory effect, with 31, 59, and 63 having the most significant impact. Oxidized flavones 22, 31, 52, and 59 increased the relative halftime of Aβ₄₂ the most, while 1, 30, 32, 37 did not affect the relative halftime nor the relative ThT fluorescence intensity.

Figure 3

UV-visible absorbance spectra of flavones, recorded at 0 h (black), 5 h (red), 40 h (blue), and 100 h (green). Spectra were baseline corrected at 800 nm. Numbers 3, 6, 13, 14, 19, 23, 33 experienced the most significant decrease in the magnitude of the spectrum, while 4, 18, 27, 34, 36 had no notable change over the course of the experiment.

Figure 4

Effects of non-incubated and incubated flavones on insulin aggregation kinetics (A) and relative ThT fluorescence intensity (B). Effect of incubated flavones on Aβ₄₂ aggregation kinetics (C) and relative ThT fluorescence intensity (D). Error bars are for one standard deviation (n = 4). The non-incubated and incubated flavones did not impact insulin and Aβ₄₂ relative halftime, while incubated flavones 34, 35, 41, and 50 had the most significant impact on the relative ThT fluorescence intensity of Aβ₄₂.

Figure 5

Atomic force microscopy images of Aβ₄₂ formed without (A,B) and with 50 µM of oxidized 2′,3′-DHF (C,D), 6,2′,3′-THF (E,F), 3,6,2′,3′-TeHF (G,H), 3,6,3′,4′-TeHF (I,J) and 5,7,3′,4′,5′-PHF (K,L) flavones. Fibril and oligomeric species height distribution (M), where box plots indicate mean ± SD and error bars are in the 5%–95% range (n = 100). FTIR spectra (N) of Aβ₄₂ fibrils formed alone and with 50 µM of 2′,3′-DHF. The AFM images of Aβ₄₂ aggregates formed with all inhibitors showed a similar distribution in height and revealed round shape structures that were not present in the image of the control sample. The FTIR spectrum of the sample with 2′,3′-DHF had less expressed β-sheet-related band at 1629 cm⁻¹ than the control sample.