The Environment Is a Key Factor in Determining the Anti-Amyloid Efficacy of EGCG

Abstract

Millions of people around the world suffer from amyloid-related disorders, including Alzheimer's and Parkinson's diseases. Despite significant and sustained efforts, there are still no disease-modifying drugs available for the majority of amyloid-related disorders, and the overall failure rate in clinical trials is very high, even for compounds that show promising anti-amyloid activity in vitro. In this study, we demonstrate that even small changes in the chemical environment can strongly modulate the inhibitory effects of anti-amyloid compounds. Using one of the best-established amyloid inhibitory compounds, epigallocatechin-3-gallate (EGCG), as an example, and two amyloid-forming proteins, insulin and Parkinson's disease-related α-synuclein, we shed light on the previously unexplored sensitivity to solution conditions of the action of this compound on amyloid fibril formation. In the case of insulin, we show that the classification of EGCG as an amyloid inhibitor depends on the experimental conditions select, on the method used for the evaluation of the efficacy, and on whether or not EGCG is allowed to oxidise before the experiment. For α-synuclein, we show that a small change in pH value, from 7 to 6, transforms EGCG from an efficient inhibitor to completely ineffective, and we were able to explain this behaviour by the increased stability of EGCG against oxidation at pH 6.

The Aggregation Conditions Define Whether EGCG Is an Inhibitor or Enhancer of α-Synuclein Amyloid Fibril Formation

“…pH 7), corresponding to the solution condition most often investigated in previous studies, EGCG is highly unstable. EGCG oxidises rapidly under the investigated buffer conditions within several hours (Sneideris et al, 2019) in a similar time scale as the aggregation process itself in presence of glass beads. A decrease of the pH value to pH 6 leads to a significant increase in the stability of EGCG and accordingly to a less strong inhibitory effect on α-synuclein aggregation.…”

“…We summarize our findings in Figure 11. First of all, EGCG itself seems to be very ineffective as inhibitor, whereas its oxidation products are much more efficient (Sneideris et al, 2019). This finding explains why in general EGCG is a rather inefficient inhibitor in de novo experiments under pH conditions where EGCG is stable, i.e.…”

“…The effects of different ratios (1:1 and 5:1 with respect to protein) of EGCG and EGCG ox on the aggregation kinetics of α-synuclein at different pH values (pH 3 to pH 7), monitored in high-binding surface plates in the presence (A) and absence (B) of glass beads. A small subset of this data (pH 6 and pH 7, 1:1 EGCG and 1:1 EGCG ox ) is from (Sneideris et al, 2019). of glass beads the enhanced fragmentation leads to a t of 9 h, whereby the absence of glass beads leads to an almost complete absence of secondary processes and a t 50 of 96 h. Due to the spectroscopic features of the ThT to increase its fluorescence intensity upon binding to amyloid fibrils, it is possible to consider the enhanced ThT fluorescence intensity as an increase in the concentration of aggregates that are formed.…”

“…In particular the oxidised EGCG strongly quenches ThT fluorescence, rather than inhibiting amyloid fibril growth. The compound either interferes with ThT-fluorescence (Buell et al, 2010) or binds to amyloid fibrils, preventing the binding of ThT (Palhano et al, 2013;Sneideris et al, 2019).…”

“…Most of the studies are performed at physiological pH, where EGCG is unstable and auto-oxidises fast into various products (Wei et al, 2016). Decreasing the pH to slight acidic pH values (≤ pH 6), results in a considerable increase in EGCG stability, which we have recently shown to lead to a strongly impaired ability of EGCG to inhibit α-synuclein aggregation (Sneideris et al, 2019). Considering that both α-synuclein and EGCG are likely to encounter such pH environments in vivo, the aim of the present study is to characterise in more detail the dependence of EGCG inhibition of α-synuclein aggregation on the solution conditions.…”