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.

Keywords: EGCG; amyloid aggregation; inhibition; insulin; α-synuclein.

Figure 1

Effects of EGCG and EGCG${}_{ox}$ on insulin aggregation kinetics (A) and maximum ThT fluorescence intensity (B). Abbreviations PB and AC represent environmental conditions (100 mM phosphate buffer and 20% acetic acid, respectively), while Q and A denote the agitation conditions (quiescent and agitated, respectively), under which the insulin aggregation reactions were performed. Error bars represent standard deviations.

Figure 2

AFM images of insulin amyloid fibrils formed in PB or AC in the absence and presence of EGCG or EGCG${}_{ox}$. Abbreviations Q and A denote agitation conditions (quiescent and agitated, respectively), under which the insulin aggregation reactions were performed. The height scale (z) is in nm.

Figure 3

Second derivative FTIR spectra of insulin amyloid-like aggregates formed in PB and AC under quiescent and agitated conditions. Abbreviations PB and AC represent environmental conditions (100 mM phosphate buffer and 20% acetic acid, respectively), while Q and A denote agitation conditions (quiescent and agitated, respectively), under which the insulin aggregation reaction was performed.

Figure 4

The effects of EGCG and EGCG${}_{ox}$ on the aggregation kinetics of $\alpha$-synuclein monitored at pH 7 (A) and pH 6 (B). (C) $\alpha$-synuclein concentration measured in the supernatant after centrifuging the end product of the aggregation reactions at pH 7 and pH 6, respectively.

Figure 5

AFM images of $\alpha$-synuclein aggregates formed at pH 7 or pH 6 in the absence and presence of EGCG or EGCG${}_{ox}$.

Figure 6

Schematic depiction of possible effects of potential anti-amyloid compounds on the amyloid aggregation reaction performed under distinct environmental conditions. Different environmental conditions can lead to the formation of distinct aggregate species of which only some are targeted by the compound (A), as in case of insulin aggregation in PB-Q and AC-Q. Certain compounds can also interfere with ThT’s fluorescence intensity (B), suggesting inhibition, which was not confirmed by other experiments, such as the quantification of soluble protein at the final plateau of ThT intensity. An example is EGCG and $\alpha$-synuclein at pH 6. Moreover, specific environmental conditions can induce modifications of the compound, which results in the generation of products that target aggregation prone species (C), as in the case of $\alpha$-synuclein aggregation at neutral pH. The compound modification can only manifest itself if it occurs with kinetics comparable to, or faster than the kinetics of aggregation. Distinct background colours represent different environmental conditions. Different shapes of aggregates represent distinct pathways reflecting the observed polymorphism of amyloid fibrils.