The triphenylmethane dye brilliant blue G is only moderately effective at inhibiting amyloid formation by human amylin or at disaggregating amylin amyloid fibrils, but interferes with amyloid assays; Implications for inhibitor design.

Rehana Akter,Bingqian Zheng,Alexander Zhyvoloup,Daniel P Raleigh,Surita R Bhatia

doi:10.1371/journal.pone.0219130

Rehana Akter, Bingqian Zheng + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0219130

Copy DOI

Abstract

The development of inhibitors of islet amyloid formation is important as pancreatic amyloid deposition contributes to type-2 diabetes and islet transplant failure. The Alzheimer’s Aβ peptide and human amylin (h-amylin), the polypeptide responsible for amyloid formation in type-2 diabetes, share common physio-chemical features and some inhibitors of Aβ also inhibit amyloid formation by h-amylin and vice versa. Thus, a popular and potentially useful strategy to find lead compounds for anti-amylin amyloid agents is to examine compounds that have effects on Aβ amyloid formation. The triphenylmethane dye, brilliant blue G (BBG, Sodium;3-[[4-[(E)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-N-ethyl-3-methylanilino]methyl]benzenesulfonate) has been shown to modulate Aβ amyloid formation and inhibit Aβ induced toxicity. However, the effects of BBG on h-amylin have not been examined, although other triphenylmethane derivatives inhibit h-amylin amyloid formation. The compound has only a modest impact on h-amylin amyloid formation unless it is added in significant excess. BBG also remodels preformed h-amylin amyloid fibrils if added in excess, however BBG has no significant effect on h-amylin induced toxicity towards cultured β-cells or cultured CHO-T cells except at high concentrations. BBG is shown to interfere with standard thioflavin-T assays of h-amylin amyloid formation and disaggregation, highlighting the difficulty of interpreting such experiments in the absence of other measurements. BBG also interferes with ANS based assays of h-amylin amyloid formation. The work highlights the differences between inhibition of Aβ and h-amylin amyloid formation, illustrates the limitation of using Aβ inhibitors as leads for h-amylin amyloid inhibitors, and reinforces the difficulties in interpreting dye binding assays of amyloid formation.

Highlights

The pathological aggregation of proteins to form amyloid or other aggregates contributes to a range of devastating human diseases including Alzheimer’s disease, Huntington’s disease, Parkinson’s disease and type-2 diabetes (T2D) [1,2,3]
We examined the ability of an equimolar amount of BBG to inhibit h-amylin amyloid formation using fluorescence detected thioflavin-T binding assays and transmission electron microscopy (TEM)
The experiments described above clearly demonstrate that BBG interferes with thioflavin-T assays if both compounds are added together at the start of an experiment, and reveal that BBG modulates h-amylin amyloid formation We examined the effect of adding BBG to pre-formed h-amylin amyloid fibrils

Summary

Introduction

The pathological aggregation of proteins to form amyloid or other aggregates contributes to a range of devastating human diseases including Alzheimer’s disease, Huntington’s disease, Parkinson’s disease and type-2 diabetes (T2D) [1,2,3]. Aβ and h-amylin have 25% sequence identity and approximately 50% similarity, with the higher identity and similarity in regions that have been proposed to be critical for amyloid formation (Fig 1) Both polypeptides are natively unfolded in their monomeric states. Aβ can seed amyloid formation by h-amylin in a mouse model and h-amylin has been reported in brain plaques in Alzheimer’s disease while Aβ has been reported to form pancreatic deposits in T2D [19, 26, 27]. These observations indicate that studies of known Aβ inhibitors are a potentially promising strategy for finding h-amylin amyloid inhibitors

Methods

Results

Conclusion