Pitfalls associated with the use of Thioflavin-T to monitor anti-fibrillogenic activity
Pitfalls associated with the use of Thioflavin-T to monitor anti-fibrillogenic activity
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DOI：
10.1016/j.bmcl.2014.04.072
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发表时间：
2014-07-15
影响因子：
2.7
通讯作者：
Follmer, Cristian
中科院分区：
文献类型：
--
作者：
Coelho-Cerqueira, Eduardo;Pinheiro, Anderson S.;Follmer, Cristian
Thioflavin-T (ThT) is a cationic benzothiazole dye that displays enhanced fluorescence upon binding to amyloid fibrils. This property makes ThT the current reagent of choice for the quantification of amyloid fibrils. Herein, we investigate the main pitfalls associated with the use of ThT-based assays to monitor the fibrillation of alpha-synuclein (alpha-syn), a protein linked to Parkinson's disease and other alpha-synucleinopathies. We demonstrated for the first time that ThT interacts with alpha-syn disordered monomer and accelerates the protein fibrillation in vitro. As a consequence, misleading conclusions may arise from the use of ThT-based real-time assays in the evaluation of anti-fibrillogenic compounds. Interestingly, NMR experiments indicated that C-terminal domain of alpha-syn is the main region perturbed by ThT interaction, similarly to that found for the pesticide paraquat, a well-documented accelerator of alpha-syn fibrillation. Moreover, we demonstrated that certain potent inhibitors of (alpha-syn fibrillation, such as oxidized catechol-amines and polyphenols, undergo spontaneous oxidation in aqueous solution, generating compounds that strongly quench ThT fluorescence. In light of these findings, we alert for possible artifacts associated to the measure of the anti-fibrillogenic activity based only on ThT fluorescence approach. (C) 2014 Elsevier Ltd. All rights reserved.
