Chemistry Department and Coimbra Chemistry Centre-Institute of Molecular Sciences (CQC-IMS), University of Coimbra, 3004-535 Coimbra, Portugal.
Centre for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal.
Int J Mol Sci. 2021 Dec 30;23(1):391. doi: 10.3390/ijms23010391.
Several degenerative amyloid diseases, with no fully effective treatment, affect millions of people worldwide. These pathologies-amyloidoses-are known to be associated with the formation of ordered protein aggregates and highly stable and insoluble amyloid fibrils, which are deposited in multiple tissues and organs. The disruption of preformed amyloid aggregates and fibrils is one possible therapeutic strategy against amyloidosis; however, only a few compounds have been identified as possible fibril disruptors in vivo to date. To properly identify chemical compounds as potential fibril disruptors, a reliable, fast, and economic screening protocol must be developed. For this purpose, three amyloid fibril formation protocols using transthyretin (TTR), a plasma protein involved in several amyloidoses, were studied using thioflavin-T fluorescence assays, circular dichroism (CD), turbidity, dynamic light scattering (DLS), and transmission electron microscopy (TEM), in order to characterize and select the most appropriate fibril formation protocol. Saturation transfer difference nuclear magnetic resonance spectroscopy (STD NMR) was successfully used to study the interaction of doxycycline, a known amyloid fibril disruptor, with preformed wild-type TTR (TTRwt) aggregates and fibrils. DLS and TEM were also used to characterize the effect of doxycycline on TTRwt amyloid species disaggregation. A comparison of the TTR amyloid morphology formed in different experimental conditions is also presented.
几种退行性淀粉样变疾病，目前尚无完全有效的治疗方法，影响着全世界数以百万计的人。这些病理学——淀粉样变——已知与有序蛋白质聚集和高度稳定且不溶的淀粉样纤维的形成有关，这些淀粉样纤维沉积在多种组织和器官中。破坏预先形成的淀粉样聚集物和纤维是治疗淀粉样变性的一种可能的治疗策略；然而，迄今为止，只有少数几种化合物被确定为体内可能的纤维破坏剂。为了正确地将化学化合物鉴定为潜在的纤维破坏剂，必须开发一种可靠、快速且经济的筛选方案。为此，使用转甲状腺素蛋白（TTR）研究了三种淀粉样纤维形成方案，TTR 是几种淀粉样变中的一种血浆蛋白，使用硫黄素-T 荧光测定法、圆二色性（CD）、浊度、动态光散射（DLS）和透射电子显微镜（TEM）来研究和选择最适合的纤维形成方案。饱和转移差核磁共振波谱（STD NMR）成功地用于研究已知的纤维破坏剂强力霉素与预先形成的野生型 TTR（TTRwt）聚集物和纤维的相互作用。DLS 和 TEM 也用于表征强力霉素对 TTRwt 淀粉样物质解聚的影响。还提出了不同实验条件下形成的 TTR 淀粉样形态的比较。
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