Abstract
Protein aggregation is the principal component of numerous protein misfolding pathologies termed proteinopathies, such as Alzheimer’s disease, Parkinson’s disease, prion disease, and AA amyloidosis with unmet treatment needs. Protein aggregation inhibitors have great potential for the prevention and treatment of proteinopathies. Here we report the development of an automated real-time microliter-scale high throughput screening (MSHTS) system for amyloid aggregation inhibitors using quantum-dot nanoprobes. Screening 504 crude extracts and 134 low molecular weight aromatic compounds revealed the relationship of amyloid-β (Aβ) aggregation inhibitory activities of plant extracts using a plant-based classification. Within the eudicots, rosids, Geraniales and Myrtales showed higher activity. Screening low molecular weight aromatic compounds demonstrated that the structure of tropolone endows it with potential Aβ aggregation inhibitory activity. The activity of the most active tropolone derivative was higher than that of rosmarinic acid. MSHTS also identified three chaperone molecules as tau aggregation inhibitors. These results demonstrate that our automated MSHTS system is a novel and robust tool that can be adapted to a wide range of compounds and aggregation-prone polypeptides.
Highlights
Proteinopathies, which include most neurodegenerative diseases, are characterized by the aggregation of misfolded proteins[1]
Crude extracts of many plants have chlorophyll-derived absorption at 650– 700 nm (Fig. 1a, bottom). Since this absorption may affect the quantification of amyloid aggregation inhibitory activity (Supplementary Fig. S1), we used Qdot[605], which does not overlap with the absorption of chlorophylls (Fig. 1a), in this automated microliter-scale high-throughput screening (MSHTS) system
We estimate the amount of Aβ aggregates from the variation in brightness of each pixel, as a standard deviation (SD) value according to a previous report[19]
Summary
We developed an automated MSHTS system using quantum-dot nanoprobes for Aβ aggregation inhibitors that can accurately and highly efficiently evaluate inhibitory activity even for crude extracts from natural products. We are currently trying to develop an evaluation method of oligomerization inhibitory activity by applying real-time quantification of Aβ aggregation using the Qdot nanoprobe By using this novel method and oligomer-specific antibodies, the inhibition stage of many inhibitors being found by the automated MSHTS system will be elucidated, and a list of more useful inhibitors, including the inhibitory stage, will be created. We showed that the mixing conditions significantly affect Aβ www.nature.com/scientificreports aggregation and its inhibition (Fig. 1d) This result implies that more careful examination of mixing conditions is important in research on oligomer formation, which has attracted attention in recent years. The ability to analyze the inhibitor group using this screening system would allow for the development of therapeutic and preventive drugs, as well as functional foods, that could be used to prevent proteinopathies
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