A Robust and Scalable High-Throughput Compatible Assay for Screening Amyloid-β-Binding Compounds

Area of Science:

• Biochemistry
• Neuroscience
• Drug Discovery

Background:

• Amyloid aggregation is a hallmark of neurodegenerative diseases.
• Developing effective amyloid-binding molecules is crucial for therapeutic strategies.
• Existing screening methods may lack the physiological relevance needed for accurate drug discovery.

Purpose of the Study:

• To develop and optimize a robust fluorescent readout assay for screening amyloid-binding molecules.
• To enhance the physiological relevance of the assay by using endogenous amyloid.
• To identify novel amyloid-binding agents from a diverse compound library.

Main Methods:

• Utilized topologically-sensitive dyes for fluorescent readout.
• Incorporated endogenous amyloid sourced from 5XFAD mouse brains.
• Optimized assay conditions for high-throughput screening (HTS) achieving Z-prime values >0.6.
• Screened a library of 3,500 compounds, including known drugs, natural products, and random molecules.

Main Results:

• Established a robust and realistic fluorescent assay for amyloid-binding molecule screening.
• Achieved high assay performance with Z-prime values >0.6, indicating suitability for HTS.
• Identified 8 unique molecules demonstrating potential amyloid-binding activity.
• The identified molecules represent diverse chemical classes, including known drugs and natural products.

Conclusions:

• The developed fluorescent assay is a powerful tool for identifying novel amyloid-binding agents.
• The use of endogenous amyloid significantly increases the assay's physiological relevance.
• The identified 8 compounds warrant further investigation as potential therapeutics for amyloid-related diseases.