High-throughput discovery of fluoroprobes that recognize amyloid fibril polymorphs

Area of Science:

• Biochemistry
• Neuroscience
• Chemical Biology

Background:

• Neurodegenerative tauopathies are characterized by the aggregation of microtubule-associated protein tau into distinct fibril conformations.
• Existing fluorescent probes (fluoroprobes) often fail to differentiate between these amyloid fibril polymorphs, limiting research.
• A high-throughput screening method is needed to assess diverse chemical libraries for novel fibril-binding probes.

Purpose of the Study:

• To develop and apply a high-throughput screening method for identifying fluorescent probes that can discriminate between amyloid fibril polymorphs.
• To discover novel fluoroprobes capable of recognizing specific fibril conformations, including those relevant to tauopathies.

Main Methods:

• Utilized protein-adaptive differential scanning fluorimetry to screen the Aurora collection of over 300 fluoroprobes.
• Tested probes against synthetic fibril polymorphs of tau, α-synuclein, and islet amyloid polypeptide.
• Employed excitation-multiplexed bright-emission recording (EMBER) imaging and orthogonal secondary assays for validation.

Main Results:

• Identified both pan-fibril-binding chemotypes and fluoroprobes selective for specific fibril subsets.
• Discovered a fluoroprobe that successfully recognized tau pathology in ex vivo brain slices from Alzheimer's disease and rodent models.
• Demonstrated the capability of the screening platform to identify conformationally sensitive fibril-binding agents.

Conclusions:

• The developed screening approach enables high-throughput identification of fibril-selective fluorescent probes.
• The identified fluoroprobe scaffolds offer promising starting points for developing ligands targeting specific amyloid fibril conformations.
• This work advances the tools available for studying the heterogeneity of protein aggregation in neurodegenerative diseases.