Detection and Characterization of Small Molecule Interactions with Fibrillar Protein Aggregates Using Microscale Thermophoresis

Neurodegenerative diseases such as Parkinson's and Alzheimer's disease share the pathological hallmark of fibrillar protein aggregates. The specific detection of these protein aggregates by positron emission tomography (PET) in the patient brain can yield valuable information for diagnosis and disease progression. However, the identification of novel small compounds that bind fibrillar protein aggregates has been a challenge. In this study, microscale thermophoresis (MST) was applied to assess the binding affinity of known small molecule ligands of α-synuclein fibrils, which were also tested in parallel in a thioflavin T fluorescence competition assay for further validation. In addition, a MST assay was also developed for the detection of the interaction between a variety of small molecules and tau fibrils. The results of this study demonstrate that MST is a powerful and practical methodology to quantify interactions between small molecules and protein fibrillar aggregates, which suggests that it can be applied for the identification and development of PET radioligands and potentially of therapeutic candidates for protein misfolding diseases.