Fluorescently labeled α-synuclein to examine destabilization of synuclein fibrils by small molecules and therapeutics for neurodegenerative diseases
Misfolding of the neuronal protein α-synuclein triggers oligomerization and the formation of amyloid fibrils that are the pathological hallmarks of Parkinson’s disease. The molecular mechanism of synuclein misfolding has not been elucidated, and therefore, screening of compounds that block or reverse aggregation can establish novel mechanisms of action.
Previous efforts to employ small molecule probes, such as Thioflavin T and Congo Red, in drug screening experiments have been unfavorable, as the probes compete with candidate compounds for binding sites on synuclein. There is no cure for these progressive neurodegenerative diseases, although therapeutics and biologics are in development. A high-throughput screening method to examine fibril disaggregation as a measure of therapeutic efficacy would be advantageous to the pharmaceutical industry developing the next generation of therapeutics.
Researchers in the Petersson Lab have developed a minimally-perturbative fluorescence polarization assay to monitor the local conformational dynamics of α-synuclein. Synuclein has been site-specifically labeled with the fluorophores, chromatographically purified, and characterized with mass spectrometry. The non-perturbing nature of the labels has been validated by in vitro kinetics experiments, quantification of the incorporated labeled protein, and TEM imaging of the synuclein fibrils. The labeled synuclein can be used to examine fibril formation and destabilization in in vitro and cellular models of neurodegenerative disease propagation and progression.
- Site-specific, homogeneous fluorophore attachment
- Non-perturbative to protein
- High-throughput screening for small molecules
- Optimization of lead compounds for Parkinson’s disease and other amyloidogenic neurodegenerative diseases
- Study misfolding and fibrillization of synuclein in vitro and in cells
Stage of Development:
In vitro proof-of-concept
Tangible research material
- Haney CM et al. Org. Biomol. Chem., 2016, 14, p. 1584-1592.
- Haney CM et al. JACS, manuscript submitted, 2016.