Mechanistic In Vitro Dissection of the Inhibition of Amyloid Fibrillation by n-Acetylneuraminic Acid: Plausible Implication in Therapeutics for Neurodegenerative Disorders

A variety of neurodegenerative disorders including Parkinson's disease are due to fibrillation in amyloidogenic proteins. The development of therapeutics for these disorders is a topic of extensive research as effective treatments are still unavailable. The present study establishes that n-acetylneuraminic acid (Neu5ac) inhibits the amyloid fibrillation of hen egg-white lysozyme (HEWL) and alpha-synuclein (SYN), as observed using various biophysical techniques and cellular assays. Neu5ac inhibits the amyloid formation in both proteins, as suggested from the reduction in the ThT fluorescence and remnant structures in transmission electron microscopy micrographs observed in its presence. In HEWL fibrillation, Neu5ac decreases the hydrophobicity and resists the transition of the alpha-helix to a beta-sheet, as observed by an ANS binding assay, circular dichroism (CD) spectra, and Fourier transform infrared measurements, respectively. Neu5ac stabilizes the states that facilitate the amyloid formation in HEWL and SYN, as demonstrated by an enhanced intrinsic fluorescence in its presence, which is further confirmed by an increase in T-m obtained from differential scanning calorimetry thermograms and an increase in the near-UV CD signal for HEWL with Neu5ac. However, the increase in stability is not a manifestation of Neu5ac binding to amyloid facilitating (partially folded or native) states of both proteins, as verified by isothermal titration calorimetry and fluorescence binding measurements. Besides, Neu5ac also attenuates the cytotoxicity of amyloid fibrils, as evaluated by a cell toxicity assay. These findings provide mechanistic insights into the Neu5ac action against amyloid fibrillation and may establish it as a plausible inhibitor molecule against neurodegenerative disorders.

Automated summary

The study confirmed that N-acetylneuraminic acid (Neu5ac) inhibits amyloid fibrillation of proteins and reduces cytotoxicity. Neu5ac stabilizes states that promote amyloid formation and attenuates the toxicity of amyloid fibrils.