Cyclic-NDGA Effectively Inhibits Human γ-Synuclein Fibrillation, Forms Nontoxic Off-Pathway Species, and Disintegrates Preformed Mature Fibrils

Parkinson's disease arises from protein misfolding, aggregation, and fibrillation and is characterized by LB (Lewy body) deposits, which contain the protein alpha-synuclein (alpha-syn) as their major component. Another synuclein, gamma-synuclein (gamma-syn), coexists with alpha-syn in Lewy bodies and is also implicated in various types of cancers, especially breast cancer. It is known to seed alpha-syn fibrillation after its oxidation at methionine residue, thereby contributing in synucleinopathy. Despite its involvement in synucleinopathy, the search for small molecule inhibitors and modulators of gamma-syn fibrillation remains largely unexplored. This work reveals the modulatory properties of cyclic-nordihydroguaiaretic acid (cNDGA), a natural polyphenol, on the structural and aggregational properties of human gamma-syn employing various biophysical and structural tools, namely, thioflavin T (ThT) fluorescence, Rayleigh light scattering, 8-anilinonaphthalene-1-sulfonic acid binding, far-UV circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR) spectroscopy, atomic force microscopy, ITC, molecular docking, and MTT-toxicity assay. cNDGA was observed to modulate the fibrillation of gamma-syn to form off-pathway amorphous species that are nontoxic in nature at as low as 75 mu M concentration. The modulation is dependent on oxidizing conditions, with cNDGA weakly interacting (K-d similar to 10(-5) M) with the residues at the N-terminal of gamma-syn protein as investigated by isothermal titration calorimetry and molecular docking, respectively. Increasing cNDGA concentration results in an increased recovery of monomeric gamma-syn as shown by sodium dodecyl sulfate and native-polyacrylamide gel electrophoresis. The retention of native structural properties of gamma-syn in the presence of cNDGA was further confirmed by far-UV CD and FTIR. In addition, cNDGA is most effective in suppression of fibrillation when added at the beginning of the fibrillation kinetics and is also capable of disintegrating the preformed mature fibrils. These findings could, therefore, pave the ways for further exploring cNDGA as a potential therapeutic against gamma-synucleinopathies.