Inhibition of Lysozyme Fibrillation by Natural and Synthetic Melanins

Abstract

Amyloidogenesis, a multistep process in which protein mis‐folding results in the formation of aggregates with cross beta sheet structures, is associated with more than 20 diseases including diabetes, AL amyloidosis, Alzheimer's disease, and prion diseases. Hence, there is an ongoing interest in the development of drugs that inhibit amyloid formation or deposition. In this study, we tested the ability of natural and synthetic polyketides to inhibit lysozyme fibrillation in vitro. Solutions of lysozyme (Hen egg, 20 mg/mL) were incubated at 58°C in 20 mM glycine HCl buffer at 58°C for 8 days in the presence of one of the following compounds (concentration tested): 1. Kraft lignin (125 μg/mL), 2. sulfonated Kraft lignin (190 μg/mL), 3. allomelanin extracted from Echinacea purpurea (210 μg/mL), 4. ES‐EM (166 μg/mL), 5. ES‐CAM (190 μg/mL), 6. C11 (130 μg/mL), or 7. C12 (150 μg/mL). Compounds 4–7 are synthetic melanins obtained From BioActive Polymers.com, (La Grange Park, Illinois, 60526). The incubation mixtures were sampled repeatedly and the kinetics of lysozyme fibrillation were evaluated by Thioflavin T fluorescence, native PAGE, and fluorescence and dark field microscopy. Compounds 6, 4, 3, and 7 inhibited the development of THT fluorescence to levels that were 16%, 42%, 52%, and 61%, respectively of the untreated control mixtures. PAGE analysis of the time points taken during the incubation containing C11 showed that this compound decreased the generation of lysozyme oligomers during the first 48 hours of incubation and delayed the progression of the assembly of lysozyme monomers and oligomers into lysozyme fibers and aggregates. The data suggests that natural and synthetic melanins may be of therapeutic value as fibrillogenesis inhibitors.[Supported in part by a gift from the Charles and Lee Finkl Amyloidosis Foundation]