Benzimidazole-based fluorophores for the detection of amyloid fibrils with higher sensitivity than Thioflavin-T

Protein aggregation into amyloid fibrils is a key feature of a multitude of neurodegenerative diseases such as Alzheimer's, Parkinson's, and Prion disease. To detect amyloid fibrils, fluorophores with high sensitivity and better efficiency coupled with the low toxicity are in high demand even to date. In this pursuit, we have unveiled two benzimidazole-based fluorescence sensors ([C15H15N3] (C1) and [C16H16N3O2] (C2), which possess exceptional affinity toward different amyloid fibrils in its submicromolar concentration (8 x 10(-9) M), whereas under a similar concentration, the gold standard Thioflavin-T (ThT) fails to bind with amyloid fibrils. These fluorescent markers bind to alpha-Syn amyloid fibrils as well as amyloid fibrils forming other proteins/peptides including A beta 42 amyloid fibrils. The(1)H-N-15 heteronuclear quantum correlation spectroscopy nuclear magnetic resonance data collected on wild-type alpha-Syn monomer with and without the fluorophores (C1andC2) reveal that there is weak or no interactions betweenC1orC2with residues in alpha-Syn monomer, which indirectly reflects the specific binding ability ofC1andC2to the alpha-Syn amyloid fibrils. Detailed studies further suggest thatC1andC2can detect/bind with the alpha-Syn amyloid fibril as low as 100 x 10(-9) M. Extremely low or no cytotoxicity is observed forC1andC2and they do not interfere with alpha-Syn fibrillation kinetics, unlike ThT. BothC1/C2not only shows selective binding with amyloid fibrils forming various proteins/peptides but also displays excellent affinity and selectivity toward alpha-Syn amyloid aggregates in SH-SY5Y cells and A beta 42 amyloid plaques in animal brain tissues. Overall, our data show that the developed dyes could be used for the detection of amyloid fibrils including alpha-Syn and A beta 42 amyloids with higher sensitivity as compared to currently used ThT.