Synthesis and in vitro quantification of amyloid fibrils by barbituric and thiobarbituric acid-based chromene derivatives

Abstract

Neurodegenerative disease is caused by the abnormal build-up of proteins in and around cells called amyloid. The amyloid fibril formation and its mechanism have been investigated with various techniques, including dye-binding assay. Thioflavin T (ThT) has been one of the most widely used dyes for quantifying amyloid deposits, but ThT has a weak fluorescence signal especially at low concentration of amyloid fibrils, low lipophilicity and positive charge that makes it unable to cross the blood-brain barrier (BBB) to detect amyloid fibrils in vivo. Hence, there is a strong motivation for designing and developing the new compounds for in vitro amyloid quantification and in vivo amyloid imaging. The need for new probes to detect amyloid fibrils, especially within the cell, is highlighted by the fact that an accurate understanding of the molecular details of amyloid fibril formation is required to design and develop strategies for controlling the amyloid formation, and this needs more reliable probes for amyloid identification. In this work, we synthesized and applied barbituric and thiobarbituric acid-based chromene derivatives, as new fluorescent dyes to quantitatively detect the amyloid fibrils of bovine serum albumin (BSA) and human insulin in comparison with native soluble proteins or amorphous aggregation. Our results showed that among the 14 synthesized compounds, five compounds 4a, 4h, 4j, 4k, and 4l could selectively and specifically bind to amyloid fibrils while other compounds demonstrated a low-affinity binding. Furthermore, according to the cell viability experiment, compounds 4a, 4j and 4l at low concentration of compounds are not toxic, especially compound 4j which could be used as a suitable candidate for in vivo study. Further studies are needed to determine all the properties of compounds, especially in vivo experiments.

Keywords: Amorphous; Amyloid; BSA; Insulin.