Brazilin inhibits fibrillogenesis of human islet amyloid polypeptide, disassembles mature fibrils, and alleviates cytotoxicity

摘要

Fibrillogenesis of human islet amyloid polypeptide (hIAPP) is a pathological hallmark of type II diabetes mellitus (T2DM), and the inhibition of hIAPP fibrillogenesis is an important strategy for the prevention and treatment of T2DM. In this study, the inhibitory effects of brazilin on the fibrillization and cytotoxicity of hIAPP were examined using the thioflavin T fluorescence (ThT) assay, transmission electron microscopy (TEM), circular dichroism (CD) spectroscopy, cytotoxicity assays, and molecular dynamics simulations. Both the ThT and TEM results have shown that brazilin inhibits hIAPP fibrillogenesis in a dose-dependent manner. CD studies revealed that brazilin delays the conformational transition of hIAPP from its initial ahelical to the beta-sheet form. As a result, brazilin greatly alleviates hIAPP-induced cytotoxicity. Moreover, we also found that brazilin disassembles preexisting hIAPP fibrils, and alleviates the cytotoxicity of hIAPP aggregates. The results of free energy decomposition studies calculated using molecular mechanics-Poisson-Boltzmann surface area analysis revealed that hydrophobic interactions contribute more than 75% of the free energy of binding in the brazilin-hIAPP complex, while electrostatic interactions (i.e., hydrogen bonds) play a secondary role (<25%). Two binding sites of brazilin on the hIAPP pentamer were identified, encompassing the N-terminal region and the turn region. There are 11 important residues of hIAPP that strongly interact with brazilin - Asn3, Thr4, Thr9, Arg11, Asn14, Phe15, His18, Ser19, Ser20, Asn21 and Phe23. The findings presented here will contribute to a comprehensive understanding of the inhibitory effect of brazilin on the fibrillogenesis of hIAPP, which is critical for the search for more effective agents that can inhibit hIAPP fibrillogenesis.