Biophysical and in silico studies on inhibition of protein aggregation by small molecules

Abstract

The extracellular amyloid deposits of distinct polypeptide that dominates its newlinecomposition in the form of highly ordered cross and#946; structure, is the pathological basis for protein newlinemisfolding diseases, such as Alzheimer s disease with high social and medical relevance and newlineother systematic amyloidosis, i.e. type II diabetes, non-neuropathic lysosomal amyloidosis, etc. newlineAs amyloid fibrils and prefibrillar soluble oligomers are cytotoxic, plentiful efforts have been newlinemade to inhibit fibrillation process as a therapeutic strategy. Several natural small molecules newlinehave recently been investigated to check for their potency as fibrillation inhibitors. The National newlineInstitutes of Health (NIH) list several clinical trials (ClinicalTrials.gov) for natural small newlinemolecule fibrillation inhibitors which are either ongoing or completed, but the final results have newlinenot yet been published (Velander et al., 2017; Yamada et al., 2015). The results from only three newlineclinical trials with Curcumin and Epigallocatechin gallate for their potency against amyloid newlinediseases have been published (Baum et al., 2008; Kristen et al., 2012; Ringman et al., 2012; newlineVelander et al., 2017). Presently resveratrol, genistein, and rosmarinic acid are under clinical newlinetrial for their effectiveness in AD or mild cognitive impairment patients (Ringman et al., 2012; newlineYamada et al., 2015). Here, we have shown inhibitory effect of small molecules on the amyloid newlinefibrillation of hen egg white lysozyme (HEWL) and human insulin (HI), the model proteins for newlineamyloid formation. The effect of small molecules on amyloid fibrillation of model proteins was newlineinvestigated using Thioflavin T (ThT) and 8-Anilinonaphthalene-1-sulfonic acid (ANS) newlinefluorescence, Congo red absorbance, circular dichroism (CD), and transmission electron newlinemicroscopy (TEM). Fluorescence quench titrations, computational docking and molecular newlinedynamics (MD) was also carried out to analyse the binding parameters. These studies showed newlinethat most small molecules significantly attenuates nucleation and inhibits amyloid fibrillation newlinein a dose dependent manner. Small molecules interacts with partially unfolded conformations newlineand/or early species of fibrillation pathway and inhibit further fibrillation. Small molecules newlineshow fairly strong binding at physiological pH suggesting better protection against misfolding. newlineComputational docking with steric zipper structures suggest that small molecules may also bind newline