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

Abstract

The extracellular amyloid deposits of distinct polypeptide that dominates its composition in the form of highly ordered cross β structure, is the pathological basis for protein misfolding diseases, such as Alzheimer’s disease with high social and medical relevance and other systematic amyloidosis, i.e. type II diabetes, non-neuropathic lysosomal amyloidosis, etc. As amyloid fibrils and prefibrillar soluble oligomers are cytotoxic, plentiful efforts have been made to inhibit fibrillation process as a therapeutic strategy. Several natural small molecules have recently been investigated to check for their potency as fibrillation inhibitors. The National Institutes of Health (NIH) list several clinical trials (ClinicalTrials.gov) for natural small molecule fibrillation inhibitors which are either ongoing or completed, but the final results have not yet been published (Velander et al., 2017; Yamada et al., 2015). The results from only three clinical trials with Curcumin and Epigallocatechin gallate for their potency against amyloid diseases have been published (Baum et al., 2008; Kristen et al., 2012; Ringman et al., 2012; Velander et al., 2017). Presently resveratrol, genistein, and rosmarinic acid are under clinical trial for their effectiveness in AD or mild cognitive impairment patients (Ringman et al., 2012; Yamada et al., 2015). Here, we have shown inhibitory effect of small molecules on the amyloid fibrillation of hen egg white lysozyme (HEWL) and human insulin (HI), the model proteins for amyloid formation. The effect of small molecules on amyloid fibrillation of model proteins was investigated using Thioflavin T (ThT) and 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence, Congo red absorbance, circular dichroism (CD), and transmission electron microscopy (TEM). Fluorescence quench titrations, computational docking and molecular dynamics (MD) was also carried out to analyse the binding parameters. These studies showed that most small molecules significantly attenuates nucleation and inhibits amyloid fibrillation in a dose dependent manner. Small molecules interacts with partially unfolded conformations and/or early species of fibrillation pathway and inhibit further fibrillation. Small molecules show fairly strong binding at physiological pH suggesting better protection against misfolding. Computational docking with steric zipper structures suggest that small molecules may also bind and stabilize protofibrils and/or mature fibrils by hydrogen bonding and/or hydrophobic forces throughout the surface, stabilize them and inhibit the release of prefibrillar oligomeric species which could be nuclei or template for further fibrillation. From the perspective of an effective therapeutic strategy against protein aggregation diseases, following findings provide an understanding of anti-amyloid property of small molecules to readers from the prospect of biochemistry. In another study, the ligand and structure based approaches were used to identify novel human islet amyloid polypeptide (hIAPP) fibrillation inhibitors and fibril binding compounds. hIAPP is a natively unfolded polypeptide hormone of glucose metabolism, which is co-secreted with insulin by the β-cells of the pancreas. In patients with type 2 diabetes, hIAPP forms amyloid fibrils because of diabetes-associated β-cells dysfunction and increasing fibrillation, in turn, leads to failure of secretory function of β-cells. This provides a target for the discovery of small molecules against protein aggregation diseases. However, the binding mechanism of small molecules with monomers, oligomers and fibrils to inhibit fibrillation is still an open question. The best generated pharmacophore model was used as a 3D search query for virtual screening of a compound database to identify novel small molecules having the potential to be therapeutic agents against protein aggregation diseases. Molecular docking and MD simulation studies were used to explore the binding of small molecules with natively folded hIAPP structure and its aggregation prone conformation as well as fibril forming segments. We observed that catechins with galloyl moiety bind to the side chain of residues on the surface of steric zipper structures with slightly greater binding affinity compared to catechins without galloyl moiety while the identified hits have slightly greater binding affinity than all catechins. Small molecules interact with side chain of residues from helices and prevent hIAPP from adopting β-sheet structure. Residue Phe23 should play an important role in inhibiting hIAPP fibrillation. The differences in binding affinity of small molecules against steric zipper structures of several fibril forming segments indicate nonspecific binding which suggest a cocktail of active small molecules targeting various amyloid proteins should be required for treatment of aggregation diseases.