Genistein: A Dual Inhibitor of Both Amyloid β and Human Islet Amylin Peptides

Abstract

Abnormal misfolding and aggregation of amyloid peptides into amyloid fibrils are common and critical pathological events in many neurodegenerative diseases. Most inhibitors or drugs have been developed to prevent amyloid aggregation of a specific peptide, showing sequence-dependent inhibition mechanisms. It is more challenging to develop or discover inhibitors capable of preventing the aggregation of two or more different amyloid peptides. Genistein, a major phytoestrogen in soybean, has been widely used as an anti-inflammation and cerebrovascular drug due to its antioxidation and antiacetylcholinesterase effects. Herein, we examine the inhibitory effects of genistein on the aggregation of amyloid-β (Aβ, associated with Alzheimer's disease) and human islet amylin (hIAPP, associated with type 2 diabetes) and Aβ- and hIAPP-induced neurotoxicity using a combination of experimental and computational approaches. Collective experimental results from thioflavin T (ThT), atomic force microscopy (AFM), and circular dichroism (CD) demonstrate that genistein shows strong inhibition ability to prevent the conformational transition of both Aβ and hIAPP monomers to β-sheet structures, thus reducing final amyloid fibrillization from Aβ and hIAPP monomer aggregation by 40-63%. Further 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and large unilamellar vesicle (LUV) assays show that genistein helps to increase cell viability, decrease cell apoptosis, and reduce cell membrane leakage, where the cell protection effect of genistein is likely correlated with its reduced membrane leakage. Comparative molecular dynamics (MD) simulations reveal that genistein prefers to bind the β-sheet groove, a common structural motif of amyloid fibrils, of both Aβ and hIAPP oligomers to interfere with their self-aggregation. This work for the first time demonstrates genistein as a dual inhibitor of Aβ and hIAPP aggregation. Further structural optimization and refinement of genistein may generate a series of effective sequence-independent inhibitors against the aggregation and toxicity of different amyloid peptides.

Computational Insight into the Effect of Natural Compounds on the Destabilization of Preformed Amyloid-β(1-40) Fibrils

“…However, it realizes moderate binding free energies (Gbind ~ -10 kcal/mol) in all sites, with the most stable complexes (Gbind ~ -20 kcal/mol) involving the β-2, and β-1 sites. These results are in agreement with the finding of a computational study recently reported by Ren et al [20] for a structurally homologous compound, genistein. They showed that genistein prefers to bind the β-sheet grooves to interfere with their self-aggregation.…”

“…Atomistic computer simulations are well suited to provide molecular-level details of amyloid oligomer and fibril interactions with ligands, helping in the future development and characterization of druggable modalities [12]. Basically four aspects of the flavonoids-amyloid interaction have been studied by computational methods: 1) the effect of ligands on the conformational transitions of Aβ monomers from initial random coil or α-helix into β-sheet structures [13,14] and ligand-mediated conformational change on Aβ dimer [15] by means of Replica Exchange Molecular Dynamics (REMD) simulations; 2) the effect of ligands on the aggregation of Aβ (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36) using Coarse-Grained 2 of 15 Simulations [16]; 3) effect of ligands on the conformation and stability of amyloid-beta mutants [17] by Molecular Dynamics (MD) simulations; 4) preferential binding sites of ligands and their effect on amyloid structure-dynamics [18] on Aβ fragments and full-length single Aβ protofilament [19][20][21][22][23][24][25] by means of Docking experiments, MD simulations and Free Energy calculations.…”

γ-AApeptides–based Small Molecule Ligands That Disaggregate Human Islet Amyloid Polypeptide

“…We have previously reported an inhibitor of Aβ aggregation by HW-155 from combinatorial screening. The possibility of cross inhibitory activity of amyloid inhibitors [32][33][34][35] toward multiple classes of protein fibrils prompted our effort to investigate its inhibition of hIAPP aggregation. To probe the importance of side chains on the aggregation of hIAPP, we synthesized a few derivatives of HW-155.…”

“…Hence, there is an urgent need for potent inhibitors of the process producing these pathogenic fibrils. In line with this, some inhibitors, including the peptides derived from the various secondary recognition elements along the amylin peptide chain [15][16][17] , engineered peptides [18][19][20] , constrained peptides [21][22][23][24][25][26][27] , small molecules 28,29 and natural products [30][31][32] , have been reported. Many of these inhibitors are specific in their activity; however, a good number of them have exhibited dual inhibitory activity such that they can inhibit amyloid aggregation of multiple classes of proteins non-specifically [32][33][34][35] .…”

“…HW-155 is a γ-AApeptide 38 tetramer (comparable to an 8-mer peptide in length) with little structural similarity to KLVFF, the core amyloid-forming unit of Aβ. Inspired by the findings on molecules that show cross-inhibition toward multiple amyloidosis [32][33][34][35] , and based on the fact that HW-155 was identified from an OBOC library, we hypothesized that HW-155 could be a non-specific amyloid inhibitor, and as such it may exhibit inhibitory activity toward the aggregation of hIAPP. To test our hypothesis, we synthesized HW-155 and eight (8) analogues (1)(2)(3)(4)(5)(6)(7)(8) with methyl group substitution on each position (similar to an alanine scan), and investigated their ability to prevent the fibrillization of amylin (hIAPP), as well as to disrupt preformed hIAPP aggregates.…”