Aggregation of amyloid β-protein (Aβ) into amyloid oligomers and fibrils is pathologically linked to Alzheimer's disease (AD). Hence, the inhibition of Aβ aggregation is essential for the prevention and treatment of AD, but the development of potent agents capable of inhibiting Aβ fibrillogenesis has posed significant challenges. Herein, we designed Ac-LVFFARK-NH2 (LK7) by incorporating two positively charged residues, R and K, into the central hydrophobic fragment of Aβ17-21 (LVFFA) and examined its inhibitory effect on Aβ42 aggregation and cytotoxicity by extensive physical, biophysical, and biological analyses. LK7 was observed to inhibit Aβ42 fibrillogenesis in a dose-dependent manner, but its strong self-assembly characteristic also resulted in high cytotoxicity. In order to prevent the cytotoxicity that resulted from the self-assembly of LK7, the peptide was then conjugated to the surface of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) to fabricate a nanosized inhibitor, LK7@PLGA-NPs. It was found that LK7@PLGA-NPs had little cytotoxicity because the self-assembly of the LK7 conjugated on the NPs was completely inhibited. Moreover, the NPs-based inhibitor showed remarkable inhibitory capability against Aβ42 aggregation and significantly alleviated its cytotoxicity at a low LK7@PLGA-NPs concentration of 20 μg/mL. At the same peptide concentration, free LK7 showed little inhibitory effect. It is considered that several synergetic effects contributed to the strong inhibitory ability of LK7@PLGA-NPs, including the enhanced interactions between Aβ42 and LK7@PLGA-NPs brought on by inhibiting LK7 self-assembly, restricting conformational changes of Aβ42, and thus redirecting Aβ42 aggregation into unstructured, off-pathway aggregates. The working mechanisms of the inhibitory effects of LK7 and LK7@PLGA-NPs on Aβ42 aggregation were proposed based on experimental observations. This work provides new insights into the design and development of potent NPs-based inhibitors against Aβ aggregation and cytotoxicity.
One of the key factors of Alzheimer's disease (AD) is the conversion of amyloid beta-peptide (Abeta) from its soluble random coil form into various aggregated forms. (-)-Epigallocatechin-3-gallate (EGCG) has been proved effective in preventing the aggregation of Abeta, but the thermodynamic mechanisms are still unclear. In this work, isothermal titration calorimetry (ITC) was utilized to study the interactions between Abeta42 and EGCG at different temperatures, salt concentrations, pH values, and EGCG and Abeta42 concentrations. Molecular dynamics (MD) simulations were performed to study the hydrogen bonding between Abeta42 and EGCG. The results indicate that the binding stoichiometry N is linearly related to the EGCG/Abeta42 ratio. Hydrophobic interaction and hydrogen bonding are both substantial in the binding process, but the extent of their contributions changes with experimental conditions. Namely, the predominant interaction gradually shifts from a hydrogen bonding to a hydrophobic interaction with the increase of the EGCG/Abeta42 ratio, resulting in a transition of the binding from enthalpy-driven to entropy-driven. This experimental observation is validated by the MD simulations. The binding of EGCG to Abeta42 can be promoted by increasing temperature and salt concentration and changing pH away from Abeta42's pI. The findings have provided new insight into the molecular interactions between Abeta42 and EGCG from a thermodynamic perspective and are expected to facilitate the research on the inhibition of Abeta42 aggregation.
Soluble amyloid β-protein (Aβ) oligomers, the main neurotoxic species, are predominantly formed from monomers through a fibril-catalyzed secondary nucleation. Herein, we virtually screened an in-house library of natural compounds and discovered brazilin as a dual functional compound in both Aβ42 fibrillogenesis inhibition and mature fibril remodeling, leading to significant reduction in Aβ42 cytotoxicity. The potent inhibitory effect of brazilin was proven by an IC50 of 1.5 ± 0.3 μM, which was smaller than that of (−)-epigallocatechin gallate in Phase III clinical trials and about one order of magnitude smaller than those of curcumin and resveratrol. Most importantly, it was found that brazilin redirected Aβ42 monomers and its mature fibrils into unstructured Aβ aggregates with some β-sheet structures, which could prevent both the primary nucleation and the fibril-catalyzed secondary nucleation. Molecular simulations demonstrated that brazilin inhibited Aβ42 fibrillogenesis by directly binding to Aβ42 species via hydrophobic interactions and hydrogen bonding and remodeled mature fibrils by disrupting the intermolecular salt bridge Asp23-Lys28 via hydrogen bonding. Both experimental and computational studies revealed a different working mechanism of brazilin from that of known inhibitors. These findings indicate that brazilin is of great potential as a neuroprotective and therapeutic agent for Alzheimer's disease.
Considerable experimental evidence indicates that (-)-epigallocatechin-3-gallate (EGCG) inhibits the fibrillogenesis of Aβ(42) and alleviates its associated cytotoxicity. However, the molecular mechanism of the inhibition effect of EGCG on the conformational transition of Aβ(42) remains unclear due to the limitations of current experimental techniques. In this work, molecular dynamics simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) analysis were coupled to better understand the issue. It was found that the direct interactions between EGCG and the peptide are the origin of its inhibition effects. Specifically, EGCG molecules expel water from the surface of the Aβ(42), cluster with each other, and interact directly with the peptide. The results of free energy decomposition calculated by MM-PBSA indicate that the nonpolar term contributes more than 71% to the binding free energy of the EGCG-Aβ(42) complex, while polar interactions (i.e., hydrogen bonding) play a minor role. It was identified that there are 12 important residues of Aβ(42) that strongly interact with EGCG (Phe4, Arg5, Phe19, Phe20, Glu22, Lys28, Gly29, Leu34-Gly37, and Ile41), while nonpolar interactions are mainly provided by the side chains of some hydrophobic residues (Phe, Met and Ile) and the main chains of some nonhydrophobic residues (Lys28 and Gly29). On the contrary, polar interactions are mainly formed by the main chain of Aβ(42), of which the main chains of Gly29 and Gly37 contribute greatly. The work has thus elucidated the molecular mechanism of the inhibition effect of EGCG on the conformational transition of Aβ(42), and the findings are considered critical for exploring more effective agents for the inhibition of Aβ(42) fibrillogenesis.
β-Amyloid (Aβ) can form aggregates through self-assembly and produce neurotoxicity in the early stage of Alzheimer's disease (AD). Therefore, the inhibition of Aβ assembly is considered as the primary target for AD therapy. In this study, we reported that fucoxanthin, a marine carotenoid, potently reduced the formation of Aβ fibrils and oligomers. Moreover, the fucoxanthin-triggered modification significantly reduced the neurotoxicity of Aβ oligomers in vitro. Molecular dynamics simulation analysis further revealed a hydrophobic interaction between fucoxanthin and Aβ peptide, which might prevent the conformational transition and self-assembly of Aβ. Most importantly, fucoxanthin could attenuate cognitive impairments in Aβ oligomer-injected mice. In addition, fucoxanthin significantly inhibited oxidative stress, enhanced the expression of brain-derived neurotrophic factor, and increased ChAT-positive regions in the hippocampus of mice. On the basis of these novel findings, we anticipated that fucoxanthin might ameliorate AD via inhibiting Aβ assembly and attenuating Aβ neurotoxicity.
Soluble amyloid oligomers are a cytotoxic species in Alzheimer's disease, and the recent discovery that trehalose can prohibit aggregation of amyloid beta-peptide (Abeta) has received great attention. However, its inhibition mechanism remains unclear. In order to investigate the molecular mechanism of the inhibition effect, molecular dynamics simulations of Abeta(16-22) and Abeta(40) peptides at different trehalose concentrations (0-0.18 mol/L) are performed using an all-atom model. The simulations confirmed that Abeta(16-22) aggregation is prevented by trehalose in a dose-dependent manner, and it is found that the preferential exclusion effect of trehalose is the origin of its inhibition effects. Namely, there is preferential hydration on the peptide surface (3 A), and trehalose molecules cluster around the peptides at a distance of 4-5 A. At high trehalose concentrations, the preferential exclusion of trehalose leads to three sequential effects that prevent the nucleation and elongation of Abeta(16-22) oligomers. First, the secondary structures of Abeta(16-22) monomers are stabilized in the turn, bend, or coil, so the beta-sheet-rich structure that is prone to forming peptide oligomers is prevented. Second, the thin hydration layer and trehalose clusters can weaken hydrophobic interactions that lead to Abeta(16-22) aggregation. Third, more direct and indirect H-bonds form between trehalose and Abeta(16-22), which suppress the interpeptide hydrogen bonding. Analyses of the simulation data for a single Abeta(40) peptide indicate that trehalose can inhibit the nucleation and elongation of Abeta(40) by a similar mechanism with that on Abeta(16-22) oligomerization. The work has thus elucidated the molecular mechanism of trehalose on the inhibition of Abeta oligomeric aggregation.
Molecular dynamics simulations of chymotrypsin inhibitor 2 in different polyols (glycerol, xylitol, sorbitol, trehalose, and sucrose) at 363 K were performed to probe the molecular basis of the stabilizing effect, and the data in water, ethanol, and glycol were compared. It is found that protein protection by polyols is positively correlated with both the molecular volume and the fractional polar surface area, and the former contributes more significantly to the protein's stability. Polyol molecules have only a few direct hydrogen bonds with the protein, and the number of hydrogen bonds between a polyol and the protein is similar for different polyols. Thus, it is concluded that the direct interactions contribute little to the stabilizing effect. It is clarified that the preferential exclusion of the polyols is the origin of their protective effects, and it increases with increasing polyol size. Namely, there is preferential hydration on the protein surface (2 A), and polyol molecules cluster around the protein at a distance of about 4 A. The preferential exclusion of polyols leads to indirect interactions that prevent the protein from thermal unfolding. The water structure becomes more ordered with increasing the polyol size. So, the entropy of water in the first hydration shell decreases, and a larger extent of decrease is observed with increasing polyol size, leading to larger transfer free energy. The findings suggest that polyols protect the protein from thermal unfolding via indirect interactions. The work has thus elucidated the molecular mechanism of structural stability of the protein in polyol solutions.
The fibrillogenesis of amyloid-β protein (Aβ) is considered a crucial factor in the pathogenesis of Alzheimer’s disease (AD). Hence, inhibiting Aβ fibrillogenesis is regarded as the primary therapeutic strategy for the prevention and treatment of AD. However, the development of effective inhibitors against Aβ fibrillogenesis has faced significant challenges. Previous studies have shown that pristine single-walled carbon nanotubes (SWNTs) can inhibit fibrillogenesis of some amyloid proteins. However, the poor dispersibility of SWNTs in an aqueous environment greatly hinders their inhibitory efficacy. Here, we examined the inhibitory activity of hydroxylated SWNTs (SWNT-OH) on the aggregation and cytotoxicity of Aβ42 using thioflavin T (ThT) fluorescence, atomic force microscopy (AFM), cellular viability assays, and molecular dynamics (MD) simulations. ThT and AFM results showed that SWNT-OH inhibits Aβ42 fibrillogenesis and disaggregates preformed amyloid fibrils in a dose-dependent manner. Furthermore, the ratio of hydroxyl groups in SWNT-OH is crucial for their effect against Aβ42 aggregation. SWNT-OH exerted cytoprotective effects against Aβ42 fibrillation-induced cytotoxicity. The results of free-energy decomposition studies based on MD simulations revealed that nonpolar interactions, and especially van der Waals forces, contributed most of the free energy of binding in the SWNT-OH–Aβ complex. Two regions of the Aβ pentamer were identified to interact with SWNT-OH, spanning H13–Q15 and V36–G38. The findings presented here will contribute to a comprehensive understanding of the inhibitory effect of hydroxylated nanoparticles against Aβ fibrillogenesis, which is critical for the search for more effective agents that can counteract amyloid-mediated pathologies.
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