Study On The Designing、Efficiency And Interaction Mechanism Of A Peptide Inhibitor Of β-Amyloid Aggregation

Alzheimer’s disease (AD) is a neurodegenerative disorder associated with the loss of memory, cognitive decline, and behavioral disability, leading to dementia and death ultimately. The accumulation of the deposits of β-amyloid (Aβ) peptide is considered to be one of the histopathological criterions for AD. The aggregation and misfolding of Aβ lead to series of pathologies in the brain tissue of AD patients and dementia eventually. Available drugs mainly relieve AD symptoms, and no effective drugs and treatments could cure this disease fundamentally. Our work is just based on the design of inhibitors against Aβ aggregation.Recently, many studies targeted the hydrophobic region of Aβ (Aβ16-20, KLVFF), and designed various inhibitors against Aβ aggregation based on the hydrophobic interaction. Although the studies have yielded encouraging results, some problems remain existing and unresolved. First, the β-sheet breakers, such as LPFFD and KLVFF, require a very large molar excess relative to Aβ to inhibit Aβ aggregation. Second, some inhibitors delay but not prevent the aggregation, merely affecting the lag time of Aβ aggregation. Third, the self-aggregation of inhibitors may reduce their effective concentrations. In our view, the main cause of these phenomena is that most of the inhibitors only combine with Aβ by hydrophobic interation, and cannot form a stable inhibitor-Aβ complex to persist for a long time.In our study, firstly we expanded the targeting from Aβ16-22to Aβ11-23(EVHHQKLVFFAED), which could offer more interaction sites, such as the electrostatic, hydrophobic interactions and hydrogen bonding. Based on the structure of Aβ11-23, we designed a decapeptide inhibitor RYYAARRARR (RR) with the help of computer simulation, which is expected to interact with Aβ11-40molecule in multiple ways, including via hydrophobic and electrostatic interactions and hydrogen bond, and our objective is that RR could interact with Aβ1-40with high affinity and ultimately inhibit the aggregation of AP1-40effectively.Then, thioflavin T (ThT) fluorescence, circular dichroism (CD) spectra, transmission electron microscopy (TEM), and a cell viability assay (MTT) were used to evaluate the inhibitory and disassembly effect of RR. The results showed that RR inhibited the aggregation of Aβ1-40by nearly75%at an equimolar concentration, and that a1:4ratio of Aβ:RR almost inhibited the aggregation completely, while LPFFD only caused a inhibition of11%at the same concentation. The results showed that compared to the β-sheet breaker LPFFD, RR could inhibit the aggregation more effectively, which reflects the superiority of multiple weak interactions. Isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) system and Autodock were used to further analyze the interaction mechanism between RR and Aβ1-40.The results revealed that RR exhibits stronger binding affinity for Aβ1-40(KD=1.10μM) than the known β-sheet breaker LPFFD (KD=156μM) and that RR binds to Aβ1-40because of its strong affinity for Aβ11-23, which is mainly driven by hydrophobic and electrostatic interactions and hydrogen bonding. Moreover, our data indicated that RR can disassemble the Aβ1-40mature fibrils into short fragmented Aβ fibrils, which may be beneficial to the removal of small aggregations of Aβ.At last, the affinity adsorbents based on agarose were prepared. The capaility for Aβ1-40was studied by static adsorption experiments, which indicated that the adsorbents has a certain adsorption capacity for Aβ1-40, and confirmed the interaction between RR and Aβ1-40. The results of this study should be helpful in designing new and effective peptide inhibitors to control the aggregation of Aβ as an AD therapy.