Design Of Peptide-derivatized Inhibitors For Protein Aggregation And PH Effects On The Inhibition Of EGCG

Fibrillogenesis ofβ-amyloid protein（Aβ）is the main pathological manifestation of Alzheimer’s disease（AD）,and the presence of Cu²⁺can promote Aβaggregation and produce reactive oxygen species（ROS）,which would enhance and amplify neuronal damage and further promoting neuronal death.Hence,development of functional agents that inhibit Aβfibrillation as well as chelate Cu²⁺has been considered as an effective strategy for AD therapy and prevention.Previously,a heptapeptide（Ac-LVFFARK-NH₂,LK7）was designed in our laboratory based on the sequence of Aβ_16-21 to inhibit the formation of Aβfibrils.Although LK7 inhibited Aβfibrillogenesis,there are defects in poor solubility,propensity to self-assembly and the strong cytotoxicity of self-assembled aggregates.It was also a single-function inhibitor.In order to improve the anti-aggregation effects of peptide-based inhibitors and to add new functions on the peptide targeting Aβ,we have herein designed the peptide-derivatized inhibitors by LK7 modifications and investigate the effect of p H on the inhibition of small molecule inhibitors（EGCG）.First,LK7 was conjugated toβ-cyclodextrin（βCy D）and studied the inhibitory effect of the LK7-βCy D conjugate on Aβaggregation.The conjugation significantly improved the peptide solubility.The results showed that LK7-βCy D significantly inhibited Aβ₄₀ conformational transition and fibrillogenesis,mitigated amyloid cytotoxicity.The conjugation ofβCy D changed the conformation of LK7,thereby enhancing its hydrophobic interaction with Aβ₄₀(30%increase of the binding affinity of LK7 for Aβ₄₀).Therefore,it exhibited superior inhibition.Then,the endogenic water-soluble carnosine（β-alanyl-L-histidine,Car）was ligated to the N-terminus of the LK7 to obtain a bifunctional inhibitor Carnosine-LVFFARK-NH₂（Car-LK7）.Analysis showed that Car-LK7 not only exhibited conformational stability and biocompatibility similar to that of natural Car,but also possess a bifunctional feature,inhibiting both Aβ₄₀ fibrillization and Cu²⁺-mediated Aβ₄₀ aggregation.Particulary,the results confirmed the prominent inhibition activity of Car-LK7 against Cu²⁺-Aβ₄₀ aggregation,which almost eliminate Cu²⁺-Aβ₄₀ aggregation at an equimolar dose.Furthermore,Car-LK7 showed higher activity on arresting Aβ₄₀-Cu²⁺-catalyzed ROS production than Car.Mechanism analysis showed that although Car-LK7 could not deprive Cu²⁺from Aβ₄₀-Cu²⁺complex,it could form ternary Aβ₄₀–Cu²⁺～Car-LK7 complexes.Formation of the ternary complexes directed the aggregation into small unstructured aggregates with very low cytotoxicity.Finally,because p H is a key parameter affecting Aβaggregation,and rapid nucleation of Aβin an acidic environment is one of the main causes of early AD symptoms,the inhibitory effect of EGCG on Aβ₄₂ aggregation was investigated under acidic conditions.The results showed that EGCG could effectively inhibit Aβ₄₂aggregation and cytotoxicity at p H 5.0～7.4,but the interactions between EGCG and Aβ₄₂ were remarkably affect by p H.In this study,new peptide-derivatized inhibitors with higher inhibitory effects were obtained by peptide modifications.The research provided new insight and theoretical basis for further development of potent Aβinhibitors.The research of p H effects on the inhibition of EGCG is of significance for understanding the in vivo roles of inhibitors.