Carnosine-LVFFARK-NH₂ Conjugate: A Moderate Chelator but Potent Inhibitor of Cu²⁺-Mediated Amyloid β‑Protein Aggregation

Abstract

Aggregation of amyloid-β (Aβ) protein stimulated by Cu²⁺ has been recognized as a crucial step in the neuro­degenerative process of Alzheimer’s disease. Hence, it is of significance to develop bifunctional agents capable of inhibiting Aβ aggregation as well as Cu²⁺-mediated Aβ toxicity. Herein, a novel bifunctional nona­peptide, carnosine-LVFFARK-NH₂ (<i>Car</i>-LK7), was proposed by integrating native chelator carnosine (<i>Car</i>) and an Aβ aggregation inhibitor, Ac-LVFFARK-NH₂ (LK7). Results revealed the bifunctionality of <i>Car</i>-LK7, including remarkably enhanced inhibition capability on Aβ aggregation as compared to LK7 and a moderate Cu²⁺ chelating affinity (<i>K</i>_D = 28.2 ± 2.1 μM) in comparison to the binding affinity for Aβ₄₀ (<i>K</i>_D = 1.02 ± 0.13 μM). The moderate Cu²⁺ affinity was insufficient for <i>Car</i>-LK7 to sequester Cu²⁺ from Aβ₄₀-Cu²⁺ species, but it was sufficient to form ternary Aβ₄₀-Cu²⁺-<i>Car</i>-LK7 complexes. Formation of the ternary complexes directed the aggregation into small, unstructured aggregates with little β-sheet structure. <i>Car</i>-LK7 also showed higher activity on arresting Aβ₄₀-Cu²⁺-catalyzed reactive oxygen species production than <i>Car</i>. Cell viability assays confirmed the prominent protection activity of <i>Car</i>-LK7 against Cu²⁺-mediated Aβ₄₀ cytotoxicity; <i>Car</i>-LK7 could almost eliminate Aβ₄₀ cytotoxicity at an equimolar dose (cell viability increased from 59% to 99%). The research has thus provided new insight into the design of potent bifunctional agents against metal-mediated amyloid toxicity by conjugating moderate metal chelators and existing inhibitors