RTHLVFFARK-NH₂: A potent and selective modulator on Cu²⁺-mediated amyloid-β protein aggregation and cytotoxicity

Dysfunctional accumulation of amyloid-β (Aβ) protein stimulated by Cu²⁺ is considered as a key process in the pathogenesis of Alzheimer's disease (AD). Thus, bifunctional substances capable of chelating Cu²⁺ and inhibiting Aβ aggregation are promising therapeutic agents against AD. Herein, a novel bifunctional decapeptide RTHLVFFARK-NH₂ (RK10) was developed by integrating a metal chelating tripeptide (RTH) and an Aβ aggregation inhibitor Ac-LVFFARK-NH₂ (LK7). The high selectivity of RK10 for Cu²⁺ over other biologically relevant metal ions was demonstrated by isothermal titration calorimetry. RK10 bound Cu²⁺ with a dissociation constant of 0.02 μM. This enabled RK10 to sequester Cu²⁺ from Aβ₄₀-Cu²⁺ species and to arrest the production of reactive oxygen species (ROS) catalyzed by Cu²⁺ or Aβ₄₀-Cu²⁺ species. Extensive physical, biophysical and biological studies indicate that RK10 targeted free and Cu²⁺-bound Aβ₄₀ species, suppressed Aβ₄₀ aggregation, and diminished the cytotoxicity induced by Aβ₄₀ and Cu²⁺-mediated Aβ₄₀ in cultured SH-SY5Y cells. Taken together, the results proved the excellent selective roles of RK10 in inhibiting Cu²⁺-mediated Aβ₄₀ aggregation and eliminating ROS generation catalyzed by Cu²⁺/Aβ₄₀-Cu²⁺ species. Thus, this work provided new insight into the design and development of potent bifunctional inhibitors against Aβ aggregation and cytotoxicity.