Inhibition Of Bifunctional Inhibitor RK10 On Cu²⁺-Mediated Amyloid-β Protein Aggregation

The accumulation of amyloid-β（Aβ）peptide has been considered as one of the hallmarks of Alzheimer’s disease（AD）.In addition,the interactions of metal ions,especially Cu²⁺,with Aβproduce toxic aggregates and reactive oxygen species（ROS）further promoting neurotoxicity.Thus,bifunctional agents with the abilities to chelate metal ions and inhibit Aβaggregation are promising therapeutic agents for AD.Particularly,to reduce side effects,it is necessary to develop molecules with the ability to specifically chelate Cu²⁺rather than systemic metal ions.Tripeptide RTH was chosen as the Cu²⁺chelator,which is the binding site of Human protamine HP2(HP2_1-15)and Cu²⁺.The ability of RTH to selectively chelate Cu²⁺and prevent ROS production was explored.The results indicate that RTH has excellent selectivity for Cu²⁺over other biologically relevant metal ions(e.g.,K⁺,Ca²⁺,Mg²⁺,and Zn²⁺).The binding affinity of RTH to Cu²⁺（K_d=0.01μM）is far greater than Aβ₄₀（K_d=2.79μM）。RTH could inhibit Aβ₄₀ rapid aggregation induced by Cu²⁺and eliminate ROS generation catalyzed by Cu²⁺or Aβ₄₀-Cu²⁺complex.Next a bifunctional decapeptide RTHLVFFARK-NH₂（RK10）was designed based on RTH and an Aβaggregation inhibitor Ac-LVFFARK-NH₂（LK7）.The results investigate RK10 could inhibit Aβfibrillogenesis in the presense of Cu²⁺(the ThT fluorescence of Aβ₄₀-Cu²⁺system reduced to²2%by adding of RK10 when the molar ratio of Aβ₄₀:Cu²⁺:RK10 was 1:0.4:4).RK10 was capable of changing the morphology of Aβ₄₀ aggregates in the presense of Cu²⁺from mature fibrils to spherical aggregates.RK10 could prevent the production of reactive oxygen species（ROS）catalyzed by Cu²⁺or Aβ-Cu²⁺complex,and the catalytic activity of Cu²⁺was completely suppressed when four times of RK10 was added.Furthermore,RK10 was effective in inhibiting Cu²⁺-mediated Aβcytotoxicity.Results shown that the cell viability was increased from 46%(Aβ₄₀-Cu²⁺-treated group)to 86%by adding RK10.Isothermal titration calorimetry（ITC）measurements indicate that RK10 could also selectively chelate Cu²⁺.Moreover,RK10 chelates Cu²⁺with a dissociation constant of 0.02μM,making it has the ability to sequester Cu²⁺from Aβ₄₀-Cu²⁺complex.For the bifunctional design,RK10 could suppress Aβ₄₀ aggregation after chelating Cu²⁺,and then change the Aβ₄₀ aggregation pathway in the presense of Cu²⁺,reduce the cytotoxicity.We designed the bifunctional peptide inhibitor RK10,which could chelate Cu²⁺with high selectivity,suppress Aβaggregation,reduce oxidative stress induced by Cu²⁺and the cytotoxicity from Aβ₄₀ aggregation.Hence,our work will benefit in the future design of multifunctional inhibitors for AD treatment.