Effects of ruthenium complexes on prion amyloid peptide PrP115-135 aggregation and their interaction mechanism

Abstract

Protein conformational change and amyloid fibrils deposition are associated with severe diseases. Those proteins include amyloid- β protein (A β , Alzheimer’s disease), human islet amyloid polypeptide (hIAPP, type 2 diabetes mellitus, T2DM), and prion protein (PrP, transmissible spongiform encephalopathies). Scientists have focused on studying these amyloid proteins to develop potential drugs against the diseases. Prion disease is a type of chronic progressive fatal neurodegenerative disease, whose pathogenic mechanism is the conformational conversion of normal prion protein PrPC to a pathogenic type PrPSc. The transition produces amyloid deposition and consequent neurotoxicity. PrP115-135 (115-AAAAGAVVGGLGGYMLGSAMS-135) is an N-terminal fragment of PrP with self-aggregation and cytotoxicity. Metal complexes act as traditional antitumor agents, and non-platinum-based metallodrugs are developed to improve clinical effectiveness in terms of their general toxicity and spectral activity. Besides, they interact with various proteins, enzymes and bioactive peptides. Ruthenium complexes can effectively bind to proteins, such as apotransferrin, apolactoferrin, and serum albumin, resulting in the altered protein structure and binding ability toward other molecules. They are also considered as potential inhibitors of amyloid proteins because of their low cytotoxicity and disaggregation ability to amyloid fibrils. This work studied the interactions of PrP115-135 with two ruthenium complexes NAMI-A and KP418 in order to compare the action mechanisms between different prion neuropeptides and ruthenium complexes. The intrinsic fluorescence method and cyclic voltammetry (CV) were used to study the binding mode and binding affinity of the two complexes to PrP15-135. In addition, we used Thioflavin T fluorescence assay to display the effects of ruthenium complexes on amyloid peptide aggregation. Furthermore, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to reveal the effects of ruthenium complexes on amyloid peptide-induced cytotoxicity. The change in peptide fluorescence upon metal complex addition could be assumed to reflect the amounts of peptide–ruthenium complex produced and the conformational change of the peptide. The dissociation constant K d values were (6.2±1.4) and (6.4±1.1) μ mol/L for KP418 and NAMI-A respectively, indicating a strong binding affinity between the peptide and ruthenium complexes. CV method showed that, the reduction potential of the complexes changed with the addition of the peptide, reducing the peak current as well. The change of reduction potential indicated that the interaction between them was attributed to electrostatic interaction, and the decrease of the reduction peak current implied that there were other binding forces, such as hydrophobic interaction existed. ThT assay showed that, after incubating ruthenium complexes with aggregated PrP115-135, the fluorescence intensity decreased dramatically. It suggested that the two complexes effectively inhibited the aggregation of peptide. The aggregation kinetics indicated that the complex could significantly prolong the nucleation period and aggregation time of amyloid peptide. Ruthenium complexes could bind to PrP115-135 and inhibit their aggregation, forming oligomers or monomers, thereby reducing the cytotoxicity of PrP115-135 and improving the survival rate of cells. The inhibitory effect of the two complexes on the aggregation of PrP115-135, and their rescue of peptide induced cytotoxicity make it possible for them to be inhibitors of peptide aggregation and potential drugs against amyloidosis. Exploring metal complexes with low toxicity, better disaggregation ability and good water solubility that can penetrate the blood-brain barrier is a principal research direction in this field. So our next step will keep on configuration design of the complexes so as to find potential metallodrugs against prion disease.