Construction Of Ultraefficient Inhibitors Of Amyloid Aggregation Based On Multivalent Effect And Their Applications In Protein Conformational Diseases Therapy

Misfolding and aggregation of amyloidogenic peptides and proteins into amyloid fibrils is essential to the pathogenesis of protein conformational diseases,such as type 2diabetes(T2D),Parkinson’s disease(PD)and Alzheimer’s disease(AD).Developing inhibitors that can effectively prevent protein misfolding and aggregation is considered potential therapeutics for the prevention and treatment of amyloid diseases.The inhibitors currently being developed include natural molecules,peptides,antibodies,and nanoparticles.Most inhibitors are currently only effective at near stoichiometric ratios or even higher concentrations,which show poor biosafety at high concentrations.Therefore,it is highly desired to develop amyloid inhibitors that are effective at biologically safe dose concentrations.The simultaneous binding of multivalent ligands to receptors increases the binding affinity and stability of the ligand and the receptor,thereby enhancing the biological effect of the ligand molecule,which is called "multivalent effect".Therefore,enhancing the interaction of drug candidates with amyloid via multivalent effects would provide a strategy to promote the inhibitory activity of inhibitors.In this thesis,oligopeptides and natural polysaccharides with polyhydroxy structures were developed to prevent amyloid aggregation.In addition,two multivalent complexes were constructed based on oligopeptides and natural polysaccharides that could efficiently inhibit amyloid aggregation.As a result,the binding affinity of polyhydroxy compounds to amyloid was greatly improved,and its effect on the biological toxicity caused by amyloid aggregation was verified in vivo.The main research contents are as follows:1.Inhibition of human islet amyloid polypeptide(hIAPP)aggregation by oligotyrosinesInspired by the phenolic hydroxyl structure of polyphenol inhibitors,oligotyrosine inhibitors with polyphenolic characteristics were developed.It demonstrated that oligotyrosines(Y,2Y,3Y,4Y and 6Y)regulated the aggregation of hIAPP in a tyrosine number-dependent manner.3Y had the best inhibitory effect.When the molar ratio of hIAPP to 3Y was 1:2.5,the aggregation of hIAPP could be completely inhibited,and 3Y could effectively alleviate the cytotoxicity induced by hIAPP.The molecular mechanism of oligotyrosine inhibition of hIAPP was further explored through molecular docking.2.Development of conformation-restricted multivalent macromolecules based on oligotyrosineTo further improve the stability and effectiveness of oligotyrosine inhibitors,a strategy for activating 4Y inhibitory activity in a graded manner was developed.First,4Y was grafted to the third complementarity determining region(CDR3)of the nanobody to construct the nanobody N4 Y,which restricted the conformation of 4Y and initially enhanced the inhibitory effect of 4Y.Molecular dynamics simulation showed that the binding energy of4 Y and hIAPP was-49 kcal/mol,while the binding energy of N4 Y and hIAPP was increased to-148 kcal/mol.Then,N4 Y was chemically coupled with N,N-dimethylethylenediaminemodified polyacrylic acid(PAA-DMA or PD)to obtain the complex PDN4 Y,and the secondary activation of 4Y was realized through the multivalent effect.It was verified that the multivalent complex PDN4 Y could hierarchically vitalize 4Y in mitigating hIAPP amyloidogenesis,effectively inhibited reactive oxygen species(ROS)production,attenuated hIAPP-mediated cytotoxicity and toxicity in zebrafish embryos.3.Regulating of hIAPP fibrillation by polyhydroxy-structured lentinanShiitake-derived polysaccharide lentinan(LNT)with a polyhydroxy structure was developed to prevent the aggregation of amyloid.It was found that LNT manipulated hIAPP fibrillation and modulated hIAPP-induced cytotoxicity in a conformation-dependent manner.hIAPP aggregation was completely inhibited when the mass ratios of hIAPP and triple-helical LNT were 1:3.2 and 1:1.6,while LNT was less effective in a random-coil conformation.Further investigation showed that the interaction between triple-helical lentinan and monomeric hIAPP was more favorable than the intermolecular binding of hIAPP.Finally,the inhibitory effect of LNT on β-amyloid polypeptide(Aβ)was explored,and the results showed that LNT has universality as an inhibitor of amyloid aggregation.4.Design of multivalent nanogels based on lentinanAβ aggregation was an important pathological feature of AD.Based on the ability of lentinan to effectively prevent Aβ aggregation and considering the complex pathological environment of AD,this study further developed LNT-based multivalent nanogels for the treatment of AD.LNT was cross-linked with conjugated polymer material with active oxygen scavenging effect to form PDDA-LNT nanogel(PLNG),and the multivalent effect of PLNG on the enhancement of LNT inhibitory effect was studied.The effects of PLNG on scavenging reactive oxygen species,restoring the normal immune environment of brain tissue,reducing inflammation,and restoring cognitive and memory function in AD mouse models were evaluated.In conclusion,a series of amyloid inhibitors have been developed based on oligotyrosines and LNT.In addition,multivalent complex PDN4 Y with conformationally constrained nanobodies as multivalent ligands and LNT-based multivalent nanogels PLNG were designed,which greatly enhanced the inhibitory effect of oligotyrosines and LNT and improved their bioavailability.These inhibitors effectively inhibited the formation of amyloid fibrils in vivo,and slowed down the occurrence of diseases,which provided a new strategy to develop effective therapeutics for protein conformational diseases.