Multivalent Complexes Based On Conformational Restriction For The Inhibition Of Amyloid Aggregation

Amyloidosis is a general disease with different clinical symptoms caused by the aggregation of amyloid,resulting in damage to associated cells.The development of inhibitors that can effectively prevent amyloid aggregation is of great significance for the prevention and treatment of amyloidosis.Peptides containing amyloid core sequences with high specificity and good safety can effectively inhibit the aggregation of amyloid through self-recognition,which is an ideal inhibitor of amyloid aggregation.However,further optimization is urgently needed to improve its activity against amyloidosis due to the high effective inhibitory concentration.Conformational restriction can reduce the freedom of peptide,thereby reducing the consumption of entropy and enhancing the binding affinity.The multivalent effect can also enhance the binding affinity between molecules by increasing the local concentration of the ligand.Therefore,a series of multivalent complexes based on conformation-constrained nanobody have been constructed to enhance the binding affinity between amyloid core sequence and amyloid,as well as enhance its inhibition on amyloid aggregation.The main research contents are as follows:（1）Inspired by the rigid structural scaffold of antibody and the convex ring structure bound to antigen,the first work used AuNPs as the structural scaffold,the amyloid core sequence of IAPP（PEP）related to T2D were simultaneously fixed on the surface of AuNPs by Au-S bond to obtain the inhibitor SPEPS@Au.Molecular dynamics simulations revealsed that the conformational restriction could increase the exposure of the action site,thus enhancing the binding affinity of PEP to IAPP and the inhibition on IAPP aggregation.The results of cytotoxicity experiments and zebrafish embryotoxicity showed that SPEPS@Au could effectively alleviate toxicity induced by IAPP aggregation at a IAPP:PEP molar ratio of 10:1.（2）Considering that SPEPS@Au inhibitory activity was affected by PEP density and lack of controllability and stability of conformational restriction,the high antigen binding and stable convex ring structure of the CDR3 were further utilized.PEP was directly expressed in the CDR3 to limit its conformation to develop the nanobody M1 with clear conformational restriction,which also achieved complete inhibition of IAPP aggregation at a IAPP:PEP molar ratio of 10:1.Meanwhile,M1 was modified on the surface of AuNPs to prepare M1-AuNP multivalent complex,which further enhanced the inhibitory activity of M1 through multivalent effect.Both in vitro and cytotoxicity experiments verified that M1-AuNP could effectively inhibit the aggregation and toxicity of IAPP even at a molar concentration 50 times lower than that of IAPP.（3）Based on the principle of His-tagged protein purification,Zn²⁺as the second largest essential element in human body was selected to coordinate directly with the imidazole group fused at the C-terminal His-tag of M1,and another conformation-constrained nanobody multivalent complex M1@Zn²⁺was developed.By adjusting the molar ratio of M1 and Zn²⁺to 1:360,all M1 in the solution could be assembled to form a microparticles,and soluble nanoparticles M1@Zn²⁺could be obtained after resuspension and dispersion.The preparation process was simple and the assembly site was clear.In vitro and cytotoxicity experiments showed that M1@Zn²⁺had good biosafety and could significantly enhance the affinity of M1 binding to IAPP by nearly three times through multivalent effect,and effectively inhibit the aggregation and cytotoxicity of IAPP.（4）To verify the universality of the conformational restriction of nanobody,the amyloid core sequence of Aβrelated to AD was expressed in the CDR3 to limit its conformation,and the immune stimulating fragment of human interleukin-1βwas then fused at the C-terminus.A fusion nanobody NBIL with dual functions of inhibiting Aβaggregation and promoting Aβclearance was prepared.Furthermore,NBIL was modified into PDDA to generate multifunctional complex PNBIL.In addition to inhibiting Aβaggregation through multivalent effect,PDDA skeleton was used to remove reactive oxygen species and regulate oxidative stress in the microenvironment.In this work,PNBIL could not only effectively inhibit Aβaggregation and remove Aβaggregates,but also effectively alleviate oxidative stress and inflammation,thus greatly improving learning and cognitive impairment in mice.In summary,aiming at the problems of low affinity and high inhibitory concentration of peptides containing amyloid core sequences on amyloid aggregation,a series of high-efficiency amyloid inhibitors were constructed through the nanobody conformational restriction and multivalent effect,which provided new insight for the prevention and treatment of amyloidosis.