| Alzheimer’s disease（AD）is a neurodegenerative disease and the most common cause of senile dementia.The existing drugs can only alleviate the symptoms of the disease,and there is no effective treatment.Over the years,AD research has focused onβ-Amyloid（Aβ）plaque and tau fiber entanglement.However,studies in recent years have shown that Aβis not the main cause of AD.The real pathogenic factor of AD is that tau protein is separated from microtubules,misfolded and abnormally aggregated in nerve cells to form paired helical filaments（PHFs）,and finally form nerve fiber tangles（NFTs）.The microtubule binding domain of tau protein is the key segment to determine the abnormal aggregation of tau protein.This paper starts with the interaction of aggregation inhibitor and tau protein microtubule binding domain.Based on the data of test tube experiment and cell model experiment,supplemented by computer simulation such as docking and fitting between inhibitor molecules and proteins,this paper discusses the interaction between drug molecules and tau protein,clarifies its pharmacodynamic mechanism,and then screens tau aggregation inhibitors with high efficiency and low toxicity,to provide lead compounds and corresponding theoretical basis for the research and development of AD drugs.The main research results are summarized as follows:1.In the design and screening of drug molecules targeting proteins,the literature mostly focuses on the cavities or grooves formed by the orderly stacking of peptide chains,which is based on the docking relationship between ligand molecules and protein receptors.Tau protein lacks the ordered structure of general proteins.That means most peptide chains are randomly coiled,highly loose and dynamically variable.A new strategy is proposed to inhibit tau protein aggregation from a kinetic way by forming molecular scaffolds through multi-point binding of inhibitor molecules to prevent protein conformation turnover and misfolding.In this paper,a series of glucose gallate derivatives:1-O-galloyl-β-D-glucose,1,3,6-trigalloyl glucose,pentagalloyl glucose and tannic acid,these star molecules are composed of an intermediate glucose ring and a different number of gallic acid polyphenol side arms.These polyphenol side arms can be combined with specific sites of tau protein peptide chain through hydrogen bond andπ-πaccumulation,as if a molecular scaffold with different shapes is attached next to the peptide chain,to effectively prevent the overturning and folding of tau peptide chain.From the perspective of kinetic process,this is equivalent to improving the energy barrier of tau peptide chain isomerization in the way of tau peptide chain folding and turnover through the clamping effect of inhibitors on protein molecules,to effectively inhibit the conformation required for the formation of orderly accumulation of tau peptide chain,and trigger the process of nucleation,extension and aggregation.Through micro thermophoresis（MST）,Th S fluorescence kinetics,transmission electron microscopy and other instruments and methods,it was observed that with the increase of gallic acid polyphenol side arm,the binding force between these glucose esters and tau increased（Kd decreased）,and the inhibitory effect on tau polypeptide aggregation increased(IC50 decreased),which confirmed the rationality of this design idea.Based on test tube experiment,tau aggregation in cells was induced by tau peptide aggregate for the first time,and human neuroblastoma cell（SK-N-SH）model and human neural stem cell/human neuron model were successfully constructed.It was observed for the first time at the nerve cell level that with the increase of polyphenol side arm of glucose gallate,its inhibitory effect was significantly enhanced,but the corresponding cytotoxicity was also enhanced,and the penetration of blood-brain barrier（BBB）was also decreased.Based on balancing aggregation inhibition effect and cytotoxicity,the paper also observed that compared with tannic acid,the inhibitory effect of pentagalloyl glucose is basically the same,but the cell permeability and toxicity are less.These results provide evidence for the potential application of glucose esters as tau aggregation inhibitors.2.Aiming at the defects of high molecular weight,strong hydrophilicity,easy oxidation and difficult to penetrate the blood-brain barrier（BBB）,a drug loading system based on liposome was designed.Through lecithin/β-Sitosterol encapsulates tannic acid in non-neurotoxic liposomes and coated with Tween 80.The encapsulation of tannic acid by the liposomes can isolate oxygen and ensure the stability of tannins in the process of transmission in vivo.Using Transwell device,it is proved that tannic acid liposomes can successfully reduce tau aggregation induced by R3 aggregates in lower human neuroblastoma cells（SK-N-SH）through the upper BBB model composed of mouse brain microvascular endothelial cells（b End.3）.This result shows that the liposome can load tannins to successfully penetrate BBB and ensure the stability of tannins in the process of transmission in vivo,to finally inhibit the insoluble neurofibrotic tangles（NFTs）caused by tau misfolding in SK-N-SH cells.3.Azo compounds are a kind of light excitation responsive molecular switch,which has the phenomenon of cis-trans isomerism driven by light.This paper is devoted to exploring the inhibitory effect of these azo compounds on the aggregation of R3peptides under the condition of avoiding light.The trans to cis isomerization of azobenzene crosslinker drives the expansion of tau protein aggregation peptide chain under natural light conditions,to realize the possible depolymerization of R3 aggregates by these compounds.Using the structure of azonaphthalene benzene,we screened a series of azo dye compounds that can pass through BBB,such as allure red,congo red,sirius rose and direct red 23.This series of small molecules contain similar azonaphthalene benzene structure,but the connection direction is different.These combined changes of azonaphthalene benzene structure are used to identify tau protein chain at different points,in different orders and in different orientations,to produce hairpin binding,prevent tau protein peptide chain from overturning,and prevent it from further nucleation and aggregation into filaments.The experimental results show that the spatial configuration of the molecule does affect its affinity for protein and has different inhibitory effects on tau aggregation.Under the condition of avoiding light,these small molecules mainly inhibit the aggregation of tau.Direct red 23 is the best inhibitor in vitro,and sirius rose is the best inhibitor in cell experiment.The difference between the best inhibitor screened in vitro and cell experiment reveals a problem which has been neglected for a long time:tau protein exists in cells as a normal physiological functional protein.If we blindly pursue the strongest inhibition of tau aggregation,it is likely to destroy its ability to form microtubules in cell bone scaffold,and then affect the survival of the whole cell.Under natural light conditions,these small molecules not only show the original inhibition effect,but also show the depolymerization effect driven by light.Sirius rose has a good depolymerization effect,and has the lowest cytotoxicity compared with direct red 23.4.Compounds with similar structures have similar biological activities.We put together molecules with different biological activities like building blocks to obtain the effect of 1+1>2.J acid is an inhibitor of protein arginine methyltransferase（PRMT1）.P-aminoazobenzene-4-sulfonic acid（S1）and 4-aminoazobenzene-3,4’-disulfonic acid（P1）have structures to azobenzene acetylcholinesterase（AchE）inhibitors.We optimized the structure of J acid and obtained saturn red by adding azobenzene molecule S1 to J acid.Taking bisazonaphthalene benzene in saturn red structure as the basic pharmacophore,we coupled J acid with P1 to obtain picrosirius red.Picrosirius red contains two bisazonaphthalene benzene structures.Through molecular docking,test tube experiment and cell model experiment,their effects on inhibiting R3polypeptide aggregation in vitro and tau aggregation in cells were compared.The IC50of saturn red and picrosirius red on R3 polypeptide aggregation was 260 nm and 129nm,respectively.Under the condition of avoiding light,they showed a good inhibitory effect on tau aggregation.Under natural light conditions,they not only inhibit,but also show the depolymerization effect of tau aggregates driven by light.Cell research results show that picrosirius red is a better tau aggregation inhibitor than saturn red.It can not only inhibit tau aggregation and depolymerization of aggregated R3 infected cells,but also promotes the proliferation of human neurons.Through comparative experiments,we confirmed that picrosirius red can clamp tau protein chain and form hairpin structure at multiple points,prevent tau conformation reversal,and then prevent tau abnormal aggregation caused by tau protein misfolding.Picrosirius red can be used as a potential lead compound in the treatment of AD.In short,we have independently carried out systematic exploration on the problems involved in the experimental scheme of tau inhibitors,from the screening of tau inhibitors,the construction of tau cell model,the preparation of brain drug carrier,the construction of BBB model,to the computer docking and fitting of inhibitors and protein molecules.The relevant research results laid a theoretical foundation for the next step to promote the research and innovation of AD drugs and explore effective treatment methods,and provided new ideas for overcoming the problems of AD prevention and treatment. |
