Screening And Evaluation Of Active Compounds Inhibiting Protein Amyloid Fibrosis And Study On The Mechanism Of Action

Protein amyloid fibrosis means that under the condition of metabolic disorder or pathology,some proteins will undergo abnormal conformational transformation induced by endogenous or exogenous factors,and misfold to form insoluble amyloid fibers or aggregates.The deposition of these fiber aggregates can induce apoptosis,cause a variety of pathological features,and eventually lead to a variety of amyloid-related diseases such as Alzheimer’s disease(AD),Parkinson’s disease(PD)and Type 2 diabetes mellitus(T2DM).Therefore,the inhibition of protein amyloid fibrosis is considered to be an important strategy for the treatment of amyloid-related diseases.In recent years,there are many studies on protein amyloid fibrosis inhibitors,among which small molecular compounds are considered to have a wide range of applications.At present,the research on small molecular inhibitors is mainly focused on the field of natural compounds of polyphenols and flavonoids,but rarely related to the re-evaluation of listed chemical drugs.Compared with some natural compounds,chemical drugs on the market have been widely studied in terms of metabolic kinetics,safety,toxicity and so on.Therefore,based on the concept of "new use of old drugs",the repositioning of listed chemical drugs for anti-amyloid fibrosis and treatment of amyloid-related diseases can effectively save research and development time and cost,and have a higher prospect of drug formation.In this paper,a model of amyloid fibrosis in vitro was constructed by using classical model protein human lysozyme(HL),and the inhibitory activity of chemical drugs on the market was screened,and three active compounds of Entacapone(Ent),Captopril(CAP)and Cimetidine(Cim)were obtained.Then,the inhibitory effects of Ent,CAP and Cim on HL amyloid fibrosis were systematically evaluated by spectroscopic experiments and microscopic imaging techniques.Finally,the interaction between Ent,CAP,Cim and HL was studied by fluorescence spectroscopy,two-dimensional nuclear magnetic resonance spectroscopy and molecular docking simulation,and the potential mechanism of inhibition was discussed.This paper mainly includes the following three parts:Part one: The effect of Ent on HL amyloid fibrosis was studied by thioflavin-T(Th T)fluorescence,8-Anilino-1-naphthalenesulfonic acid(ANS)fluorescence,Congo red(CR)ultraviolet spectroscopy,Rayleigh light scattering,dynamic light scattering and atomic force microscope(AFM)imaging techniques.The results of spectroscopic studies show that Ent can inhibit the formation of HL amyloid fibers by reducing the formation ofβ-sheet structure,the exposure of hydrophobic regions and the particle size of fiber aggregates,and reduce the rate constant of HL fibrosis kinetics and prolong the lag time of fiber formation.AFM can directly observe that the number and length of HL fibers decrease obviously in the presence of Ent.In addition,fluorescence quenching and molecular docking simulation showed that Ent interacted with Glu35,Asp53,GIn58,Trp64,Ala108 and Trp109 amino acid residues of HL active pocket,and directly bound to Glu35 and Asp53 amino acid residues of HL by hydrogen bonding force.The results of two-dimensional nuclear magnetic resonance spectroscopy further found that the amino acid residues of Trp64 and Trp109 of HL play an important role in the binding to Ent and have a high correlation with the results of molecular docking.Part two: The effect of CAP on amyloid fibrosis of HL was studied by a variety of biophysical and microscopic imaging methods.The results of Th T and CR spectroscopy showed that CAP prevented the formation of HL amyloid fiber in a concentration-dependent manner,and circular dichroism(CD)spectra showed that CAP weakened the transformation of HL from natural α-helix conformation to HL fiber rich inβ-sheet structure.Through dynamic light scattering,Rayleigh light scattering and turbidimetry,we found that CAP can reduce the particle size of HL fiber aggregates and delay the formation of HL fiber.Based on the visual observation of AFM and transmission electron microscope(TEM),it was further confirmed that the number and length of HL fibers decreased significantly in the presence of CAP.Through MTT experiment,we found that CAP reduced the cytotoxicity induced by HL fibers and increased the survival rate of cells.The results of protein-ligand interaction show that CAP interacts with HL by static quenching and directly binds to Glu35 and Ala108 amino acid residues in the active pocket of HL through hydrogen bonding force to form a stable CAP-HL complex.Part three: The effect of Cim on amyloid fibrosis of HL was studied by spectroscopy experiment,microscope imaging and molecular docking simulation.The results of various spectroscopic experiments showed that Cim inhibited the amyloid fibrosis of HL in a concentration-dependent manner.The conclusion of spectroscopy is further confirmed by TEM visualization.The addition of Cim can significantly shorten the length of HL fibers and significantly reduce the number of straight and long fibers.The interaction between Cim and HL was studied by fluorescence quenching and molecular docking.The results show that Cim interacts with HL in the form of static quenching,and hydrogen bond and van der Waals interaction play an important role in the formation of Cim-HL complex.In summary,based on the in vitro model,the amyloid fibrosis activity of the drugs on the market was re-evaluated,and the action mechanism of Ent,CAP and Cim was deeply studied.The results showed that Ent,CAP and Cim could bind to the key amino acid residues in the aggregation tendency region of HL through a variety of forces,and the protein-ligand complex formed maintained the conformational stability of HL,thus inhibiting the amyloid fibrosis of HL.The results obtained in this paper will be helpful to the development and application of potential drugs for the treatment of amyloid-related diseases.