Study On The Interaction And Mechanism Between Several Natural Polyphenols And The MPTP Opening Regulatory Protein CypD

As a currently recognised regulatory protein of the mitochondrial permeability transition pore,Cyclophilin D（CypD）is involved in regulating not only mitochondrial function but also the expression of mitochondrial genes.And CypD is closely associated with apoptosis and necrosis.It has emerged as a potent target for the treatment of a variety of diseases.Natural polyphenolic compounds including magnolol,curcumin and epimedium have a variety of pharmacological activities and are potential preventive and therapeutic agents for a wide range of clinical conditions.However,most of the available literatures has only investigated the effects of magnolol,curcumin,icariin and derivatives of these three drugs on mitochondria at the cellular level.And on the basis of which it has been postulated that the drugs may act on CypD,a mitochondrial permeability transition pore regulatory protein.In vitro models of these drugs and CypD are scarce,and definitive studies of the molecular interactions and mechanisms between drugs and CypD are lacking.Therefore,the interactions and mechanisms between CypD and several natural polyphenol drugs and their respective derivatives were investigated separately using multiple spectroscopic,molecular docking and molecular dynamics simulations in this paper.The research content of this paper consists of the following four parts:ChapterⅠ:The association of mitochondria with disease,the relationship between mitochondrial permeability transition pore and CypD with apoptosis and necrosis are presented in a more comprehensive manner.The progress of pharmacological research on natural polyphenolic compounds is reviewed.The significance and research methods of small molecule drugs and proteins are reviewed,and the background and research ideas of this thesis are clarified.ChapterⅡ:The CypD plasmid was independently constructed,expressed and purified.We obtained human CypD with high purity and homogeneous particle size in the end.The interactions between and honokiol and magnolol and CypD were studied using UV-Vis absorption spectroscopy,circular dichroism,isothermal titration calorimetry and computer simulation at 298 K,respectively.The results of the study showed that both honokiol and magnolol formed the corresponding drug-protein complexes during their interaction with CypD and that both drugs had only one binding site on CypD.Honokiol/magnolol has a strong binding capacity to CypD,with honokiol binding more strongly to CypD than magnolol.The interaction process with honokiol and magnolol with CypD is enthalpy-driven and the interaction forces are mainly hydrogen bonds and van der Waals forces.Honokiol have a greater effect on the conformation of CypD than magnolol,but as a whole,these two drugs do not actually affect CypD much.Comparing the structures of honokiol and honokiol,it can be seen that the different hydroxyl positions have a certain impact on the interaction ability of honokiol/magnolol with CypD.ChapterⅢ:The interactions of curcumin（CUR）,demethoxycurcumin（DMC）and bis-demethoxycurcumin（BDMC）with CypD were studied by using fluorescence spectroscopy,UV-Vis absorption spectroscopy,circular dichroism,molecular docking and computer simulations at 298 K,respectively.The results showed that the fluorescence quenching of CypD by curcumin and its derivatives was in the form of static quenching.They all bound to CypD to form drug-protein complexes,and the order of binding strength was bis-demethoxycurcumin>demethoxycurcumin>curcumin,and the complexes formed by bis-demethoxycurcumin and CypD were more stable.The binding of the three compounds to CypD resulted in a slight change in the conformation of CypD,with bis-demethoxycurcumin having a relatively greater effect on the conformation of CypD.The specific binding sites of curcumin and its derivatives to CypD are almost the same.At the same time,hydrogen bonding and hydrophobic interactions are the main forces.The site-labelling experiments demonstrated that these compounds may have a synergistic effect with Cyclosporin A（CsA）and that the binding site to CypD may be in the vicinity of the CsA binding site.The methoxy on the benzene ring in curcumin and its analogues has an effect on the interaction of the compounds with CypD,resulting in a difference in the strength of the interaction of the three compounds with CypD.ChapterⅣ:The interactions between icariin（ICA）,icariside Ⅰ（ICA Ⅰ）and icariside Ⅱ（ICAⅡ）and CypD were investigated by fluorescence spectroscopy,UV-Vis absorption spectroscopy,circular dichroism,molecular docking and molecular dynamics simulations.The spectral results showed that the three compounds quenched the intrinsic fluorescence of CypD by static quenching and formed 1:1 drug-protein complexes with CypD.The binding constants were 7.10×10⁵ M^-1,1.29×10⁴ M^-1 and 4.79×10⁴ M^-1,respectively.The molecular docking results show that CypD forms a suitable cavity in which to embed the icariin and its derivative molecules,respectively,and that the formation of the complex mainly depends on hydrogen bonding and hydrophobic interactions.The site labeling experiment with CsA also indicate that the binding site of icariin and its derivatives to CypD is likely to be located near the binding site of CsA.In addition,circular dichroism spectroscopy and molecular dynamics simulation showed that all three compounds had some effect on the conformation of CypD and that the stability of the complex formed by icariin and CypD was relatively high.Analysis of the structures of the three compounds revealed that the position and type of glycoside substituent had a greater influence on the interaction of icariin and its derivatives with CypD.