Study On The Mechanism Of Interaction Between Several Natural Small Molecule Drugs Targeting Mitochondria And CypD

Targeted therapy is already a promising modality when treating diseases.There have been many research studies on cellular autophagy,diseases caused by mitochondrial dysfunction,and apoptosis.One of the recent research hotspots is the targeting of cyclophilin D(CypD)to induce mitochondrial permeability transition pore opening leading to mitochondrial dysfunction.Natural small molecule drugs are promising substances for research as therapeutic agents for various diseases.For example,natural anthraquinone chemistry,flavonoids,and terpenoids are unique in their anticancer,antibacterial,and antioxidant activities,and therefore,they are sources of drugs for the treatment of various diseases.Current studies on mitochondrial dysfunction due to the opening and closing of mitochondrial permeability pores are mainly at the cellular or subcellular organelle level,with few studies at the molecular level;therefore,in vitro studies on the interaction of CypD with natural small molecule drugs are of great significance.In this paper,we investigated the stabilization conditions and properties of CypD,the interaction of three anthraquinones,rhein,emodin and aloe emodin,and three flavonoids,quercetin,luteolin and phloretin,with CypD in vitro.1.Briefly introduced the therapeutic means and pathways for cancer,and detailed the therapeutic mechanism of targeting mitochondria and the important role of the mitochondrial permeability transition pore for disease treatment,focusing on the components of the mitochondrial permeability transition pore and its key regulator CypD.The important role of natural drugs in the treatment of diseases is reviewed,and several methods of in vitro protein probing are introduced,namely UV-Vis absorption spectroscopy,fluorescence spectroscopy,circular dichroism spectroscopy,molecular docking simulations,and molecular dynamics simulations.The idea of selecting the topic for this thesis and its innovation are illustrated.2.Plasmids were constructed for the amino acid residues of CypD as well as the characteristics of the expression system.The target protein was expressed and purified using the constructed plasmids,and the quality of the purified CypD was examined by SDS gel electrophoresis and nanoparticle size testing.Data on the hydrophobicity of CypD,the distribution of amino acids on the surface,the secondary structure of CypD and the possible active sites were obtained by simulation experiments.The experimental results showed that CypD is a soluble protein,and the CypD obtained by expression and purification is homogeneous and stable;its surface amino acids include tyrosine and tryptophan,etc.The secondary structure contains α-helix with spinodal properties,and there are four possible active pockets.3.Based on these properties of CypD,the interactions of Rhein,Emodin,and Aloe emodin with CypD were investigated.The results of UV-vis-absorption and fluorescence spectroscopy experiments showed that their interaction with CypD not only caused static quenching of fluorescence but also affected the structure of the protein,but both circular dichroism spectroscopy and molecular dynamics simulations were able to show that this effect was very small and did not affect the activity of CypD.Site-labelling experiments have shown that Cs A does affect their binding to CypD,most intuitively by increasing or decreasing their binding energy and shifting the direction and location of binding to interact with CypD.Overall,they all have the potential to be CypD targeting drugs.The reasons for their strong and weak binding ability to CypD were investigated by five structurally similar small molecule drugs.It was found that the binding ability of small molecule drugs interacting with CypD was generally greater for carboxyl compounds than for hydroxyl compounds when other conditions were the same,and the more phenolic hydroxyl groups in hydroxyl compounds the stronger the drug binding ability.4.Based on the mechanism of the action of quercetin,luteolin and phloretin with CypD,the in vitro interaction of quercetin,luteolin and phloretin with CypD was investigated.The results show that all three drugs have the potential to be targeted by CypD.In the presence of Cs A,the active site they occupy on the surface of CypD remains pocket I,but their affinity for interacting with CypD is increased for all three drugs.Among them,the ability of quercetin and luteolin to interact with CypD was significantly greater than that of phloretin,and their ability to interact with CypD was quercetin >luteolin > phloretin,respectively.The pattern of the magnitude of their ability to interact with CypD verifies the conclusion that compounds with more hydroxyl groups have a stronger ability to interact with CypD,and that the ring structure(pyran ring)in small molecule drugs is closely related to the magnitude of their binding ability to CypD,which provides clues for the search of CypD-targeted drugs.