Study On The Interaction Between Asterric Acid And Its Derivatives And Ele AChE

Alzheimer’s disease（AD）,commonly known as dementia,is a neurodegenerative disorder that is characterized by memory loss and other cognitive impairments.Currently,the main stay drugs for the clinical management of AD are still acetylcholinesterase inhibitors（AChEI）.The United States Food and Drug Administration（FDA）approved 5 drugs in the treatment of senile dementia,including4 kinds of AChEI,but these drugs have the disadvantages of great toxic side effect,high daily dosage,greatly stimulate the digestive tract and higher production cost,etc.Therefore,seeking new AChEI with high-efficiency from microorganisms and medicinal plants has become a hot topic for researchers.Our research group previously have isolated a number of endophytic fungi from Huperzia serrata.Among them,the endophytic fungus strain LF5 has a significant inhibitory effect on AChE.In this study,the AChEI tracking separation in LF5strain was carried out.Ellman’s colorimetric method was employed to study the in vitro inhibitory activity of three purified compounds with the Ele AChE.Then,the three-dimensional structure of Ele AChE was constructed through homology modeling basing on the template PDB ID:1F8U.Using the three-dimensional structure of Ele AChE as the receptor,molecular docking was performed to explore mechanism of action of three inhibitors with the receptor.The results provide a theoretical basis for empolder novel AChE inhibitors and the further structural modification of new AChE inhibitors to obtain highly active compounds.The contents are as followed:Section 1:The alcohol extract of LF5 thallus had a high AChE inhibitory activity by the AChE inhibitory activity tracking analysis.Subsequently,the active site was isolated by gradient extraction,column chromatography and other separation techniques,and the active compounds were obtained as follows:asterric acid,methyl asterrate and ethyl asterrate.AChE inhibition activity of the three compounds against Ele AChE were evaluated by Ellman’s spectrophotometric method.The IC₅₀ values for AChE inhibitions are 66.68μM、23.34μM、20.10μM respectively.The results showed that asterric acid,methyl asterrate and ethyl asterrate exhibited potent AChE inhibitory activities and the size and variety of the esterification substituent at C-8’on the parent nucleus may contribute to AChE inhibitory activities improvement of these compounds.Section 2:In order to study the mechanism of the three small molecules with the Ele AChE,we predicted the three-dimensional structure of the Ele AChE.The three-dimensional structure of Ele AChE was constructed through homology modeling basing on the template PDB ID:1F8U.The initial model was refined by MD simulations,and then evaluated by using PROCHECK,Verify₃D and ProSA.The PROCHECK evaluation results shown as the Ramachandran plot that the amino acid probability of Ele AChE structure in the reasonable region residues is as high as99%.The results of Verify₃D showed that all amino acid residues score were higher than 0,and more than 80%of the amino acid residues score was greater than 0.2,indicating that the location of amino acids are reasonable.The ProSA evaluation results,the model Z-core value of-10.7,within a reasonable range.The three-dimensional structure of Ele AChE contains twelve major helices,ten psheets and the connecting loops.The model of Ele AChE was used for molecular docking.Section 3:Molecular docking software AutoDock 4 was applied to investigate the binding mode include interaction energy,combining sites and reaction types in the interaction.The results indicated that three small molecules could interact with peripheral sites and catalytic active sites of AChE by hydrogen bond,hydrophobic interaction andπ-πstacking.One of the benzene rings in the structure of the asterric acid interacted with the main amino acid residues of the peripheral sites of AChE by hydrogen bond andπ-πstacking.The other benzene ring and the substituent on the benzene ring interacted with catalytic active sites of AChE by hydrogen bond and hydrophobic interaction.The docking results showed that the changing of the esterification substituent at C-8’on the parent nucleus may affect the type of binding force with the number of the peripheral sites.The binding energies of asterric acid,methyl asterrate and ethyl asterrate exhibited with AChE were-7.89kcal/mol、-9.72kcal/mol、-9.74 kcal/mol respectively.Furthermore,the results were compatible with the detected IC₅₀ values.The results show that the binding energy of the docking system reflects the binding ability of the compound to AChE and the IC₅₀ value of acetylcholinesterase inhibition.