Virtual Screening And Structural Modification Of GABA-AT Inhibitory Components In Zhenxin Pill And Evaluation Of Antiepileptic Activity

Epilepsy is a chronic neurological disease,which is characterized by spontaneous convulsive behavior and abnormal discharge of the brain.It has severe adverse effects on patients' physical and mental health and socioeconomic status,and severe cases can be life-threatening.The most commonly used treatment is still drug therapy,and it is still urgent to find more safe and effective anticonvulsant drugs.At present,there are still few drugs on the market with independent intellectual property rights in China.Therefore,it is a feasible strategy to develop anticonvulsant drug through the modification of effective monomers from natural products like traditional Chinese medicine(TCM).TCM has been used in the prevention and the treatment of epilepsy for thousands of years in China.Currently published studies have also revealed that a variety of monomers have exerted anticonvulsant activity in different models.Among.them,substituted cinnamic acids which contain the skeleton of phenylpropanoid were the potential fragments of anticonvulsant agents like ilepcimide.Chemical modification aiming on cinnamic acids is an important method to acquire novel anticonvulsant drugs.GABA-amino transaminase(GABA-AT)is an important target for anticonvulsant drugs.Inhibiting the activity of GABA-AT can effectively increase the content of GABA in the brain to achieve the anticonvulsant potency.This research aims to find compounds with potential GABA-AT inhibitory activity from the classic prescription Xiao Zhen Xin Wan by virtual screening,then modify the skeleton fragments,so as to improve the pharmacophore matching and the anticonvulsant activity of the synthesized derivatives was preliminarily evaluated.The main contents and the results of the investigations are listed as follows:1.The classic prescription Xiao Zhen Xin Wan for the treatment of epilepsy syndrome was selected as the research object.The library of the prescription was constructed by collecting the information of constituents in the herbs from the databases including Web of Science,PubChem,Scifinder and CNKI.A total of 199 compounds containing volatile oils,flavones and triterpenes were collected.Subsequently,the existing GABA-AT inhibitors reported in the literature were used as a training set to extract their chemical characteristics to set up a HipHop pharmacophore.The established library was used to match with the HipHop pharmacophore.Additionally,the drug-like properties of the matched compounds were evaluated.Based on the results of pharmacophore matching and drug-like properties,molecular docking of the 34 compounds were carried out with GABA-AT.The results of ligand receptor interaction showed that gingerol containing a phenylpropanin fragment and a rotatable hydrophobic long chain had a high matching degree with pharmacophore,and the results of molecular docking showed a good interaction between the target and ligand.Therefore,the gingerol was used as a lead for structural modification.2.Based on the results of the virtual screening,this study maintained the phenylpropanin fragment in gingerol,cinnamic acid with different substituents as the research object,and added the fructose diacetone hydrophobic fragment in the anticonvulsant drug topiramate.This change is to promote the FitValue with pharmacophore model and to be helpful for the designing of potential GABA-AT inhibitors.In this dissertation,a novel series of diacetonefructose cinnamate derivatives 119 were designed and synthesized using 4-dimethylaminopyridine(DMAP)and 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(EDCI)as catalytic with all the identification data of prepared analogues with LC-HRMS and NMR spectrum presented.3.Among the synthesized compounds,compounds 3,4 and 6 showed significant anticonvulsant activity in primary evaluation through the maximal electroshock(MES)model in vivo,with the ED50 values for 31.3,33.5 and 37.8 mg/kg.Furthermore,in subsequent tests,the anticonvulsant effect of active compounds 3,4 and 6 were evaluated using sc-pentylenetetrazol(sc-PTZ)model test,the neurotoxicity was investigated as well.Additionally,the molecular modeling analysis predicted good binding interactions of the most promising compounds 3,4 and 6 with GABA transferase(PDB ID:1OHW),and the ligand-based HipHop pharmacophore model was used to evaluate the initial structure-activity relationship.Therefore,it could be concluded that compounds 3,4 and 6 would represent useful precursors for further investigation in the development of anticonvulsant and analgesic agents.In this study,potential leads were obtained through virtual screening of classical TCM prescriptions and further structural modifications were made.This research provides a theoretical basis for the discovery of new clinical candidate drugs from TCM.