| Oligopeptide drug is an important part of the drug,which have pharmacological advantages and commercial advantages.Oligopeptide drug have good efficacy,safety,and tolerability,as well as short time to market and easy synthetic protocols.From the application of ejiao over two thousand years ago to the recent preparation of hirudin and the marketing of soybean peptides,the application of protein and bioactive peptides from traditional Chinese medicine(TCM)have a long history,exact pharmacological and clinical data.Studies have shown that the TCM of commonly used plants,animals and fungi from the land or the sea has rich protein and peptide components,which is a good carrier for the research of oligopeptide drugs.Compared with the traditional extraction,hydrolysis and isolation method of TCM oligopeptides,in silico proteolysis could obtain TCM oligopeptides effectively.In silico proteolysis is based on bioinformatics approach to explore cleavage sites of protein sequences under specific conditions,specific proteases or chemicals.According to in silico gastrointestinal tract proteolysis,the database of TCM oligopeptides was constructed to explore the chemical structure of active oligopeptides.Furthermore,the active mechanism of oligopeptides can be analyzed systematically by molecular simulation and biological network technology.In this paper,six TCM from gramineae seeds and two TCM from shells were used as the research carrier to explore the application of in silico proteolysis in the study of TCM protein and peptides.TCM oligopeptides databases were constructed for TCM from gramineae seeds and shells.According to six key targets of hypertension and dyslipidemia,chemical constituents and the bioactive peptides from TCM were identified for reducing blood pressure and regulating lipids based on molecular simulation techniques,including pharmacophore and molecular docking.Combined with protein interaction network(PIN)and multi-target screening study,multi-target mechanism of TCM oligopeptides was analyzed for hypertension and dyslipidemia.Finally,the biological activities of some TCM oligopeptides were verified by biological assay and molecular dynamics simulation.This study formed a TCM design methods of oligopeptides with "dry and wet combination" by a combination of in silico proteolysis and virtual screening1.Construction of TCM oligopeptides databaseTCM oligopeptides database was constructed by in silico proteolysis.TCM from gramineae seeds was commonly used and has rich protein content,which is a good carrier of plant TCM protein.TCM from shells is also rich in protein content,which is a good carrier of animal TCM protein.In this paper,the protein sequences of six TCM from gramineae seeds and two TCM from shells were collected,including Coix seed,malt,japonica rice,wheat,sorghum and maize,as well as oyster and mother of pearl.The main protein sequences included gramineae seed storage protein of gliadin and glutenin,oyster collagen,and pearl-level protein.According to in silico proteolysis,cleavage sites of the protein sequence were analyzed.Based on simulating gastrointestinal tract proteolysis,protein sequences were hydrolyzed by pepsin,trypsin and chymotrypsin,and the hydrolyzates was used to construct TCM oligopeptides database.TCM oligopeptides database contains 4913 TCM oligopeptides,including 1180 oligopeptides from gramineae seeds,3523 oyster oligopeptides and 458 pearl oligopeptides.Furthermore,according to prediction model of PepTl substrates,the absorption properties of TCM oligopeptides were predicted,which is beneficial for explaining the mechanism of TCM oligopeptides2.Construction of pharmacological identification models of hypertension and dyslipidemiaIn order to explore the mechanism of TCM for the treatment of hypertension and dyslipidemia,this paper constructed pharmacological identification models of key targets for hypertension and dyslipidemia.According to the different data situation and research objectives of different targets,this paper explored and innovated varieties of new methods of identification of TCM effective ingredients based on combinatorial molecular simulation technology.These methods could improve efficiency and accuracy of identification of TCM effective ingredients based on molecular simulation.1)According to three typical crystal structures of angiotensin converting enzyme(ACE),three groups of molecular docking models were constructed.Combined with established pharmacophore model,combinatorial virtual screening was implemented for ACE inhibitors from TCM.2)Agonists and antagonists of angiotensin II Receptor(AT1)have similar chemical structures and binding sites.To distinguish their differences,ligand-based pharmacophore model of the antagonists,structure-based pharmacophore model of agonists and docking models of antagonists were constructed.The compounds hit by antagonists models and not hit by agonists models were retained,which is beneficial to improve the accuracy of AT1 antagonist screening.3)In order to screen the peptide antagonist of endothelin receptor A(ETA),this paper explored the construction of peptide pharmacophore model for ETA peptide antagonists.According to homologous modeling technology,ETA three-dimensional structure was constructed for molecular docking.Based on consistent scoring method,TCM oligopeptides were identified with ETA antagonist activity.4)In view of the loss of three-dimensional structure of Endothelin receptor B(ETB)protein,the three-dimensional structure of ETB was constructed by homologous modeling technology.According to active pocket recognition,molecular docking model was constructed for the identification of ETB TCM antagonists.5)According to synthesizing the advantages of ligand-and structure-based pharmacophore models,merged pharmacophore methods was presented for peroxisome proliferator-activated receptor α(PPARα)agonists.Comparing with the screening efficiency of ligand-and structure-based pharmacophore models,merged pharmacophore,molecular docking and combinatorial screening,merged pharmacophore has good screening efficiency.6)The selective inhibitor of acetyl-coenzyme A:cholesterol acyltransferase-2(ACAT-2)was difficult to identify and ACAT-2 protein was unclear.Therefore,identification methods for selective inhibitor were designed based on multiple ligand-based drug design.According to selectivity index,ACAT-2 and ACAT-1 selective pharmacophore models were constructed.Compounds hit by ACAT-2 and not hit by ACAT-1 were retained with higher subtype selectivity.Then,based on selective SVM model and bioactive SVR model,potential selective ACAT-2 TCM inhibitors were identified by combinatorial screening.3.Analysis on multi-target mechanism of TCM for anti-hypertension and lipid regulationIn this paper,reverse target identification based on pharmacophore was utilized to identify the key target of TCM oligopeptides.PIN of TCM from gramineae seeds,oyster and mother of pearl were constructed by PPIs of key targets.Enrichment analysis was implemented to analyze functional modules,especially anti-hypertension and lipid regulation modules for TCM oligopeptides.According to three target combinations of ACE/ETB,AT1/NEP and PPARα/PepTl,multi-target effects of TCM compounds and oligopeptides were analyzed.Top 200 active compounds were analyzed and the potential TCM active ingredients were selected from screening results.According to virtual screening,three potential ACE inhibitors,one ETB potential antagonist,and two dual-target ACE/ETB inhibitors were identified.We also discovered two potential AT1 antagonists and two dual-target AT1/NEP inhibitors.Lipid regulation mechanism of Panax notoginseng and Ganoderma lucidum,as well as TCM combination of Panax notoginseng and Salvia miltiorrhiza were analyzed based on target combination of PPARα/PepT1.Finally,two potential PPARa agonists,one potential ETA antagonist and five potential AC AT-2 inhibitors were also discovered from TCM.4.Mechanism of Active Components from Traditional Chinese MedicineAccording to screening results of TCM compounds and active peptides for ACE AT1 and NEP,biological assay was implemented for evaluating the results.ACE inhibitors from TCM were evaluated by RP-HPLC.TCM oligopeptide of NPATY was identified as ACE inhibitor with IC50 of 61.88μM from Coix lacryma-jobi.At the same time,the activity of AT1 antagonists was evaluated by calcium flow detection.The active ingredient of BUCM-SRS was was identified as AT1 antagonist with IC50 of 29.76μM from Parmeliasaxatilis.Further,based on the fluorescence assay method,that the active ingredient of BUCM-JLA derived from Bos taurus domesticus had a good NEP inhibitory activity with IC50 of 15.67μM.Then,the interaction of protein-ligand complex was studied by molecular dynamics simulation.In this paper,in silico proteolysis was utilized to the study of TCM oligopeptides.The novel research strategy was presented to analyze the effect and mechanism of TCM oligopeptides based on the combinatorial method of in silico proteolysis and virtual screening.This strategy could be beneficial to improve identification of effective ingredients from TCM and reveal anti-hypertensive and lipid regulated mechanism of TCM oligopeptides,which provided an important technical support TCM design. |
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