| The occurrence and development of the disease involves many links of the complex network system of the human body.In the face of complex system disease such as cardiovascular disease,single target drugs would appear to be "unable to do so",which have the shortcomings,for example,high side effects and high resistance.In the study of single target and high affinity research mode,the multi-target research model came into being which could overcome the limitation of many single target drugs from multi-target,multi-link and multi-angle regulation of the pathological links.Hyperlipidemia is abnormally elevated levels of any or all lipids and/or lipoproteins in the blood,which is an important pathogenic factor of atherosclerosis or other cardiovascular diseases.The researches of blood lipid are clear now,including metabolic pathway,the key targets of marketed drugs,and so on.Thus,hyperlipidemia is a good study carrier for multi-target Chinese medicine design.The aim of this paper is to study the lipid-lowering drug design from traditional Chinese medicine based on the identification of key targets combination,which is to provide new ideas and models for multi-target Chinese medicine design.This paper included the following contents:identification lipid-lowering Chinese medicine ingredient based on molecular simulation and cell experiment;identification the key targets combination based on the bipartite graph;the study of component compatibility based on the key targets combination.1.Identification lipid-lowering Chinese medicine ingredient based on molecular simulation and cell experimentFirstly,the methodology of identification lipid-lowering Chinese medicine ingredient based on molecular simulation and cell experiment was established.Because the 3D structure of the active pocket is clear,thus,the allosteric active site of CDK2 was selected as carrier to clarify the method.Due to the allosteric active site was near to the othosteric active site,the pharmacophore model and the molecular docking model of the two sites were constructed.And the optimal models were used to screening the TCMD.The screened compounds which can interact with both two binding site(othosteric site and allosteric site)were discarded,and the potential pure allosteric compounds were chosen from the remaining compounds.The growth inhibitory effect of the most promising compounds was determined on human HepG2 cell lines which improved the accuracy of the virtual screening.Secondly,the identification models of anti-hyperlipidemia target FXR were built to discover potential active compounds from TCM and the lipid-lowering activity of the most promising compounds was evaluated on cell level.Based on the structural characteristics of the active compounds,HipHop pharmacophore models were built.The best model was selected based on the test set,which was used to screen the TCMD.There are 21 crystal structures of FXR complexed initial ligand in the RCSB Protein Data Bank(http://www.rcsb.org/pdb/home/home.do)and their resolution are less than 3 A.In order to identify novel and structurally diverse inhibitors,the crystal structures were clustered based on the amino acid residues which formed hydrogen bond between receptor and initial ligand.Three cluster method,average linkage,longest distance method and sum of squares of deviation from mean,were used to cluster the amino acid residues.Twenty-one crystal structures were grouped in 2 clusters including 10 and 11 respectively.From cluster 1,the crystal structure with PDB ID 3OMM was considered as the representative structure and in cluster 2,3P89 was regarded as the representative structure based on the correlation coefficient between the docking score and the activity value of the initial ligand.The represent crystal structures 3OMM and 3P89 were used to screen the compounds which filtered by the best pharmacophore model.Two set of 54 and 56 candidate compounds which fitvalue and docking score were in the top 100 were obtained by 3OMM and 3P89,respectively.The lipid-lowering activity of FXR-Comp.1(the represent compound of cluster 1)and FXR-Comp.2(the represent compound of cluster 2)was evaluated on HepG2 cell.FXR-Comp.1 could significantly reduce triglyceride(TG)content in HepG2 cells at a concentration of 30μmol/L and FXR-Comp.2 could significantly reduce triglyceride(TG)content in HepG2 cells at a concentration of 10μmol/L.In addition,the interaction modes of the two compounds were verified by using molecular dynamics simulation and the results were stable,which explained the action mechanism of the compounds at molecular level.In this study,we combined the method of molecular simulation and cell experiment to obtain the targeted lipid-lowering traditional Chinese medicine component,which provides a basis for the future design of lipid-lowering Chinese medicine.2.Identification the key targets combination based on the bipartite graphIn this study,the relationship between the target and the lipid-lowering biological index was abstracted,and the target combination problem was transformed into the collection problem of graph theory and combinatorial problem.First,the target-biological index response graph was constructed based on the lipid-lowering metabolic network.Under the certain premise and assumption,according to the shortest path number of the target and the lipid-lowering biological index,the corresponding target was given to different weights.The target combinations of CETP-NPC1L1and HMG-CoA-NPC1L1 were found by using the idea of weighted greedy algorithm in the state of high cholesterol,high triglycerides and low HDL.In addition,the specifications of drugs already on the market were collected from the National Drug Code Database,including four lipid-lowering targets.Based on the instructions of the target drug,the relationship between the target and the biological index was obtained.Based on this,the target-biological index response map was constructed,and the corresponding target weight was given according to the data already available in the manual.The target combinations of HMG-CoA-PPAR-α were found by using the idea of weighted greedy algorithm.The corresponding Western medicine of the target which was in the target combination was used to verify the rationality of the target combination at the cell level.The concentration of the single drug is the best concentration and the half.The combined concentrations of Western medicine are the sum of the best concentration of individual drugs and the sum of the half.The results showed as follows:For the target combination of CETP-NPC1L1,when the combination of Western medicine concentration was a half of the best concentration of a single drug,the lipid-lowering effect is significant and better than a single drug group;For the target combination of HMG-CoA-NPC1L1,when the combination of Western medicine concentration was the sum of the best concentration of a single drug,the lipid-lowering effect is significant and better than a single drug group;For the target combination of HMG-CoA-PPAR-α,when the combination of Western medicine concentration was a half of the best concentration of a single drug,the lipid-lowering effect is significant.But compared with a group of a single drug,there was not statistically significant.Therefore,the target combination of CETP-NPC1L1 and HMG-CoA-NPC1L1 was reasonable.According to the principle of "small dose",the effect of target combination CETP-NPC1L1 was better than that of HMG-CoA-NPC1L1.In this study,based on the bipartite graph and weighted greedy algorithm to identify the combination of lipid-lowering key target and verify its rationality,it is provide guidance for multi-target lipid-lowering Chinese medicine design based on target combination.3.The study of component compatibility based on the key targets combinationBased on the optimal target combination CETP-NPC1L1,the compatibility of lipid-lowering Chinese medicine ingredients was discussed at cell level.Dauricine is the effective component of the target CETP and cynarin is the effective component of the tergert NPC1L1,so the compatibility of the two components was discussed.Dauricine could reduce TG content of 7.20%in HepG2 cells at a concentration of 5μmol/L and cynarin could reduce TG content of 9.57%in HepG2 cells at a concentration of 10μmol/L.However,the two components combination could reduce the TG content of 22.19%,which suggested that the lipid-lowering effect of the component compatibility is better than the single component and superior to the sum of the single component effect.Therefore,the component compatibility had the better lipid-lowering effect.We also discussed the compatibility of lipid-lowering Chinese medicine ingredients combined with the virtual screening results of the lipid-lowering targets and the results of literature research.Based on the virtual screening results schisandrin interacted with CETP and salvianolic acid A interacted with NPC1L1 that were the component compatibility.In addition,based on the key lipid metabolism module obtained from the blood circulation network and the analysis of the dynamic network module,naringenin interacted with the target CETP and salvianolic acid A/B interacted with the target NPC1L1 which were the component compatibility.This study from different angles to obtain the compatibility of traditional Chinese medicine based on the target combination,which provided basis for multi-target lipid-lowering Chinese medicine design.In this paper,the idea of applying the bipartite graph and the weighted greedy algorithm to the identification of key combinations of lipid-lowering targets is proposed.Based on the target-biological index response map to identify the key target combination of lipid-lowering and combined with molecular simulation techniques and literature research to found the component compatibility,it was provided new ideas and models for multi-target lipid-lowering Chinese medicine design. |
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