Jingxian Ren,Zhuoyang Cheng,Xiaoxiao Huang,Shaojiang Song

Key Laboratory of Computational Chemistry-Based Natural Antitumor Drug Research &Development,Liaoning Province,School of Traditional Chinese Materia Medica,Shenyang Pharmaceutical University,Shenyang 110016,China

Abstract Camptotheca acuminate belongs to Nyssaceae family and has been used as traditional Chinese medicine for several years.A number of compounds with anti-tumor activities have been isolated from C.acuminate.However,the targets of some compounds have not been determined.In this study,network pharmacology methods were used to analyze 74 compounds from C.acuminate and 49 target proteins of liver cancer.The results showed that the target with the largest number of directed edges was Dihydroorotate Dehydrogenase (1d3g).The compound docked with 1d3g best was AL.23 (19-O-methyangustoline).The results of this study provide further insight into the role of network pharmacology as an effective tool to illuminate the research on liver cancer.

Keywords:Camptotheca acuminate; liver cancer;network pharmacology

1 Introduction

Liver cancer is the most common cause of cancer death in the world.Most patients with liver cancer are diagnosed at the advanced stage [1].Previous studies have indicated that the standard anti-cancer therapy,which inhibits tumor growth,may be circumvented by some cancer cells.In order to ensure the therapeutic effect and the quality of life after treatment,the therapeutic methods must be innovated.Several studies have shown that some natural products have novel anti-tumor activities [2].Therefore,it is very important to screen new natural products with anti-cancer activities and clarify their mechanism of action.

Camptotheca acuminate

is a native tree species of Nyssaceae family,mainly distributed in Yunnan,Sichuan,Hunan,Zhejiang,Fujian and some other provinces in China [3].Its fruits,leaves,barks and roots have anti-tumor,heat-clearing and insectkilling activities [4].Bioactive substances from

C.acuminate

include alkaloids,glycoside compounds,ellagic acid derivatives,terpenes and fatty acids [5].Camptothecin isolated from the barks of

C.acuminate

was proved to show good cytotoxic activity [6].However,the effective targets for some compounds from

C.acuminate

are still unknown.Network pharmacology technology is a novel technology based on systematic biology [7].It allows us to predict the protein drug targets of cancer and evaluate the pharmacological effects of traditional Chinese medicine at the molecular level [8].Therefore,we can use network pharmacology technology to screen more potential targets of hepatoma carcinoma cells and more therapeutic approaches for liver cancer.

2 Material and methods

2.1 Collection of compounds and establishment of compound database

Through literature survey,we obtained a total of 74 compounds from

C.acuminate

.The structure of each molecule was imported to minimize energy for optimization using ChemDraw Ultra 14.0 software and structures of the compounds were then optimized by SYBYL-X 2.0 software.

2.2 Screening and processing of target protein

Therapeutic Target Database (TTD) and Uniport were used to search for the protein targets of human hepatoma carcinoma cells and we obtained 49 protein targets (Table 1).The crystal structure of the target protein complex was obtained from RCSB Protein Data Bank database.SYBYL-X 2.0 software was used to process the structure,including removal of the original ligand and water,hydrogenation,optimization and repair of the amino acid residues.

Table 1 The detailed information of targets and diseases for vibsane-type diterpenes

Continued table 1

2.3 Molecular docking

SYBYL-X 2.0 software was used to obtain the chemical structures of the compounds.Thereafter,we imported the molecular forms and the protein targets for docking.To ensure the accuracy of the data,molecules with a total score greater than 7.0 were screened for subsequent analysis.Finally,45 compounds and 31 protein targets were obtained for further network analysis.

2.4 Network construction and analysis

We used Cytoscape 3.6.0 software to construct the compound-target network (Fig.1)and used the“Analyze network”tool of the software to obtain the data of the degree and betweenness centrality.

Fig.1 The compound-target network.The colorized circles represent the compounds and the colorized diamonds represent the protein targets

3 Results and discussion

3.1 Prediction of potential compounds

As the result of the molecular docking showed,the compound-target network consisted of a total of 76 nodes and 219 edges.The edges revealed the action relationship between the active compounds and the liver cancer targets.Degree and betweenness centrality analysis revealed 10 potentially active compounds in

C.acuminate.

Most of the active compounds are alkaloids,named 10-methoxy-20-

O

-acetylcamitothecin(AL.12) [9],20-hexanoyl-10-methoxycamptothecin(AL.10) [10],20-hexanoylcamptothecin(AL.9) [10],strictosidinic acid (AL.18) [11],strictosidinic acid 6′-

O

-

β

-D-glucopyranoside(A L.2 7) [1 2],hyperoside (FL.2) [1 3],3’-methoxy-3,4-methylenedioxy ellagic acid-4′-

O

glucoside (EL.9) [11],(5

R

)-camptothecin 5-

O

-

β

-Dglucopyranoside (AL.26) [12],trifolin (FL.1) [13],and vincosamide 11,6′-di-

O

-

β

-D-glucopyranoside(AL.29) [12].The specific structures of these potential compounds are shown in Fig.2 and the parameters of the ten potential compounds are shown in Table 2.

Table 2 Parameters of the ten potential compounds

Continued table 2

Fig.2 Specific structures of the top ten potential compounds

Continued fig.2

3.2 Prediction of potential targets

In order to describe the action relationship between the active compounds and their liver cancer targets,the compounds-target network was constructed.As shown in Fig.1 and data analysis,the top ten targets with the higher degree value are Dihydroorotate Dehydrogenase (1d3g),Nuclear receptor coactivator 2 (3up3),TGF-beta receptor type-1 (5e8z),GMP Synthase (2vxo),ATR-interacting protein (5yz0),RAC-alpha serine/threonine-protein kinase (3o96),Tyrosine-protein kinase Lck (1qpc),Hepatocyte growth factor receptor (5eyd),Epidermal growth factor receptor(5jeb),and Histone Deacetylase 4 (2vqq).The specific parameters are shown in Table 3.

Table 3 Specific parameters of the ten potential target proteins

3.3 Further molecular docking about 1d3g

The result showed that 1d3g protein was the target with the highest degree and largest number of directed edges.1d3g was expressed by the gene DHODH.Some studies have suggested that the small molecule DHODH inhibitors may be an attractive therapeutic approach to the treatment of autoimmune diseases,immunosuppression,and cancer [14].

Previous studies have found that the compounds have the activity of inhibiting hepatocellular carcinoma cells in several different ways.Docking results showed that AL.23 (19-

O

-methyangustoline),AL.26((5

R

)-camptothecin 5-

O

-

β

-D-glucopyranoside),AL.12 (10-Methoxy-20-

O

-acetylcamitothecin),and AL.13 (20-Formylbenz[6,7]indolizino[1,2-b]quinoline-11(13)-one) had the highest docking score with the 1d3g target.These alkaloids were chosen for further molecular docking about the structureactivity relationship using iGEMDOCK v2.1 software.Results showed that AL.23 was linked to the active site of 1d3g through conventional hydrogen bonds,carbon hydrogen bonds,Pi-Sigma relation,Alkyl relation and Pi-Alkyl relation(Fig.3).Similarly,the conformational and ichnographic binding modes of the other 3 compounds are shown in Figs.4-6.Results showed that the five-ring structure was necessary for biological activity of AL.23 to AL.13.

Fig.3 Identification of the structure-activity relationship of AL.23 by molecular docking.(A) The binding mode of AL.23 in the active site of 1d3g pocket;(B) The 2D ligand interaction Diagram

Fig.4 Identification of the structure-activity relationship of AL.26 by molecular docking.(A) The binding mode of AL.26 in the active site of 1d3g pocket;(B) The 2D ligand interaction Diagram

Fig.5 Identification of the structure-activity relationship of AL.12 by molecular docking.(A) The binding mode of AL.12 in the active site of 1d3g pocket;(B) The 2D ligand interaction Diagram

Fig.6 Identification of the structure-activity relationship of AL.13 by molecular docking.(A) The binding mode of AL.13 in the active site of 1d3g pocket;(B) The 2D ligand interaction Diagram

In recent years,a number of natural products,such as camptothecin,have been isolated from

C.acuminate

,and some of the alkaloids have shown good anti-tumor activities.However,the effective targets of most bioactive substances are still unknown.In this study,network pharmacology methods such as wide-scale compound prediction,target prediction and network analysis were used to identify potential targets of bioactive compounds.The compound-target network constructed in this study could provide ideas for future research on the specific action mechanism of the compounds with anti-tumor activities.

4 Conclusion

Network-based pharmacological results predict that Dihydroorotate Dehydrogenase is an effective target for the compounds from

C.acuminate

to act on the liver cancer cells.The docking results prove that the alkaloids from

C.acuminate

might be the research focus for developing anti-tumor drugs and discovering new action modes.