Prediction of triptolide targets in colorectal cancer using network pharmacology and molecular docking


 Network pharmacology is an approach that uses bioinformatics to predict and identify multiple drug targets and interactions in disease. Here, we applied network pharmacology to investigate the potential mechanisms of action of triptolide, an active component in the traditional Chinese medicine Tripterygium wilfordii Hook F, in colorectal cancer (CRC). We first searched public databases for genes and proteins known to be associated with CRC, as well as those predicted to be targets of triptolide, and then used Ingenuity Pathway Analysis (IPA) to identify enriched gene pathways and networks. Networks and pathways that overlapped between CRC-associated proteins and triptolide target proteins were then used to predict candidate protein targets of triptolide in CRC. The following proteins were found to be expressed in both CRC-associated networks and triptolide target networks: JUN, FOS, CASP3, BCL2, IFNG, and VEGFA. Docking studies suggested that triptolide can fit in the binding pocket of the four top candidate triptolide target proteins (CASP3, BCL2, VEGFA and IFNG). The overlapping pathways were activation of neuroinflammation signaling, glucocorticoid receptor signaling, T helper (Th) cell differentiation, Th1/Th2 activation, and colorectal cancer metastasis signaling. These results show that network pharmacology can be used to generate hypotheses about how triptolide exerts therapeutic effects in CRC. Network pharmacology may be a useful method for characterizing multi-target drugs in complex diseases.


Introduction
Colorectal cancer (CRC) is the second leading cause of cancer-related death in the world [1]. Many Asian countries, including China, South Korea, Singapore, and Japan, are experiencing an escalating incidence of CRC [2][3][4][5][6][7][8][9]. A lot of effort has been conducted to enhance the treatment of CRC. Despite improved surgical techniques and advancements in radio-and chemotherapy over the past few decades, the overall survival rate of patients with CRC has not improved substantially [10]. It is therefore imperative to devise novel strategies for safe and effective treatment of CRC.
Triptolide is a diterpene triepoxide puri ed from Tripterygium wilfordii Hook F, commonly known as 'lei gong teng' or 'thunder god vine', a medicinal plant whose extracts have been used in traditional Chinese medicine for treating rheumatoid arthritis and other in ammatory diseases [11][12][13][14][15][16]. Recent studies have shown that triptolide kills CRC and other cancer cells in vitro with high potency [17,18]. Animal studies have shown that triptolide inhibits the growth of CRC cells in a mouse xenograft model [19,20]. However, the mechanisms underlying the therapeutic effects of triptolide in CRC are unclear.
Network-based drug discovery is a promising, cost-effective drug development approach based on bioinformatics, systems biology and pharmacology. Instead of the current "one target, one drug" approach, network pharmacology utilizes a "network target, multicomponent" strategy. Because network pharmacology can provide a good understanding of the principles of network theory and systems biology, it has been considered the next paradigm in drug discovery [21][22][23][24]. Herbal medicines such as triptolide are particularly promising drugs for developing new multicomponent and multitarget synergistic cancer therapeutics [25][26][27].
In this study, we investigated the potential mechanisms by which triptolide acts in CRC using network pharmacology. Our results revealed potential mechanisms that may underlie the therapeutic effects of triptolide in CRC and showed that network pharmacology is a useful tool to facilitate the development of novel CRC drugs.

Prediction of pathways and networks associated with CRC and triptolide targets
The protein symbols of candidate triptolide targets and CRC-associated genes were uploaded into Ingenuity Pathway Analysis (IPA) software version 2019 (https://www.qiagenbioinformatics.com, Redwood City, CA, US) for network and pathway analysis. IPA was used to construct pathways and networks based on known interactions between genes and proteins. Enrichment analysis of CRC target Gene Ontology (GO) enrichment and network was performed using R (version 3.6.0 for Windows) and Cytoscape version 3.6.1 (http://www.cytoscape.org). Pathways and networks were ranked according to the number of molecules in pathways and networks, and cut-off p value < 0.05 was used to identify signi cantly enriched pathways/networks. Pathways and networks involving CRC-associated genes and candidate triptolide targets were identi ed using the "Compare" module in IPA.

Construction of common networks and core target screening
Proteins that were both previously associated with CRC and predicted to be targets of triptolide were collected using Venny 2.1.0 software (http://bioinfogp.cnb.csic.es/tools/venny/), and potential proteinprotein interactions were analyzed in STRING 10.5 (https://string-db.org/) after ltering by Homo sapiens.
Target proteins whose values for the topological attributes of node degree distribution and betweenness centrality were above the means were de ned as "core targets". The degree of a node is the number of nodes to which it is linked, while betweenness re ects the extent to which nodes lie between one another.
Finally, core targets were analyzed using IPA.

Prediction of binding between triptolide and candidate target proteins
The crystal structures of candidate proteins bound by triptolide were downloaded from the RCSB Protein Data Bank (http://www.pdb.org/) and modi ed using YASARA software (http://www.modekeji.cn) to remove ligands, add hydrogen, remove water, and optimize and patch amino acids. Before docking, ChemBioDraw 3D was used to generate 3D chemical structures and minimize binding energies for all candidate triptolide targets. We used YASARA software to test the precision of docking between triptolide and candidate target proteins because it had the highest accuracy and consistency (data not shown). The best docking poses were identi ed as those showing the smallest root mean square deviation (RMSD) between the predicted conformation and the observed X-ray crystallographic conformation.
Models with an RMSD ≤ 4 Å were considered reliable and those with an RMSD ≤ 2 Å were considered accurate [34]. The network pharmacology approach in this study is summarized in Fig. 1.

Networks and enriched functions in CRC-associated genes
A total of 3,298 CRC-associated genes were identi ed in GenBank. IPA identi ed a total of 596 pathways and 25 networks associated with these genes. The top/majority of pathways were involved in molecular mechanisms of cancer, CRC metastasis signaling and Wnt/β-catenin signaling. The majority of networks were involved in cancer, cellular movement, organismal injury and abnormalities, cellular development, embryonic development, organismal development, cell-to-cell signaling and interaction, protein synthesis, and RNA damage and repair (Fig. 2).
Gene Ontology (GO) enrichment and network analysis of CRC-associated proteins showed that the top three functions were epithelial cell proliferation, ameboidal-type cell migration and regulation of vasculature development (Fig. 3).

Networks and enriched functions in triptolide target genes and proteins
A total of 33 proteins were identi ed as candidate triptolide targets, and IPA revealed a total of 294 enriched pathways and 10 networks. The most signi cantly enriched pathways were neuroin ammation signaling, glucocorticoid receptor signaling, T helper (Th) cell differentiation, Th1 and Th2 activation, and CRC metastasis signaling. The top networks identi ed were involved in gene expression, cellular function and maintenance, cell cycle, in ammatory response, organismal injury and abnormalities, cell-to-cell signaling and interaction, cell death and survival, dermatological diseases and conditions, and infectious diseases (Fig. 4).

Networks of shared proteins and special proteins targeted by triptolide
The intersections between the set of potential triptolide targets and CRC-related proteins were analyzed using Venny software, which identi ed 29 shared proteins (Fig. 5A). STRING suggested that the 29 proteins can interact with one another via 269 interactions (edges) (Fig. 5B).
Proteins linked to CRC and potentially targeted by triptolide participate in several canonical pathways involved in a range of biological activities. To demonstrate the ability of our integrative bioinformatic approach to propose speci c protein targets for further mechanistic studies, we selected the top pathway in the IPA categories "molecular mechanisms of cancer" and "colorectal cancer metastasis signaling" that were linked to CRC and targeted by triptolide. Several nodes in this pathway emerged as potential direct targets of triptolide in CRC: JUN, FOS, CASP3, BCL2, IFNG, and VEGFA ( Fig. 5C and D). Combining these results with STRING analysis, we identi ed JUN, FOS, CASP3, BCL2, IFNG, and VEGFA as particularly likely targets of triptolide in CRC.

Predicted binding of triptolide to target proteins in CRC
To further validate candidate triptolide targets in CRC, we tested the precision of docking between triptolide and the following potential target proteins by YASARA software (Fig. 6) Fig. 6, triptolide binds to the active sites of these target proteins and interacts with several amino acid residues, with most interactions being hydrophobic.
For instance, in the combination of triptolide with VEGFA, there are different hydrophobic interactions between triptolide and residues of VEGFA such as Gln-22, Tyr-25, His-27 and Pro-28. In addition, triptolide can form a hydrogen bond with a length of 1.8 Å and a bond energy of 8.40 kJ/mol with the Arg-207 residue of CASP3, and its aromatic ring forms a π-π interaction with the aromatic ring in Phe-256 of CASP3. Overall, these results provide further evidence that these four proteins may act as triptolide targets in CRC.

Discussion
The network pharmacology approach is relatively new and was rst proposed by Li et al. in 2014 [28]. Because it provides a more complete understanding of network theory and systems biology, it has been considered the next paradigm in drug discovery [29,30]. Network pharmacology has been used to study pathways of interaction between drugs and proteins or genes and diseases, and it is capable of describing complexities among biological systems, drugs and diseases from a network perspective [31][32][33][34]. Therefore, the development of network pharmacology techniques that can predict multiple drugtarget interactions may hold the key to future drug discoveries in complex diseases such as CRC. In this work, we integrated information from publicly available databases to predict interactions between triptolide and its potential targets in CRC, as well as the signaling pathways and networks involved. Pathway analysis suggested that triptolide regulates the activation of neuroin ammation signaling, glucocorticoid receptor signaling, Th cell differentiation, Th1 and Th2 activation, and metastasis signaling in CRC.
These results are consistent with several in vitro and in vivo studies. Previous work [35] suggested that triptolide is able to induce G1 cell cycle arrest by inhibiting transcriptional activation of E2F1. Triptolide was found to reduce both tumor number and tumor size in mice carrying mutations to promote growth of adenomatous polyposis coli as well as in mice treated with azoxymethane/dextran sodium sulfate to induce cancer [36]. Triptolide effectively inhibits CRC cell proliferation, colony formation, and organoid growth in vitro, and these effects are associated with down-regulation of target genes transcribed by RNA polymerase III. Moreover, triptolide inhibits cyclooxygenase-2 and inducible nitric oxide synthase expression in human colon cancer [37].
Our study presents several limitations. Although we predicted and veri ed potential triptolide target proteins by molecular docking, our results are based on bioinformatic predictions and require experimental con rmation. As next steps, we intend to use triptolide to treat CRC cells, identify its targets using transcriptomics and proteomics, and thereby elucidate its mechanisms of action.
In summary, our results predict that the therapeutic effects of triptolide in CRC are mediated, at least in part, via JUN, FOS, CASP3, BCL2, IFNG, and VEGFA. These results may be useful in guiding further research to determine the molecular targets of triptolide in CRC, and they may inspire further application of network pharmacology to drug discovery.

Funding
The present study was supported by The National Natural Science Foundation of China (grant no.     cancer" and (D) "colorectal cancer metastasis signaling" that have been linked to CRC and are predicted to be targeted by triptolide. Proteins likely to be targeted by triptolide are marked with purple boxes.
Triangles mean proteins with enzymatic activity and circles mean proteins without enzymatic activity. The thick sticks represent the triptolide molecule, and the thin lines represent residues in the protein binding site. For interpretation of the references to colors in this gure legend, the reader is referred to the web version of this article.