Pharmacognosy Magazine

ORIGINAL ARTICLE
Year
: 2022  |  Volume : 18  |  Issue : 77  |  Page : 175--182

Network pharmacology analysis with molecular docking of phytochemicals of Panax ginseng against osteosarcoma


Fahad Hassan Shah, Song Ja Kim 
 Department of Biological Sciences, College of Natural Sciences, Kongju National University, Gongju 32588, Republic of Korea

Correspondence Address:
Song Ja Kim
Department of Biological Sciences, College of Natural Sciences, Kongju National University, Gongju
Republic of Korea

Abstract

Background: Panax ginseng is a perennial medicinal herb also known as Korean ginseng or Insam (인삼), commonly found in Korean Peninsula. These herbs are used to treat different types of diseases. Recent studies have shown that phytochemicals found in P. ginseng harbor anticancer activity against various cancers. However, the biological and molecular mechanisms of these phytochemicals are still unknown in osteosarcoma (OS). Materials and Methods: This study utilized the network pharmacology method comprised target prediction, gene enrichment and ontology, KEGG pathway analysis, and gene expression studies. The obtained results were used to predict the interaction of phytochemicals with the human CDKL3 domain implicated in OS using molecular docking. Toxicity and pharmacokinetic elements of these phytochemicals were also identified. Results: Results showed that Fumarine and Inermin are bioactive phytochemicals that have a multimodal effect on multiple targets and pathways involved in the progression of OS. These compounds were able to regulate the expression of genes and interacted with human CDKL3. These compounds have good pharmacokinetic and toxicological characteristics. However, they exert a high risk of hepatotoxicity. Conclusion: The present study provided a predicted mechanism of action of bioactive phytochemicals of P. ginseng in the inhibition of OS.



How to cite this article:
Shah FH, Kim SJ. Network pharmacology analysis with molecular docking of phytochemicals of Panax ginseng against osteosarcoma.Phcog Mag 2022;18:175-182


How to cite this URL:
Shah FH, Kim SJ. Network pharmacology analysis with molecular docking of phytochemicals of Panax ginseng against osteosarcoma. Phcog Mag [serial online] 2022 [cited 2022 Nov 29 ];18:175-182
Available from: http://www.phcog.com/text.asp?2022/18/77/175/341078


Full Text



SUMMARY

The phytochemicals contained in the P. ginseng has been explored in the treatment of osteosarcoma (OS) using Network Pharmacology methodDrug likeness and pharmacokinetic screening identified six potential medicinal compounds such as Fumarine, Inermin, Frutinone A, Celabenzine, Nepetin and SuchilactoneFumarine and Inermin showed moderate anti-OS activity identified by Network Pharmacological analysisMolecular docking validation was reconfirmed that Fumarine and Inermin are potential candidate for the treatment of OS.

[INLINE:1]

Abbreviations used: ADMET: absorption, distribution, metabolism, excretion and toxicity, BATMAN-TCM: Bioinformatics Analysis Tool for Molecular mechanism of Traditional Chinese Medicine, BBB: Blood Brain Barrier, CDKL3:Cyclin dependent kinase like 3, KEGG: Kyoto Encyclopedia of Genes and Genomes, OS: Osteosarcoma, PASS: Prediction of activity spectra for biologically active substances, P. ginseng: Panax ginseng, TCMSP: Traditional Chinese Medicine System Pharmacology Database, TTD: Therapeutic Target Database.

 Introduction



Osteosarcoma (OS) is a primary skeletal tumor in which aberration in the bone-forming mesenchymal cells causing the formation of an immature osteoid matrix.[1] This type of tumor has a high malignancy rate and usually originates in soft tissues. An approximate incidence of OS reported around ~4 million cases per year in adults, whereas ~5 million cases per year in children (0–19 years).[2] The disease etiology is still elusive yet; however, some studies indicated that exposure to radiation initiates OS formation reported in 2% of cases. However, it has been largely attributed to germline mutation in p53 protein.[3]

OS is characterized as surface and central bone tumors classified by the World Health Organization system of histological classification.[3] Almost 90% of OS cases are diagnosed as central tumors. The musculoskeletal tumor society has devised a staging system (I-III) to observe and characterize the tumor progression.[4] Stage I and II are low-grade tumors, whereas Stage III are highly malignant and vulnerable to metastasis.[4] The therapy for OS is comprised of surgery, in which amputation of the affected limb is performed combined with post-therapy chemotherapy. However, in high-grade tumors, the treatment fails to avert the proliferation and expansion of OS.[5] Another arising issue with this tumor is the development of resistance to chemotherapy which makes it more challenging to avert the recurrence and metastasis.[6],[7],[8] That is due to the involvement of multiple protein factors facilitating such processes.[8] So far, no targeted treatments have been discovered for this disease to prolong the survival rate. The discovery of new therapeutic molecules able to perform multiple biological activities, including anticancer with anti-metastatic properties is of great concern to be able to deter cancer growth and development. Network pharmacology is defined as an integration of system biology, network analysis, and in silico drug discovery methods to determine the multiple pharmacological activities of a compound against various diseases.[9],[10] This method combined with medicinal phytochemicals can be beneficial to discover therapeutic with biological activity against multiple targets in the studied disease.

In our study, we used the network pharmacology method to explore the phytochemicals of Panax ginseng against OS. P. ginseng is an ethnobotanical plant of the Korean Peninsula and China that belongs to the family of Araliaceae, Genus: Panax L, and Species: P. ginseng Meyer.[11] Traditionally, the dried form of the plant, especially the roots, has been used to treat several diseases, including cancer and inflammation.[12] Recently, these plants have been utilized to extract various bioactive compounds whose biological activity is yet to be determined. The present study obtained all these compounds contained in P. ginseng and explored the activity against OS using network pharmacology combined with in silico interaction studies.

 Materials and Methods



Selection of bioactive compounds in Panax ginseng

The current study utilized P. ginseng to analyze its therapeutic effect on OS. We have accessed the traditional Chinese medicine (TCM) system pharmacology database,[13] to acquire bioactive compounds from this plant. In the search box, P. ginseng was searched as a keyword, and compounds information related to this plant was downloaded in the excel file. The compounds were selected based on drug-likeness (DL), oral bioavailability (OB), toxicity class, and Lipinski's rule of five.

Screening of target genes

Gene cards database was used to procure gene targets involved in the progression of OS. Bioinformatics Analysis Tool for Molecular mechANism (BATMAN) TCM,[14] and DIGEP-Pred,[15] webservers were accessed to predict the effect of phytochemicals on the target's genes. The canonical SMILES of these compounds were used to retrieve the prediction results of the compound's induced effect on genes.

Molecular docking

The compound's interaction was evaluated against human CDKL3 kinase is an important molecular target of OS.[16] This analysis was facilitated by IGEMDOCK, and the compounds were targeted toward the 38R active site of CDKL3. The amino acids residues present in the 38R active site are VAL18, VAL10, LYS33, PHE79, GLU80, ILE82, THR85, GLU129, LEU132, and CYS142. The interaction of compounds with these residues was considered, and interactions other these residues were discarded.

Profiling of toxic characteristics

Toxic characteristics such as acute toxicity dose, organ-specific damage, and adverse effects prediction were predicted with GUSAR,[17] ROSC-Pred,[18] and Adver-Pred database.[19] These characteristics were determined by providing the canonical SMILES of compounds.

PASS and absorption, distribution, metabolism, excretion, and toxicity prediction

Other biological activities and pharmacokinetic attributes absorption, distribution, metabolism, excretion, and toxicity (ADMET) of compounds present in P. ginseng were also determined to explain their possible mechanism of action and safety attributes in the human body. These attributes were predicted by ADMET SAR 2.0,[20] and PASS online.[21]

 Results



Compounds selection in Panax ginseng

TCM database identified 215 medicinal compounds in P. ginseng. The information of these compounds was downloaded and screened for DL, oral BO, toxicity class, and Lipinski's rule of five. Parameters set for obtaining bioactive compounds were: DL >0.3, OB >20%, toxicity class >Class 4, and Lipinski's: no violations. Results of DL and OB were already indicated by TCMSP, but toxicity class and Lipinski's evaluation were facilitated by ProTox-II,[22] and SwissADME.[23] After refinement, six compounds [Table 1] qualified the criteria, which were further used for gene expression, target prediction, biological pathway governed by these targets, and enrichment and ontological analysis along with drug-target network association.{Table 1}

Network pharmacology analysis

Identification numbers of these compounds were procured from the PubChem database and added to the dialog box of BATMAN-TCM. The score cut off was kept at 15 for target prediction, whereas for target analyses, the adjusted P ≤ 0.05. In [Table 2], Fumarine (4970) influenced 35 genes, whereas Inermin on 14 genes. Nepetin, Celabenzine, Frutinone A, and Suchilactone had no potential effect on any target [Table 2]. Gene enrichment analysis of these genes showed that seven enriched KEGG pathways (RAS signaling pathway, Rap1 signaling pathway, PI3K-AKT signaling pathway, hematopoietic cell lineage, purine metabolism, cytokine-cytokine, and neuroactive ligand-receptor interaction, respectively) [Table 3]. Significantly enriched therapeutic target database (TTD) diseases regulated by these compounds were analgesics, chronic obstructive pulmonary disease, asthma, OS, ischemia, nausea, and vomiting, cough, dyspnea, B-lineage malignancies, allergic rhinitis, multiple organ failure, acute myelogenous leukemia, chronic urticaria, sustained ventricular tachycardia, chronic myeloproliferative disease, phyllodes tumors, macular edema, and other, as mentioned in [Table 4]. Gene ontological studies revealed that these two compounds have a role in initiating molecular functions such as signal transducer activity, kinase activity, and ion binding, as summarized in [Table 5]. Biological processes include circulatory system process, cellular protein modification process, lipid metabolic process, response to stress, signal transduction, cell-cell signaling, cell proliferation, and differentiation and locomotion, homeostatic process, anatomical structure development, and neurological system process. The compound target pathway/disease network obtained from BATMAN-TCM is illustrated in [Figure 1]. Gene expression induced by these compounds was explored with DIGEP-Pred. It was observed that Fumarine upregulates the CASP2 gene, whereas Inermin downregulated PCOLCE2 and STK39 and upregulated NOTCH1 and GAS6 genes [Table 6].{Table 2}{Table 3}{Table 4}{Table 5}{Figure 1}{Table 6}

Molecular docking results

We have discarded Suchilactone, Celabenzine, Nepetin, and Frutinone A from further analysis based on their inactivity in the network pharmacological analysis. Fumarine and Inermin were then used to check the interaction with CDKL3 protein, which is highly upregulated in OS cells and provides them with proliferative properties. The standard docking algorithm of IGEMDOCK was selected, which is comprised a population size of 200, generation: 70, and a number of docked solutions = 3. Site-directed docking was performed, and the results were characterized on hydrogen interaction of compounds with the active site of CDKL3 protein. IGEMDOCK analysis revealed that Fumarine [Figure 2] and Inermin [Figure 3] established hydrogen bonding with LYS33 amino acid of CSKL3 protein, indicating a similar mechanism of action [Table 7].{Figure 2}{Figure 3}{Table 7}

Toxicity profiling results

Fumarine has acute toxicity of 135,100 mg/kg for the intraperitoneal route, 22,960 mg/kg for the intravenous route, 616,500 mg/kg for the oral route, and 224,800 mg/kg for the subcutaneous route. The acute toxicity dose of Inermin was 237,100 mg/kg for the intraperitoneal route, 56,720 mg/kg for the intravenous route, 1,150,000 mg/kg oral and 306,100 mg/kg subcutaneous routes, respectively. These compounds are characterized as class 4 chemicals and the adverse and organ damaging effects associated with these compounds are given in [Table 8].{Table 8}

PASS and absorption, distribution, metabolism, excretion, and toxicity prediction

The biological function of these compounds was further explored with PASS online. Results indicated that Fumarine stimulates the activity of caspase 8,3 and promotes apoptosis and tumor suppressor gene-53 (TP53) expression. It is also an antineoplastic alkaloid and inhibits topoisomerase-I activity. A similar type of activity was observed for Inermin except for this compound also has topoisomerase-I and topoisomerase-II activity [Table 9]. ADMET SAR 2.0 was used to predict the compound's absorption, site of metabolism, and toxicity. Fumarine has a high blood–brain barrier (BBB) and intestinal absorption and its subcellular localization is lysosome. This compound is inactive against p-glycoprotein, CYP2C9, CYP3A4, and CYP2C9 whereas active for CYP2D6 substrate and inhibit CYP2C19, CYP2D6, and CYP1A2. It is nontoxic to cells and genes but possesses high hepatotoxicity. Inermin has high intestinal and BBB permeability and it is subcellularly localized in mitochondria. Like Fumarine, Inermin also has no activity for p-glycoprotein, CYP3A4, CYP2C9, CYP2C19, CYP2D6, and CYP1A2, respectively [Table 10]. This compound is relatively more toxic than Fumarine.{Table 9}{Table 10}

 Discussion



OS is multifactorial cancer which means this disease requires the activity of various genes to drive the growth of osteoblastic cells.[24] Recent studies emphasized inhibiting a single OS target to prevent tumor proliferation.[25],[26] However, cancer cells have devised various mechanisms to circumvent the inhibited protein target by stimulating other genes to further navigate the pathway to sustain the survival of the tumor.[8],[24] These mechanisms are also responsible for developing resistance, invasiveness, and migratory properties to OS cells. Therefore, single target targeting drugs becomes obsolete considering the complex evading mechanism of this disease. In recent years, network pharmacology has changed drug discovery research by merging artificial intelligence to correlate the interaction of therapeutic drugs with cellular networks and genes. This new discipline allowed researchers to understand the effect of drug interaction with various molecular targets and cellular networks. Natural compounds are constantly being repurposed for different types of diseases. However, these natural compounds are multimodal in action and influence multiple molecular targets which were overlooked previously prior to the discovery of network pharmacology. The emergence of this discipline streamlined the process of drug discovery and allowed scientists to thoroughly evaluate the therapeutic effects of a compound on a biological system.

In this study, we used network pharmacology, in silico gene expression, molecular docking, and ADMET method to elaborately analyze P. ginseng phytocompounds against OS. Initially, we obtained 215 compounds of P. ginseng from TCMSP, which on screening yielded 6 bioactive compounds such as Fumarine, Inermin, Fruitnone A, Celabenzine, Nepetin, and Suchilactone. These compounds were subjected to network pharmacology analysis to unravel their biological activity in OS.

BATMAN-TCM database facilitated the network pharmacology analysis and the results revealed that Fumarine and Inermin had a significant interaction with different biologically active targets, whereas Fruitnone A, Celabenzine, Nepetin, and Suchilactone failed to show any discerning activity. The predicted molecular target targeted by Fumarine was 35 and 13 for Inermin. These gene targets were subjected to gene enrichment studies to establish a significant association with different disease phenotypes and to recognize the molecular functions, cellular locations, and biological processes governed by these compounds. These compounds affect purine metabolism, which allows rapid tumor cell proliferation and growth in OS. Ras,[27] Rap1,[28] and PI3K-AKT signaling pathways,[29] are implicated in providing OS cells with invasive and migratory properties that are also targeted by these compounds. The therapeutic target database showed that these compounds have a significant therapeutic influence in alleviating pain, cancer, and other physiological ailments, including OS. Gene ontological analysis revealed that these compounds have a role in regulating biological and molecular functions along with some cellular functions.

We took these compounds and analyzed them with DIGEP-pred to evaluate the effects on mRNA gene expression, which might have been overlooked BATMAN-TCM algorithm. Fumarine upregulated the expression of the CASP2 gene, which is involved in inducing tumor cell apoptosis. Whereas Inermin reduced the expression of PCOLCE2 and STK39. Both these genes equip OS cells with rapid proliferative, migratory invasiveness, and metastatic properties,[30],[31] Moreover, this compound upregulates some other regulatory genes that help in the prevention of tumor growth invasiveness and sensitize these cells to chemotherapy.[32],[33],[34] From gene ontological analysis, it was observed that these compounds have a role in regulating cell proliferation and kinases activity. To further validate these findings, we used the molecular docking method to analyze the inhibitory effects on CDKL3 kinase which is involved in OS progression and proliferation.[35] These compounds were focused on the reported active site residue of CDKL3 kinase protein to determine the protein-ligand interaction. IGEMDOCK software was used, and the docking studies were performed three times to increase confidence in the obtained results. Both these compounds used the same amino acid residues LYS33 to establish hydrogen bonding with the CDKL3 protein. This interaction shows that Inermin and Fumarine have a similar mechanism of action.

Furthermore, we utilized the PASS algorithm to identify other biological activities. The results of PASS prediction predicted that these compounds stimulate the activity of caspase 3, and 8,[36],[37] and TP53.[38] These proteins induce tumor apoptosis and prevent tumor recurrence and growth. Besides these activities, they also interact with topoisomerase I and II that provide further evidence that these compounds also prevent DNA replication in OS cells.[39]

The safety and pharmacokinetic properties of a compound is a major component in drug discovery and development. Inadequate elucidation of these properties of a compound can jeopardize human health and may lead to serious harm during a clinical trial. To determine these properties, we elaborately analyzed each compound to increase its approval rating in different animal and clinical trials. These compounds are highly soluble and readily absorbed inside the gastrointestinal tract. However, these compounds pose a significant risk of causing hepatotoxicity and can affect the stomach and liver. These challenges can hinder their therapeutic efficacy and approval in various clinical trials. There are several methods reported in the literature to solve these challenges associated with these compounds, such as nanoformulations,[40] structural modification,[41] organic synthesis,[42] and drug concentration calibration.[43]

 Conclusion



Inermin and Fumarine present in P. ginseng have significant anticancer activity in OS cells. These compounds target both genes and other molecular drug targets to reduce the proliferation and aggressiveness of these tumors. However, the toxic nature of these compounds could jeopardize their therapeutic activity that can be a challenge for other researchers to work on. Our findings provided an elaborate insight about Inermin, and Fumarine in OS treat, which require further in vitro validation.

Financial support and sponsorship

This work was supported by the National Research Foundation of Korea (NRF) funded by the Korean Government (MEST) (2020R1I1A306969912).

Conflicts of interest

There are no conflicts of interest.

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