Hepatocyte damage due to HBV infection and the pathogenesis of liver disease is not directly associated with the HBV, however it is caused by the immune-mediated host HBV interactions . The complex mechanisms that cause HBV-mediated liver disease are not fully understood due to the involvement of complex protein-protein interactions with multiple host factors . However, the utilization of already existing single drug molecules against a cluster of HBV mediated liver disease pathways may cause adverse drug effects via disrupting the host tissue mechanisms. The utilization of chemotherapeutic agents during HCC may alter hepatic metabolism, reduce the efficacy, or may cause liver toxicity by the disposition of the drug in liver tissue . Further, physicians utilize palliative treatments such as systemic chemotherapy, interferon, transarterial chemo-embolization, and hormonotherapy against HCC when there are no curative treatment options. However, the utilization of these therapies for HCC patients is challenging because HCC is highly resistant to systemic therapies . In the current study, we elucidated the molecular mechanisms and probable safe use of traditionally used herbs of Western Ghats, Karnataka against jaundice, fever, inflammation, and viral infections via cluster and enrichment analysis of potential HBV disease protein targets that participate in the progression of immune processes and inflammatory responses. The results obtained from the current study predicts that flavonoids and diterpenoids from A. paniculata and flavonoids from T. populnea could potentially prevent the disease progression and could be effective therapeutics against chronic HBV-induced HCC.
Identification of potent candidates from herbal medicines is not only the key focus of new drug discovery against HBV-induced HCC, drug safety is also a key concern. This means that ensuring that the drugs have a positive benefit-risk balance is the goal . Hepatotoxicity induced by herbs is one of the most important factors limiting treatment. The issue has often been responsible for post-marketing warnings and withdrawal . Many researchers have well documented the adverse hepatic reactions, idiosyncratic liver damage caused by herbs. The major hepatotoxic herbs reported so far are, Teucrium chamaedrys, Larrea tridentate, Cimicifuga racemosa, Scutellaria lateriflora, and Scutellaria baicalensis . In the current study, we screened 274 phytocompounds from 16 herbs for their druggable characteristics using the Lipinski rule of five and selected only compounds having positive drug-like properties. Further, these compounds were screened for major adverse drug reactions/ side effects i.e. Hepatotoxicity, Cardiac toxicity, Nephrotoxicity, and potential to cause Myocardial infraction using the ADVERpred online prediction. To minimize the toxicity, compounds having a probable toxic activity (Pa) with a p-value ≤ 0.5 were considered. Further, compounds were also predicted for their probable affinity towards potential drug targets using BindingDB (p-value ≥ 0.7) and also segregated the target associated with HBV infection and HCC with the aid of published literature, KEGG pathway, and Therapeutic Target Database (TTD).
STRING database was also utilized to identify the molecular functions, biological processes, and pathways regulated by the predicted targets, Wherein, the targets were meticulously chosen soley based on potential molecular functions, processes, and pathways with higher significance (p ≤ 0.05) and those that are only associated with HBV-induced HCC. Based on the results obtained from STRING, we constructed compound-protein-pathway network building to understand the relations between compounds, target proteins, and pathways. It is important to consider that one pathway containing many protein molecules modulated by one compound is more essential than the function of one protein that involves many pathways modulated by one drug molecule. The impact of one target in numerous pathways may be less, and the impact of one pathway having multiple protein targets modulated by the one compound could be large . Based on this theory, we chose 11 phytocompounds viz., Andrographidine C, 5-Hydroxy-7,8,2'-Trimethoxyflavone (Andrographin), Skullcapflavone I, 2'-hydroxy-5,7,8- trimethoxyflavone, 5,4'-dihydroxy-7,8,2',3'- tetramethoxyflavone, 5-hydroxy-7,8-dimethoxyflavone, 5-hydroxy-7,8,2',5'- tetramethoxyflavone, 5-hydroxy-7,8,2',3',4'- pentamethoxyflavone, Wightin, 5-Hydroxy-7,8,2',3'-Tetramethoxyflavone, and Altisin from A. paniculata [majorly are of Andrographolide derivatives] contained in A. paniculata whole plant and Gossypetin, Herbacetin and Kaempferol-7-Glucoside contained in T. populnea flowers . These phytocompounds were identified as flavonoids and diterpenoids and were found to modulate thirteen protein molecules involved in the progression of HBV infection to HCC i.e. CDK1, CDK2, CDK6, EGFR, HGF, IGF1R, TERT, STAT3, PRKCA, PRKCB, PRKCG, AKT1, and TNF. Further, these protein targets were also found to modulate 14 major molecular pathways i.e. Hepatitis B pathway, Hepatocellular carcinoma, MicroRNAs in cancer, MAPK signaling pathway, PI3K-Akt signaling pathway, Ras signaling pathway, mTOR signaling pathway, Apoptosis, Viral carcinogenesis, TNF signaling pathway, Jak-STAT signaling pathway, p53 signaling pathway, and Cell cycle. The role of each protein target associated with the identified pathways involved in the pathogenesis of HBV infection and progression of HCC is shown in Supplementary Table 8.
After gene ontology, enrichment, and network analysis, we further extended the study to examine the intermolecular interactions between phytocompounds and predicted protein targets using docking and MD simulation studies. As a result, we have chosen EGFR, a therapeutic target for cancer (identified to be involved in the MAPK, PI3-Akt, Ras, and Jak-STAT signaling pathways within the network). Earlier studies also have demonstrated that overexpression of EGFR to be common in HCC, meaning that it can play a role in the pathogenesis and treatment of the disease. From early inflammation and hepatocellular proliferation to fibrogenesis and neoplastic transformation, the EGFR signaling mechanisms have been recognized as a central player in all stages of the liver response to injury [30, 31]. Schiffer et al  reported Gefitinib, a potent EGFR tyrosine kinase inhibitor to curtail HCC development in rats exposed to diethylnitrosamine. Further, Zheng et al  reported that, Tropomodulin 3 levels to rise in HCC condition, especially when it spreads outside the liver. Tmod3 expression was also linked to aggressive malignancy and poor patient survival in HCC patients . Tmod3 enhances matrix metalloproteinase-2, -7, and − 9 transcription, which is dependent on PI3K-AKT. The interactions between Tmod3 and the EGFR, which promotes EGFR phosphorylation is required for signalling activation of PI3K-Akt. Similarly, VersicanV1 was also found to enhance EGFR–PI3K–Akt signalling pathway in HCC cells . A recent study by Liu et al  demonstrated that, in HCC cells, the EGFR-P38 MAPK axis may up-regulate PD-L1 via miR-675-5p and down-regulate HLA-ABC via HK2 and this might be responsible for the immune suppression in HCC, and they suggest that EGFR signalling might be targeted for HCC immunotherapy. Hence, based on the potential role of EGFR in HCC and also key network hub status in the current study, the phytocompounds were screened against EGFR. First, the WT EGFR x-ray crystallographic protein structure was obtained from the PDB and the missing residues were fixed by homology modelling. The structural stability of the best model was confirmed by molecular dynamics for 50ns production run. The least potential energy conformation from MD simulation was retrieved for utilization in docking studies. The molecular docking of 11 compounds from A. paniculta and 3 from T. populnea showed significant intermolecular interactions with active site residues. Further, based on the BE and highest interactions with active site residues, 5-Hydroxy-7,8,2'-Trimethoxyflavone (Andrographin) from A. paniculata and Gossypetin (T. populnea) were identified as lead hits against WT EGFR. On post docking studies, the MD method was used to delineate the effects of protein structure stability on ligand binding. To explain the structural modification of the protein upon ligand binding, we run a three replicates of 100ns MD simulation for Andrographin and Gossypetin with WT EGFR, to ensure the plausibility of complex formation. The protein-ligand interactions RMSD was found to be ~ < 0.8Å and formed stable interactions throughout 100ns production run in all three replicates.
Andrographis paniculata and Thespesia populnea
Andrographis paniculata Nees. is known as a “king of bitters”, a member of an Acanthaceae family, and native to India and Sri Lanka. In Ayurveda, the roots and leaves are used for medicinal purposes . A. paniculata is a key ingredient of many herbal formulations for the treatment of liver disease, hepatitis, diabetes, cancer, etc [47, 48]. A previous study by Tan et al  demonstrated Andrographolide to downregulate EGFR expressed in epidermoid carcinoma (A-431) cells. Andrographolide and Neoandrographolide from A. paniculata showed a protective effect against tert-butylhydroperoxide intoxication induced hepatotoxicity in mice [50, 51], and protective activity against galactosamine induced hepatotoxicity in rats (Handa et al. 1990b). The crude extract of A. paniculata showed potent inhibitory activity against HBV surface antigen . Dehydroandrographolide and Andrographolide showed potent inhibitory activity against HBV DNA replication . Neoandrographolide is a potent anti-inflammatory compound that acts via inhibition of iNOS and COX-2 expression through inhibiting p38 MAPKs activation . It also suppresses NO production in LPS activated macrophages .
Thespesia populnea Soland ex. Correa belongs to the family Malvaceae. It is mainly found in tropical regions and the coastal forests of India. Ayurvedic physicians use bark decoction for the treatment of skin and liver diseases. The bark and flower possess hepatoprotective, antioxidant, anti-inflammatory, memory improving, cholesterol-lowering activities, and have been reported for the management of Alzheimer's disease . In the current study, we identified Gossypetin, Herbacetin, and Kaempferol-7-Glucoside flavonoids from T. populnea flower as potent hepatoprotective hits against HBV-induced HCC. It supports earlier reports of these compounds as potent antiviral flavonoids [41, 57, 58]. Arthanari et al had also demonstrated that methanolic flower extracts of T. populnea have a strong antiviral potency and possessed nontoxic properties .
The idea of modulating multiple proteins by a single molecule has triggered researcher’s interest in finding the first strike on specific targets. Phytocompounds from A. paniculata and T. populnea have also been shown to have antiviral and hepatoprotective properties in the past. As a result, the current research discovered 11 compounds from A. paniculata and three from T. populnea that may have effectiveness against hepatocellular carcinoma caused by the HBV virus, and also further prompted us to investigate the binding affinity and intermolecular interactions of these compounds with active site residues of EGFR, a well-known cancer therapeutic target. Although the identified small molecules possess druggability and non-toxic profile, which ensures safety cum efficacy.