Integrating network pharmacology and in silico analysis deciphers Withaferin-A's anti-breast cancer potential via hedgehog pathway and target network interplay

Abstract

This study examines the remarkable effectiveness of Withaferin-A (WA), a withanolide obtained from Withania somnifera (Ashwagandha), in encountering the mortiferous breast malignancy, a global peril. The predominant objective is to investigate WA's intrinsic target proteins and hedgehog (Hh) pathway proteins in breast cancer targeting through the application of in silico computational techniques and network pharmacology predictions. The databases and webtools like Swiss target prediction, GeneCards, DisGeNet and Online Mendelian Inheritance in Man were exploited to identify the common target proteins. The culmination of the WA network and protein-protein interaction network were devised using Stitch and String web tools, through which the drug-target network of 30 common proteins was constructed employing Cytoscape-version 3.9. Enrichment analysis was performed by incorporating Gprofiler, Metascape and Cytoscape plugins. David compounded the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, and enrichment was computed through bioinformatics tools. The 20 pivotal proteins were docked harnessing Glide, Schrodinger Suite 2023-2. The investigation was governed by docking scores and affinity. The shared target proteins underscored the precise Hh and WA network roles with the affirmation enrichment P-value of <0.025. The implications for hedgehog and cancer pathways were profound with enrichment (P < 0.01). Further, the ADMET and drug-likeness assessments assisted the claim. Robust interactions were noticed with docking studies, authenticated through molecular dynamics, molecular mechanics generalized born surface area scores and bonds. The computational investigation emphasized WA's credible anti-breast activity, specifically with Hh proteins, implying stem-cell-level checkpoint restraints. Rigorous testament is imperative through in vitro and in vivo studies.

Keywords: in silico studies; breast cancer; computational analysis; hedgehog pathway; network pharmacology; phytoconstituents.

Figure 1

(A) Preliminary networks for the drug–target network construction. (a) Common target proteins obtained from Venny.2, (b) Network of WA. (c) PPI network of common target proteins. (B) Drug–target network of WA with common target proteins. (a) Drug–target network. (b) Enriched network highlighted with enrichment split doughnut pie charts.

Figure 2

KEGG enrichment pathway plots. (A) Bubble plot. (B) Enrichment bar plot. (C) Pathway enrichment category plot.

Figure 3

(A) Go pathway enrichment plots. (a) Bubble plot. (b) Enrichment bar plot. (B) Go pathway enrichment plots. (C) Pathway enrichment category plot.

Figure 4

Drug–target network—clusters.

Figure 5

Path view enrichment diagram for the Hedgehog pathway (Pathway ID: hsa04340) from KEGG pathways.

Figure 6

(A) Molecular docking image in 2D and 3D formats for the PRKC apoptosis WTI regulator protein (2JK9)-capecitabine complex. (B) Molecular docking image in 2D and 3D formats for the PRKC apoptosis WT1 regulator protein (2JK9)-WA complex. (C) Molecular docking image in 2D and 3D formats for the Indian hedgehog protein (3N1O)-TAM complex. (D) Molecular docking image in 2D and 3D formats for the Indian hedgehog protein (3N1O)-WA complex. (E) Molecular docking image in 2D and 3D formats for the Suppressor of fused (SUFU:4KMH)-TAM complex. (F) Molecular docking image in 2D and 3D format for the suppressor of fused (SUFU:4KMH) with WA complex.

Figure 7

(A) MD simulation analysis of 100 ns trajectories of (a) Cα backbone RMSD of 2JK9 bound to ligands CAP. WA. (b) RMSF of Cα backbone of 2JK9 bound to ligands CAP and WA. (c) Cα backbone Rg of 2JK9 bound to ligand CAP. WA. (d) Formation of hydrogen bonds in of 2JK9 bound to ligand TAM and WA. (e) Interactions of CAP. (f) Interactions of WA. (B) MD simulation analysis of 100 ns trajectories of (a) Cα backbone RMSD of 2VWE bound to ligand TAM and WA. (b) RMSF of Cα backbone of 2VWE bound to ligands TAM and WA. (c) Cα backbone Rg of 2VWE bound to ligands TAM and WA. (d) Formation of hydrogen bonds in 2VWE bound to ligands TAM and WA. (e) Interactions of TAM. (f) Interactions of WA. (C) MD simulation analysis of 100 ns trajectories of (a) Cα backbone RMSD of 3N1O bound to ligands TAM and WA. (b) RMSF of Cα backbone of 3N1O bound to ligands TAM and WA. (c) Cα backbone Rg of 4KMH bound to ligand TAM and WA. (d) Formation of hydrogen bonds in of 3N1O bound to ligand TAM and WA. (e) Interactions of TAM. (f) Interactions of WA. (D) MD simulation analysis of 100 ns trajectories of (a) Cα backbone RMSD of 4KMH bound to ligands TAM and WA. (b) RMSF of Cα backbone of 4KMH bound to ligands TAM and WA. (c) Cα backbone Rg of 4KMH bound to ligands TAM and WA. (d) Formation of hydrogen bonds in 4KMH bound to ligand TAM and WA. (e) Interactions of TAM. (f) Interactions of WA. (E) MD simulation analysis of 100 ns trajectories of (a) Cα backbone of 4WPB bound to ligands TAM and WA. (b) RMSF of Cα backbone of 4WPB bound to TAM and WA. (c) Cα-backbone Rg of 4WPB bound to ligands TAM and WA. (d) Formation of hydrogen bonds in 4WPB bound to ligands TAM and WA. (e) Interactions of TAM. (f) Interactions of WA.

Figure 7

(A) MD simulation analysis of 100 ns trajectories of (a) Cα backbone RMSD of 2JK9 bound to ligands CAP. WA. (b) RMSF of Cα backbone of 2JK9 bound to ligands CAP and WA. (c) Cα backbone Rg of 2JK9 bound to ligand CAP. WA. (d) Formation of hydrogen bonds in of 2JK9 bound to ligand TAM and WA. (e) Interactions of CAP. (f) Interactions of WA. (B) MD simulation analysis of 100 ns trajectories of (a) Cα backbone RMSD of 2VWE bound to ligand TAM and WA. (b) RMSF of Cα backbone of 2VWE bound to ligands TAM and WA. (c) Cα backbone Rg of 2VWE bound to ligands TAM and WA. (d) Formation of hydrogen bonds in 2VWE bound to ligands TAM and WA. (e) Interactions of TAM. (f) Interactions of WA. (C) MD simulation analysis of 100 ns trajectories of (a) Cα backbone RMSD of 3N1O bound to ligands TAM and WA. (b) RMSF of Cα backbone of 3N1O bound to ligands TAM and WA. (c) Cα backbone Rg of 4KMH bound to ligand TAM and WA. (d) Formation of hydrogen bonds in of 3N1O bound to ligand TAM and WA. (e) Interactions of TAM. (f) Interactions of WA. (D) MD simulation analysis of 100 ns trajectories of (a) Cα backbone RMSD of 4KMH bound to ligands TAM and WA. (b) RMSF of Cα backbone of 4KMH bound to ligands TAM and WA. (c) Cα backbone Rg of 4KMH bound to ligands TAM and WA. (d) Formation of hydrogen bonds in 4KMH bound to ligand TAM and WA. (e) Interactions of TAM. (f) Interactions of WA. (E) MD simulation analysis of 100 ns trajectories of (a) Cα backbone of 4WPB bound to ligands TAM and WA. (b) RMSF of Cα backbone of 4WPB bound to TAM and WA. (c) Cα-backbone Rg of 4WPB bound to ligands TAM and WA. (d) Formation of hydrogen bonds in 4WPB bound to ligands TAM and WA. (e) Interactions of TAM. (f) Interactions of WA.

Figure 7

(A) MD simulation analysis of 100 ns trajectories of (a) Cα backbone RMSD of 2JK9 bound to ligands CAP. WA. (b) RMSF of Cα backbone of 2JK9 bound to ligands CAP and WA. (c) Cα backbone Rg of 2JK9 bound to ligand CAP. WA. (d) Formation of hydrogen bonds in of 2JK9 bound to ligand TAM and WA. (e) Interactions of CAP. (f) Interactions of WA. (B) MD simulation analysis of 100 ns trajectories of (a) Cα backbone RMSD of 2VWE bound to ligand TAM and WA. (b) RMSF of Cα backbone of 2VWE bound to ligands TAM and WA. (c) Cα backbone Rg of 2VWE bound to ligands TAM and WA. (d) Formation of hydrogen bonds in 2VWE bound to ligands TAM and WA. (e) Interactions of TAM. (f) Interactions of WA. (C) MD simulation analysis of 100 ns trajectories of (a) Cα backbone RMSD of 3N1O bound to ligands TAM and WA. (b) RMSF of Cα backbone of 3N1O bound to ligands TAM and WA. (c) Cα backbone Rg of 4KMH bound to ligand TAM and WA. (d) Formation of hydrogen bonds in of 3N1O bound to ligand TAM and WA. (e) Interactions of TAM. (f) Interactions of WA. (D) MD simulation analysis of 100 ns trajectories of (a) Cα backbone RMSD of 4KMH bound to ligands TAM and WA. (b) RMSF of Cα backbone of 4KMH bound to ligands TAM and WA. (c) Cα backbone Rg of 4KMH bound to ligands TAM and WA. (d) Formation of hydrogen bonds in 4KMH bound to ligand TAM and WA. (e) Interactions of TAM. (f) Interactions of WA. (E) MD simulation analysis of 100 ns trajectories of (a) Cα backbone of 4WPB bound to ligands TAM and WA. (b) RMSF of Cα backbone of 4WPB bound to TAM and WA. (c) Cα-backbone Rg of 4WPB bound to ligands TAM and WA. (d) Formation of hydrogen bonds in 4WPB bound to ligands TAM and WA. (e) Interactions of TAM. (f) Interactions of WA.

Figure 7

(A) MD simulation analysis of 100 ns trajectories of (a) Cα backbone RMSD of 2JK9 bound to ligands CAP. WA. (b) RMSF of Cα backbone of 2JK9 bound to ligands CAP and WA. (c) Cα backbone Rg of 2JK9 bound to ligand CAP. WA. (d) Formation of hydrogen bonds in of 2JK9 bound to ligand TAM and WA. (e) Interactions of CAP. (f) Interactions of WA. (B) MD simulation analysis of 100 ns trajectories of (a) Cα backbone RMSD of 2VWE bound to ligand TAM and WA. (b) RMSF of Cα backbone of 2VWE bound to ligands TAM and WA. (c) Cα backbone Rg of 2VWE bound to ligands TAM and WA. (d) Formation of hydrogen bonds in 2VWE bound to ligands TAM and WA. (e) Interactions of TAM. (f) Interactions of WA. (C) MD simulation analysis of 100 ns trajectories of (a) Cα backbone RMSD of 3N1O bound to ligands TAM and WA. (b) RMSF of Cα backbone of 3N1O bound to ligands TAM and WA. (c) Cα backbone Rg of 4KMH bound to ligand TAM and WA. (d) Formation of hydrogen bonds in of 3N1O bound to ligand TAM and WA. (e) Interactions of TAM. (f) Interactions of WA. (D) MD simulation analysis of 100 ns trajectories of (a) Cα backbone RMSD of 4KMH bound to ligands TAM and WA. (b) RMSF of Cα backbone of 4KMH bound to ligands TAM and WA. (c) Cα backbone Rg of 4KMH bound to ligands TAM and WA. (d) Formation of hydrogen bonds in 4KMH bound to ligand TAM and WA. (e) Interactions of TAM. (f) Interactions of WA. (E) MD simulation analysis of 100 ns trajectories of (a) Cα backbone of 4WPB bound to ligands TAM and WA. (b) RMSF of Cα backbone of 4WPB bound to TAM and WA. (c) Cα-backbone Rg of 4WPB bound to ligands TAM and WA. (d) Formation of hydrogen bonds in 4WPB bound to ligands TAM and WA. (e) Interactions of TAM. (f) Interactions of WA.

Figure 7

(A) MD simulation analysis of 100 ns trajectories of (a) Cα backbone RMSD of 2JK9 bound to ligands CAP. WA. (b) RMSF of Cα backbone of 2JK9 bound to ligands CAP and WA. (c) Cα backbone Rg of 2JK9 bound to ligand CAP. WA. (d) Formation of hydrogen bonds in of 2JK9 bound to ligand TAM and WA. (e) Interactions of CAP. (f) Interactions of WA. (B) MD simulation analysis of 100 ns trajectories of (a) Cα backbone RMSD of 2VWE bound to ligand TAM and WA. (b) RMSF of Cα backbone of 2VWE bound to ligands TAM and WA. (c) Cα backbone Rg of 2VWE bound to ligands TAM and WA. (d) Formation of hydrogen bonds in 2VWE bound to ligands TAM and WA. (e) Interactions of TAM. (f) Interactions of WA. (C) MD simulation analysis of 100 ns trajectories of (a) Cα backbone RMSD of 3N1O bound to ligands TAM and WA. (b) RMSF of Cα backbone of 3N1O bound to ligands TAM and WA. (c) Cα backbone Rg of 4KMH bound to ligand TAM and WA. (d) Formation of hydrogen bonds in of 3N1O bound to ligand TAM and WA. (e) Interactions of TAM. (f) Interactions of WA. (D) MD simulation analysis of 100 ns trajectories of (a) Cα backbone RMSD of 4KMH bound to ligands TAM and WA. (b) RMSF of Cα backbone of 4KMH bound to ligands TAM and WA. (c) Cα backbone Rg of 4KMH bound to ligands TAM and WA. (d) Formation of hydrogen bonds in 4KMH bound to ligand TAM and WA. (e) Interactions of TAM. (f) Interactions of WA. (E) MD simulation analysis of 100 ns trajectories of (a) Cα backbone of 4WPB bound to ligands TAM and WA. (b) RMSF of Cα backbone of 4WPB bound to TAM and WA. (c) Cα-backbone Rg of 4WPB bound to ligands TAM and WA. (d) Formation of hydrogen bonds in 4WPB bound to ligands TAM and WA. (e) Interactions of TAM. (f) Interactions of WA.

Figure 8

Study-workflow representation.