Embelin targets PI3K/AKT and MAPK in age-related ulcerative colitis: an integrated approach of microarray analysis, network pharmacology, molecular docking, and molecular dynamics

Vaibhdang, an Ayurvedic treatment for Crohn’s and UC, has been used for centuries. The main component of Vaibhdang is embelin derived from Embelia ribes. However, the pharmacological and molecular mechanisms of embelin in UC remain unclear. This study investigated the molecular targets and mechanisms of action of embelin in UC using microarray analysis, network pharmacology, molecular docking, and molecular dynamics simulations. Embelin targets were obtained by Swiss Target, TargetNet, STITCH, ChEMBL, and TCMSP. Ulcerative colitis targets were mapped using DisGenNET, Genecards, TCMSP, Therapeutic targets, and GEO databases (GSE87466). Co-targets between ulcerative colitis and embelin were identified, and a PPI network was constructed using the STRING database. To identify the core targets, we used Cytoscape to analyze the topology of the PPI network. There were 545 effective Embelin targets and 5171 effective ulcerative colitis targets, including 1470 DEG targets. ShinyGo and AutoDock were used to analyze GO and KEGG enrichment pathways and docking studies, respectively. Venn diagram analysis revealed 327 core targets of embelin in UC. An enrichment study showed that embelin is involved in PI3K-AKT, MAPK, RAS, and chemokine signalling. The top ten core targets docked with embelin and AKT1, MAPK1, and SRC complexes were utilized as representations and simulated using GROMACS for 100 ns. A comparison of native proteins and their complex interactions with embelin revealed that embelin might act on various PI3K/AKT and MAPK targets to treat ulcerative colitis. This study provides insights into the molecular targets and mechanisms of action of embelin against ulcerative colitis. Communicated by Ramaswamy H. Sarma</p

A) Ball and Stick representation of 4PBA; Carbon = Grey, Hydrogen = Cyan, Oxygen = Red b) Cartoon model of HSA-MYR complex (PDB: 2BXP) showing different subdomains and major fatty acid binding sites.

<p>A) Ball and Stick representation of 4PBA; Carbon = Grey, Hydrogen = Cyan, Oxygen = Red b) Cartoon model of HSA-MYR complex (PDB: 2BXP) showing different subdomains and major fatty acid binding sites.</p

Ligand displacement assay of HSA with Quercetin, 4PBA.

<p>a) Percentage initial fluorescence of HSA (at 345 nm) upon addition of 4PBA, Palmitic acid and Quercetin (2–20 μM). b) Binding of 4PBA and Quercetin at different sites. Tryptophan fluorescence of HSA was monitored at 345 nm in the presence of 4PBA. To HSA-PBA complex (of varying PBA concentration: 2–12 μM), Quercetin was added from 2–20 μM and c) To HSA-Quercetin complex (of varying Quercetin concentration: 2–12 μM), PBA was added from 2–12 μM. HSA fluorescence was normalized to 100% in the absence of added ligands.</p

A) Ball and Stick representation of 4PBA; Carbon = Grey, Hydrogen = Cyan, Oxygen = Red b) Cartoon model of HSA-MYR complex (PDB: 2BXP) showing different subdomains and major fatty acid binding sites.

<p>A) Ball and Stick representation of 4PBA; Carbon = Grey, Hydrogen = Cyan, Oxygen = Red b) Cartoon model of HSA-MYR complex (PDB: 2BXP) showing different subdomains and major fatty acid binding sites.</p

Motion of Cα atoms for the extreme values of the principal components obtained from MD simulation trajectory.

<p>a-f) represents the motion of Cα atoms of FA site 1 to 6- 4PBA occupied complex respectively.</p

MM-PBSA Binding Free Energy components of HSA-4PBA complex.

<p>All energy values are expressed in KJM^-1.</p><p>*LJ = Lennard Jones Potential</p><p>*Coul = Coulombic Charge</p><p>∆G _{<i>binding</i>} was calculated from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133012#pone.0133012.e001" target="_blank">Eq 1</a></p><p>MM-PBSA Binding Free Energy components of HSA-4PBA complex.</p

Stability of 4PBA at FA binding sites of HSA.

<p>a) Time evolution of RMSD of the HSA backbone and PBA bound forms during 7ns MD simulation of 4PBA bound to HSA at different FA binding sites. b) Interaction profile of 4PBA at all FA binding sites c) Salt bridge formation at FA3.</p

Binding of 4PBA induces conformational changes on HSA.

<p>a) RMSF of Cα atoms of Unliganded HSA (discontinuous black lines) and 4PBA bound HSA at different FA binding sites. The demarcations show different Subdomains of HSA. Significant fluctuations can be seen at Subdomain IA and IIIB; the most mobile and hydrophobic fragments of HSA. b) 2D projection of first two principal components of different 4PBA-HSA bound models. c) Spectrum of Eigenvalues vs Eigenvector Index. d) CD absorption spectra of HSA-4PBA complex (HSA-1 μM; 4PBA-1μM).</p

Total and Residue Hydrophobicity (SASA analysis).

<p>*Values are expressed in nm²</p><p>Total and Residue Hydrophobicity (SASA analysis).</p

Displacement of Dansylglycine by 4PBA and Palmitic acid.

<p>4PBA and Palmitic acid displacement of Dansylglycine. To HSA (1 μM) and Dansylglycine (1 μM) complex, 4PBA and Palmitic acid were added incrementally from 1–8 μM.</p