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. 2023 Nov;30(11):103804.
doi: 10.1016/j.sjbs.2023.103804. Epub 2023 Sep 6.

In-vitro and computational analysis of Urolithin-A for anti-inflammatory activity on Cyclooxygenase 2 (COX-2)

Archana G Revankar  1 , Zabin K Bagewadi  1 , Ibrahim Ahmed Shaikh  2 , Basheerahmed Abdulaziz Mannasaheb  3 , Mohammed M Ghoneim  3 , Aejaz Abdullatif Khan  4 , Syed Mohammed Basheeruddin Asdaq  3
Affiliations

In-vitro and computational analysis of Urolithin-A for anti-inflammatory activity on Cyclooxygenase 2 (COX-2)

Archana G Revankar et al. Saudi J Biol Sci. 2023 Nov.

Abstract

Cyclooxygenase 2 (COX-2) participates in the inflammation process by converting arachidonic acid into prostaglandin G2 which increases inflammation, pain and fever. COX-2 has an active site and a heme pocket and blocking these sites stops the inflammation. Urolithin A is metabolite of ellagitannin produced from humans and animals gut microbes. In the current study, Urolithin A showed good pharmacokinetic properties. Molecular docking of the complex of Urolithin A and COX-2 revealed the ligand affinity of -7.97 kcal/mol with the ligand binding sites at TYR355, PHE518, ILE517 and GLN192 with the 4-H bonds at a distance of 2.8 Å, 2.3 Å, 2.5 Å and 1.9 Å. The RMSD plot for Urolithin A and COX-2 complex was observed to be constant throughout the duration of dynamics. A total of 3 pair of hydrogen bonds was largely observed on average of 3 simulation positions for dynamics duration of 500 ns. The MMPBSA analysis showed that active site amino acids had a binding energy of -22.0368 kJ/mol indicating that throughout the simulation the protein of target was bounded by Urolithin A. In-silico results were validated by biological assays. Urolithin A strongly revealed to exhibit anti-inflammatory effect on COX-2 with an IC50 value of 44.04 µg/mL. The anti-inflammatory capability was also depicted through reduction of protein denaturation that showed 37.6 ± 0.1 % and 43.2 ± 0.07 % reduction of protein denaturation for BSA and egg albumin respectively at 500 µg/mL. The present study, suggests Urolithin A to be an effective anti-inflammatory compound for therapeutic use.

Keywords: Anti-inflammatory; Cyclo-oxygenase 2; Molecular Dynamics simulation; Urolithin A.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
3D Structure of human Cyclooxygenase −2 protein.
Fig. 2
Fig. 2
2D structures of Redock of COX-2 with RCX ligand and Docking with Urolithin A.
Fig. 3
Fig. 3
Molecular Docking of Urolithin A in the binding site of Cyclooxygenase −2 (PDB ID: 5KIR) visualized using Pymol Software a) Cartoon diagram of COX-2 chain A (Blue colour) with ligand (Magenta colour); b) Surface diagram of COX-2 chain A (Grey colour) with ligand (Magenta colour).
Fig. 4
Fig. 4
Pymol Visualization of Interacting Amino acids of Molecular Docking of Urolithin A against Human Cyclooxygenase-2. Hydrogen Bonds are represented by yellow dots and 2D interaction. The Ligand is seen bounded to hydrophobic pocket residues PHE518, ILE 517, bounded by GLN192 and TYR355 at entry of active site of protein.
Fig. 5
Fig. 5
RMSD plot for the APO and Protein-Ligands [Urolithin-A, STD] complex of COX-2 protein for 500 ns of simulation time.
Fig. 6
Fig. 6
RMSF plot of amino acid residues in the APO and Protein- Ligand [Urolithin A, STD] complexes.
Fig. 7
Fig. 7
Radius of gyration plot showing the compactness of APO, Urolithin A, and STD complex throughout the simulation duration.
Fig. 8
Fig. 8
Plot of SASA value vs time for Cyclooxygenase 2- APO, Urolithin A, and STD complex.
Fig. 9
Fig. 9
Number of Hydrogen bonds formed for the trajectory of MD simulation for Cyclooxygenase-2 enzyme with Urolithin A and STD.
Fig. 10
Fig. 10
Active Site-level interaction energy (Binding energy) from MM-PBSA in the simulation.
Fig. 11
Fig. 11
Anti- inflammatory activity of Urolithin A by In-vitro COX-2 assay.
Fig. 12
Fig. 12
Anti-inflammatory activity of Urolithin A by Reduction of protein denaturation using BSA and egg albumin.
See this image and copyright information in PMC

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