Research Article |
Corresponding author: Liliia Vyshnevska ( realmanutd.ua@gmail.com ) Academic editor: Plamen Peikov
© 2022 Liliia Vyshnevska, Hanna I. Severina, Yuliya Prokopenko, Alexander Shmalko.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Vyshnevska L, Severina HI, Prokopenko Y, Shmalko A (2022) Molecular docking investigation of anti-inflammatory herbal compounds as potential LOX-5 and COX-2 inhibitors. Pharmacia 69(3): 733-744. https://doi.org/10.3897/pharmacia.69.e89400
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According to the World Journal of Gastroenterology, more than 5 million people worldwide suffer from inflammatory bowel disease. The use of phytotherapeutic remedies in treatment of chronic inflammatory processes can be an effective alternative in patient’s therapy. The advantage of herbal medicines is the ability to influence various links of pathogenesis, lack of addiction, and the absence of withdrawal syndrome with long-term use in chronic pathology. In order to develop a new combined remedy with anti-inflammatory activity for the treatment of colitis, thirteen herbs, which are used in official or traditional medicine in inflammatory processes, were selected among the Ukrainian flora members. To select the most promising drugs and optimize further pharmacological research, molecular docking of the main active substances of the selected herbs to the fundamental pro-inflammatory enzymes – lipoxygenase-5 (LOX-5) and cyclooxygenase-2 (COX-2) – was carried out. Native inhibitors AKBA and celecoxib, respectively, were used as the reference ligands. The selection of candidate structures for in silico research was carried out according to the bibliosemantic research and logical-structural analysis concerning anti-inflammatory effect of the substances, which are part of chemical composition of the selected herbs. Molecular docking results have shown a high affinity level for the active site of the LOX-5 inhibitor gallotannin, quercetin, inulin, sitosterine, and moderate for ellagic acid. High affinity level for the active site of the COX-2 inhibitor was found for inulin, quercetin, gallotannin, ellagic acid and urticin A, moderate one – for gallic acid. For the further pharmacological in vitro and in vivo studies for anti-inflammatory activity, medicinal herbs with the highest content of the mentioned compounds were selected: Inula helenium, Cichorium intybus, Capsella bursa-pastoris, Foeniculum vulgare, Equisetum arvense, Veronica officinalis. Besides, it is recommended to use aqueous extracts of the selected herbs for the further pharmacological studies.
molecular docking, herbal remedy, anti-inflammatory, LOX-5, COX-2
According to the World Journal of Gastroenterology, more than 5 million people worldwide suffer from inflammatory bowel disease. The choice of drugs with pronounced pharmacological activity, minimal side effects and the possibility of long-term use, including in chronic diseases, is one of the most important problems in therapy (
At the beginning of the study, literature sources concerning the use of medicinal herbs in treatment of inflammatory bowel disease were analyzed. It was found that Foeniculum vulgare (
Structural features and biological activity of natural polysaccharides determine their efficiency for the treatment of inflammatory bowel disease. These compounds have positive effects on the intestinal microbiota; they regulate formation of cytokines involved in inflammation development, restore the intestinal epithelial barrier (
Although etiology and pathogenesis of inflammatory bowel disease remain unknown, therapy includes the use of anti-inflammatory drugs together with an anti-inflammatory diet (
Anti-inflammatory effect of herbal polyphenols has long been known (
Molecular anti-inflammatory activity of flavonoids is realized by transcription factors inhibition, such as nuclear factor-kappa B NF-kB (NF-kB) and activating protein-1 (AP-1), which control immune and inflammatory response expression (Serafini et al. 2008). It has also been found that some flavonoids may reduce expression of proinflammatory cytokines, in particular tumor necrosis factor α (TNF α) (
Besides, the role of natural polyphenols in inflammatory bowel disease correction is recognized and characterized by many molecular mechanisms of anti-inflammatory effect, including the above mentioned (
In order to develop a new combined remedy with anti-inflammatory activity for treatment of colitis, the aim of the given research was molecular docking of the main active ingredients of herbal material to biotargets – lipoxygenase-5 (LOX-5) and cyclooxygenase-2 (COX-2), as fundamental proinflammatory enzymes. The study will optimize further pharmacological screening and predict any possible mechanism of pharmacological action. In addition, the obtained results of molecular docking can substantiate the choice of markers for standardization of the finished product or herbal material, explain pharmacological features and optimize the complex herbal remedy composition.
Undoubtedly, therapeutic effect of herbs is achieved through the synergism of different groups of natural compounds, but the problem of insufficient information about the pharmacological mechanisms of individual substances remains relevant (
13 herbal species from Ukrainian flora were selected for the study. According to the results of bibliosemantic research, the selected herbs are used in official or traditional medicine.
The hardware used is the ASUS VivoBook X530UN S15 with an Intel Core i7 8550U 8thGen and Windows 10, 64-bit operating system. Flexible molecular docking was carried out using Vina and AutoDockTools 1.5.6 software (
Ligands preparation. BIOVIADraw 2017R2 tool was used for ligand structures base construction. The structures were saved as .mol files, were optimized by Chem3D software using molecular-mechanic MM2 algorithm and then saved in .pdb format. Then, AutoDockTools-1.5.6 was used to convert the files in .pdbqt format (
Proteins preparation. Discovery Studio Visualizer 2017/R2 was used to remove the solvent and native ligand from the crystal. The proteins were saved as .pdb files. Proteins were saved as .pdb files. AutoDockTools-1.5.6. was used to add polar hydrogen to the protein structure and to save the data as .pdbqt files.
Grid box size, as well as its center, were determined according to the native ligand.
Lipoxygenase-5 (LOX-5) (PDB ID 6NCF) x = 11.6, y = -23.38, z = -18.01; size x = 30, y = 28, z = 26; Cyclooxygenase-2 5 (PDB ID 3LN1) x = 18,84, y = -52,89, z = -53,81; size x = 22, y = 24, z = 24.
AutoDock Vina software was used for molecular docking.
To validate the docking method, the reference ligands – AKBA and Celecoxib – were extracted and then reused for the redocking process after given charged, set torque and saved as .pdbqt. RMSD value is the quantitative characteristic of the technique’s validity, which characterizes higher probability of a successful docking result. RMSD was calculated using ProFit Results outsource. It was 2.023 A for AKBA and 1.987 A for celecoxib between experimental and reference conformation of the substances.
Visualization and analysis of the obtained results of the docking studies were carried out using Discovery Studio V17.2.0.16349.
The general algorithm of research on rationalization of development of phytotherapeutic remedy with anti-inflammatory activity is shown in Fig.
The selection of candidate structures for in silico research was carried out according to the bibliosemantic research and logical-structural analysis concerning anti-inflammatory effect of the substances, which are part of chemical composition of the selected herbs. The structures of the main biologically active substances from the groups of terpene compounds, polyphenols and polysaccharides are shown in Fig.
5-LOX enzyme was chosen as the main biotarget for the search of the herbal compounds’ anti-inflammatory action, due to the mentioned compound initiates biosynthesis of powerful inflammatory mediators – leukotrienes. Therefore, the search for selective inhibitors of this enzyme continues and remains relevant. Although the human enzyme LOX-5 macromolecule was isolated in 2011 (
catalytic domain: Ile126, His130, Lys133, Glu134, Thr137, Arg138; amino terminal domain: Arg68, Leu66, Glu108, Val110, Val109, Arg101.
To evaluate the efficiency of the used methodology and the docking parameters in the experimental data reproduction, the native АКВА ligand was docked into the LOX-5 allosteric site, and the affinity was – 9.1 kcal/mol. Reproducibility of placement into the active site, as well as amino acid residues interactions, is shown in Fig.
Docking results of the studied substances into the LOX-5 active site inhibitor.
Ligand | Biotarget – LOX-5 enzyme | |||
---|---|---|---|---|
Binding energy kcal/mol | Hydrophobic interactions | Hydrogen bonds | Other interactions | |
АКВА reference ligand | -10.0 | Val110 (3), Lys133 Ile126, His125*, His130(3) | Val110, Arg138(2), Val109 | Arg101(2)Pi-Cation |
Anethole cys-/ trans- isomers | -5.0/-5.0 | Lys133(2), Glu134, His130 | Arg101 | – |
Apiol | -5.9 | Pro164*, Val107*, Tyr142, Arg138 | Arg101, Arg138, Glu134, Thr137 | Arg101(2) Pi-Cation |
Carvone R/S | -5.0/-5.1 | Val107*, Pro164*, Arg165*, Arg101 | Arg165*, Asp166 | – |
Petroselinic acid | -3.8 | – | Arg138(2) | – |
Coumarin | -5.9 | Ala388(4)*, Val389(3)*, Leu111, Pro98*, Arg101(2) | Lys394*, Arg101 | Glu134 Pi-Anion |
Umbelliferone | -6.4 | Ala388(3)*, Val389(3)*, Leu111, Pro98*, Arg101 | Lys394*, Arg101 | Glu134 Pi-Anion |
Gallic acid | -6.8 | Ala388*, Arg112*, Val389* | Lys394(2)*, Arg112(2)*, Glu134(2), His130, | – |
Ellagic acid | -7.8 | Thr137, Val107 (2) | His130, Thr137(4) | – |
Quercetin | -8.2 | Val107*, Val110(2), Thr137 | His130(2), Glu134(2), Arg138, Asp166* | Arg101(2) Pi-Cation |
Gallotannin | -9.4 | Thr137, Val110(2) | Arg68, Arg101, His130, Glu108, Thr137 | Arg101(2) Pi-Cation |
Urticin А | -8.0 | Val109(2)*, Arg138 | Thr104*, Thr137, Arg138, Glu134, His130, Trp102*, Asp166*, Gln141* | – |
Urticin В | -7.9 | Val109(2), Arg138 | Thr137(3), Arg138, Tyr142*, Trp102* | Asp166 Pi-Anion |
Sitosterine | -8.3 | Val110 (4), Lys133, Leu66, Arg68, Val107(2)*, Lys133, His130 | – | – |
Inulin | -8.9 | Val110 | Arg68, Arg101, Thr104*, Val110, His130(2), Thr137(2), Trp102(2)*, Glu134(2), Asp166(2)* | – |
All necessary native interactions and one additional connection with imidazole cycle of histidine (His125) are visualized. However, the presence of interaction with isoleucine residue (Ile126) testifies the identity of the conformational arrangement relative to the native position. Docking results of the mentioned biologically active substances were evaluated according to the binding energy parameter (kcal/mol) relative to the reference ligand, according to the type and amount of interaction with amino acid residues of the active site, and according to the spatial position in the hydrophobic pocket cavity.
Compounds that are part of the essential oils – anethole, apiole, carvone – also have shown high scoring function values of about -5.0 to -5.9 kcal/mol, as well as unfavorable conformational arrangement for allosteric modulation: fixation of “small” ligands occurs at the entrance to hydrophobic pocket (interaction with Arg101, Arg138), obvious inability of deeper immersion, and majority of the interactions happen with adjacent amino acid residues that are not part of the active site (Table
The inability to penetrate the allosteric LOX-5 site was predicted for petroselinic acid: high scoring function -4.8 kcal/mol and no hydrophobic interactions were discovered. Coumarin and umbelliferone docking has resulted almost in the same manner. Despite the large number of hydrophobic interactions (Table
Phenolic acids have shown a moderate inhibitory effect probability on LOX-5, in particular gallic acid having scoring function of -6.8 kcal/mol. For ellagic acid, although formation of hydrogen bonds trihedral network with threonine (Thr137) hydroxy group and histidine carboxyl group remains possible (Table
Quercetin shows its spatial arrangement similar to ellagic acid with fixation in the front of the pocket (Fig.
Gallotannin has shown the highest affinity for the AKBA inhibitor site – the scoring function value was -9.4, versus -10.0 kcal/mol for the reference ligand. In terms of interactions with amino acids, a large amount of hydrogen ones, additionally stabilizing all fragments position of gallotannin molecule, has been discovered together with the hydrophobic bonds. Concerning co-location with the native ligand, a high affinity for the AKBA site becomes apparent, as the molecule is completely and deeply immersed into the active site, occupying almost identical to the native ligand spatial position (Fig.
Utricin А and В glycosides have shown quite a significant affinity level for LOX-5 inhibitor site – scoring function was -8.1 and -7.9, relatively. For both compounds, interaction with amino acid residues of the active site was predicted, but in conformation with the native ligand, inability to dive into the narrow pocket and pass beyond it, with fixation due to interaction with adjacent amino acid residues, becomes apparent (Fig.
A high affinity level was predicted for LOX-5 allosteric site in sitosterine: its’ binding energy was -8.3 kcal/mol. Compatible conformation with the native ligand (Fig.
High affinity level was also predicted for inulin (-8.9 kcal/mol) with an extensive hydrogen bonds network of all fragments of the molecule with amino acids of the active site (Fig.
Cyclooxygenase-2 (COX-2) became the second bio-target for predicting possible mechanism of anti-inflammatory effect realization by complex herbal preparation components. The enzyme COX-2 homotetrameric macromolecule consists of two homodimers, each of which contains a binding site of a high-affinity selective inhibitor – celecoxib (
Docking results for the studied natural compounds into the СОХ-2 inhibitor active site.
Ligand | Biotarget – COX-2 enzyme | ||
---|---|---|---|
Binding energy kcal/mol | Hydrophobic interactions | Hydrogen bonds | |
Celecoxib reference ligand | -12.1 | Val335(2), Ser339, Val509(2), Leu370, Leu345, Leu517, Tyr371, Tyr341, Trp373, Ala513(2) | Arg106, Arg499, Gln178, Leu338, Ser339 |
Anethole, cys-/trans-isomers | -6.3/-6.2 | Val509(2), His75*, Leu338 | – |
Apiol | -7.5 | Val335(3), Ala513, Val509(2), Tyr341, Tyr371, Trp373, Phe504*, Leu338, Ala513, Leu517 | Ser516 |
Carvone R/S | -6.7/-6.8 | Val509(2), Ala513, Leu338(3), Val335, Tyr371, Trp373, Phe504* | – |
Petroselinic acid | -7.0 | Ala513, Val509, Tyr341 | Arg499, GLn178, Leu338 |
Coumarin | -7.2 | Val509(2), Leu338(2), Ser339(2), Leu338(2), Leu338, Ala502* | – |
Umbelliferone | -7.2 | Val509(2), Leu338 (2), Ser339(2), Leu338, Ala502* | – |
Gallic acid | -6.3 | Ser339, Val509 (2) | Tyr341, Arg499, His75*, Ser339 |
Ellagic acid | -8.6 | Val335(3), Val509(4), Ala513(3), Leu338 (3) | Tyr341, Ser516, Leu338 |
Quercetin | -9.8 | Leu338(2), Phe504, Val509(2), Val335 | Tyr341(2), Phe504, Ser516 |
Gallotannin | -8.6 | Val509(3), Ala513, Phe191*, Leu520*, Leu338 | Arg106, Tyr341(2), Leu338, Ala513, Phe195* |
Urticin А | -8.5 | Val335, Leu338, Ala513(2), Val509, Val335, Leu517 | Tyr341(3), His75, Ser339, Ser516*, Gln178*, Ser516* |
Urticin В | -5.0 | Ala513(2), Tyr341, Val335 Leu338, Val509 | Arg106, Gln178, Tyr341 |
Sitosterine | -6.6 | Val335(3), Val509(3), Ala513(6), Leu338(2), Leu517, Leu345, Val74*, Leu78(2)*, Val102(2)*, Val102*, Tyr101(2)*, Tyr341(4), Phe504 | – |
Inulin | -6.2 | – | His337*, Arg499, Gln178(3), Ser339(2), Asp333*, Gly340*, Leu338*, Pro500*, Asp501* |
Binding energy values for all studied ligands after docking into the active site of the COX-2 inhibitor were significantly higher than the reference ligand: from -5.0 kcal/mol in urticin B to -9.8 kcal/mol in quercetin (Table
Significant ability to inhibit COX-2 enzyme was predicted for gallotannin with its scoring function of -8.6 kcal/mol (Fig.
Apiol and carvone have demonstrated fixation only at the entrance to the inhibitor binding pocket, although with many hydrophobic interactions (Fig.
Coumarin and umbelliferone were similarly fixed into the cavity space of the active site, interacting with the same amino acid residues and overlapping in the visualization of benzopyranone cycles (Fig.
A fairly deep immersion into the pocket was predicted for the mentioned compounds, as evidenced by the fixation in the area of benzenesulfonamide fragment of celecoxib. However, that type of allocation lead to the “Unfavorable Acceptor-Acceptor” formation: interaction between the tertiary nitrogen atom of imidazole histidine (His75) and benzopyranone carbonyl (Fig.
Scoring function value for ellagic acid was -8.6 kcal/mol, and the possibility of forming a branched network containing 13 hydrophobic bonds with amino acids of the active site entirely (Fig.
Urticin A and B glycosides have demonstrated ambiguous results: along with the scoring function of -8.5 kcal/mol for urticin A, -5.0 kcal/mol for urticin B was predicted. Hydrophobic fixation of all fragments was predicted for urticin A with additional stabilization of pyranoside fragment by hydrogen bonds (Fig.
The ligand was deeply immersed into the pocket, and its position made it possible to be fixed in the macromolecule in positions like celecoxib (Fig.
A low affinity level was predicted for the site of COX-2 inhibitor in sitosterine – the binding energy was -6.6 kcal/mol, despite the apparent branched network containing 26 hydrophobic interactions (Fig.
Obtained docking results also point to the inability of anethole and petroselinic acids to have an inhibitory effect on COX-2 both in terms of scoring functions and due to the features of the interactions with amino acid residues and allocation into the active site.
Results have shown that some herbal substances have rather high level of affinity for the LOX-5 active site inhibitor. Therefore, possible manifestation of anti-inflammatory effect of the following biologically active substances: gallotannin, quercetin, inulin, and sitosterine was determined. Ellagic acid has a moderate probability of manifestation by the mentioned mechanism. Possibility of anti-inflammatory effect by inhibition mechanism of COX-2 enzyme was predicted for inulin, quercetin, gallotannin, ellagic acid and urticin A. Moderate activity was detected for gallic acid. Therefore, obtained results of in silico studies for the mentioned substances predict a significant anti-inflammatory potential by a multifactorial mechanism, which may be effective in the treatment of bowel inflammatory processes.
For the further pharmacological in vitro and in vivo studies, as well as a complex herbal remedy composition optimization, six medicinal herbs with the highest content of the mentioned compounds were selected: Inula helenium, Cichorium intybus, Capsella bursa-pastoris, Foeniculum vulgare, Equisetum arvense, Veronica officinalis. Due to hydrophilic nature of the most promising natural compounds of the selected herbs, it is recommended to use aqueous extracts of them.