Molecular Docking Analysis of Selected Urtica dioica Constituents As Human Carbonic Anhydrase II (hCA-II), Human 11 Beta-Hydroxysteroid Dehydrogenases Type 1 (h11beta-HSD1), and Human Dual Specificity Phosphatase (hCDC25B) Inhibitory Agents

Background Urtica dioica (Stinging nettle)has been reported to exhibit various pharmacological activities. In the present study, we aimed to evaluate 24 selected constituents of U. dioica as potent inhibitory agents of human carbonic anhydrase II (hCA-II), human 11 beta-hydroxysteroid dehydrogenases type 1 (h11beta-HSD1), and human dual specificity phosphatase (hCDC25B) using in silico method. Methodology The 24 selected constituents of U. dioica (Stinging nettle) were studied on the docking behavior of hCA-II, h11beta-HSD1, and hCDC25B by using the Webina docking method. In addition to docking, toxicity analysis was also performed using the pkCSM free web server, respectively. Results Toxicity analysis has shown that six ligands (25%) of U. dioica (Stinging nettle) are predicted to have hERG II (Human ether-a-go-go-related gene) inhibition activity. The docking analysis showed that afzelin, stigmastane-3, 6-diol, and astragalin of U. dioica have shown the maximum binding energy (-7.2, -9.5, and -8.5 kcal/mol) with the hCA-II, h11beta-HSD1 and hCDC25B, respectively. Conclusions Thus, the current finding provides new knowledge about the 24 selected ligands of U. dioica (Stinging nettle) as potent inhibitory agents of human carbonic anhydrase II (hCA-II), human 11 beta-hydroxysteroid dehydrogenases type 1 (h11beta-HSD1) and human dual specificity phosphatase (hCDC25B).


Introduction
Carbonic anhydrase II (CA II) is one of the metalloenzymes that catalases the reversible hydration of carbon dioxide into a bicarbonate ion.It participates in a variety of physiological functions, such as bone reabsorption, respiration, and acid-base balance [1].CA II deficiency syndrome [a human autosomal recessive disorder] is characterized by i) cerebral calcification, ii) osteopetrosis, and iii) renal tubular acidosis [1].Studies on naturally occurring phytochemicals that inhibit carbonic anhydrase II activity have been reported to possess therapeutic uses.Similarly, the metabolism of glucocorticoids is catalyzed by two enzymes namely 11 beta-hydroxysteroid dehydrogenases type 1 and 2 (11 beta-HSD1 and 11 beta-HSD2).These enzymes are involved in the inter-conversion of the non-receptor binding inactive form (cortisone) and the receptor binding active form (cortisol). 11 beta-HSD1 is an NADP(H)-the dependent enzyme that converts inactive cortisone to active cortisol in the adipose tissue, brain, liver, and vasculature [2].An excessive amount of 11 beta-HSD1 enzyme activity has been reported to be involved in metabolic syndrome and its associated cardiovascular complications [2].Therefore, identifying natural substances that inhibit 11 beta-HSD1 activity has been reported to possess therapeutic applications.Another key enzyme namely, dual specificity phosphatases (DUSP) [CDC25A, CDC25B and CDC25C] are the enzymes that regulate phosphorylation of cyclin-dependent kinases (substrates) during the cell cycle progression.In contrast, (i) cancer, (ii) diabetes, and (iii) neurodegenerative disorders have been associated with dysregulation of DUSP activity [3].Therefore, identifying natural compounds that inhibit DUSP activity has been reported to possess therapeutically uses.

Toxicity analysis
Toxicity analysis was determined for 24 selected ligands of U. dioica (Stinging nettle) using the pkCSM (predicting small-molecule pharmacokinetic properties using graph-based signatures method) free web server [14].

Identification and preparation of target enzyme
The three-dimensional (3D) structure of human carbonic anhydrase II (hCA-II) (PDB ID: 1BCD with a resolution of 1.90 Aᵒ), human 11 beta-hydroxysteroid dehydrogenases type 1 (h11 beta-HSD1) (PDB ID: 2BEL with a resolution of 2.11 Aᵒ) and human dual specificity phosphatase (hCDC25B) (PDB ID: 1QB0 with a resolution of 1.91 Aᵒ) was obtained from Protein Data Bank (PDB).A chain of these enzymes was processed individually by removing other chains, ligands, and crystallographically observed water (H2O) molecules (i.e., water without hydrogen bonds) by utilizing UCSF Chimera software [14].

Docking study
A docking analysis was performed for 24 selected constituents of U. dioica (Stinging nettle) using the Webina (runs based on AutoDock Vina method) free web server [15].Webina docking method is simple and straight forward one, where it allows users to use various file formats namely mol, pdb and sdf to upload both enzymes (receptors) and ligands input files.Similarly, it also allows users to setting up docking calculations (identifying an appropriate docking box) and analyzing docking output (examining/virtualizing predicted binding poses).
According to International Regulatory Guidelines (namely ICH S7B) all new drugs under the drug development (DD) process should be assessed for effects on the human ether-a-go-go-related gene (hERG) channel prior to clinical trials [25].In the current investigation, six ligands (afzelin, cycloolivil, estra-1,3,5(10)-trien-17B-ol, kephton, 14-octacosanol and stigmastane-3, 6-diol) of U. dioica (Stinging nettle) have predicted to exhibit hERG II inhibition activity, thus six ligands have failed to obey with the above said regulatory guideline (ICH S7B).Gansser and Spiteller (1995) reported for the first time the presence of 14octacosanol from U. dioica (Stinging nettle) root extract and shown to have weak aromatase inhibition activity [26].Mares and colleagues have reported that inhibitors of human carbonic anhydrase II (hCA-II) have been used for treating glaucoma, neoplasms, and neurodegenerative diseases [27].The current investigation showed that all 24 ligands of U. dioica (Stinging nettle) have docked successfully with the human carbonic anhydrase II (hCA-II) enzyme, which was in good agreement with a recent report [28].
Figure 2 shows the docking image, where the afzelin ligand docked with the human dual specificity phosphatase (hCDC25B).

Limitations and future recommendations
The current study findings are based on in silico analysis which provides detailed information about these 24 ligands of U. dioica (Stinging nettle) as hCA-II, h11beta-HSD1, and hCDC25B enzyme inhibition activities and are considered initial research work.Furthermore, in vitro and in vivo experiments are required to confirm these 24 phytoconstituents of U. dioica (Stinging nettle) as good enzyme inhibitors against hCA-II, h11beta-HSD1, and hCDC25B activities.

Conclusions
The present study showed that all 24 ligands of U. dioica (Stinging nettle) have dock effectively with the three target enzymes namely human carbonic anhydrase II (hCA-II), human 11 beta-hydroxysteroid dehydrogenases type 1 (h11beta-HSD1) and human dual specificity phosphatase (hCDC25B).Interestingly, 1, 2, and 3-butanetriol showed the least binding energy (-4.3 and -5.6 kcal/mol) with both the h11 beta-HSD1 and hCDC25B enzymes, respectively.Thus, the results of the present investigation have shown good knowledge of these 24 ligands of U. dioica (Stinging nettle) as potential suppressers against hCA-II, h11beta-HSD1, and hCDC25B concerning the treatments of atherosclerosis, glaucoma, metabolic syndrome (including obesity), neoplasms, neurodegenerative diseases, renal disease, and type 2 DM.

FIGURE 2 :
FIGURE 2: The docking image where afzelin ligand docked with the human dual specificity phosphatase (hCDC25B).