Molecular docking studies on rocaglamide , a traditional Chinese medicine for periodontitis

Purpose: To undertake an in silico assessment of rocaglamide as a potential drug therapy for periodontitis (dental arthritis). Method: Lamarckian algorithm-based automated docking approach using AutoDock4.2 tool was applied for calculating the best possible binding mode of rocaglamide to IL-23p19 and IL-17, the targets of anti-inflammatory drugs in periodontal disease. Results: The top two interactions of rocaglamide with IL-17 (ΔG = -5.45 and -4.83 kcal/mol) were more spontaneous, and the physical interactions (two hydrogen bonds and one π-πbond) generated in the two IL-17rocaglamide complexes were higher in number than in IL-23p14-rocaglamide complexes. Conclusion: In silico analysis of rocaglamide, a known antimicrobial and anti-inflammatory agent, is a promising natural candidate for periodontitis therapy, and should be further subjected to in vitro and in vivo anti-periodontitis investigations.


INTRODUCTION
Periodontal disease or periodontitisis is a chronic, bacterial infection-associated autoimmune disease characterized by anomaly in the teeth-supporting structure, tissue destruction and bone loss [1].Some of the bacterial pathogens involved in the infection leading to periodontitis are Bacteroides gingivalis, Bacteroides intermedius and Actinobacillus actinomycetemcomitans [2].The autoimmune basis of this disease was first postulated nearly fifty years ago by Brandtzaeg and Kraus [3].The innate immune system has a general role against bacterial pathogens [4,5].Indeed, it is the subsequent inflammatory and immune processes associated with the bacterial pathogens that are harmful to the host and contribute to the tissue injury observed in periodontitis [6].Cell-mediated immune response plays a pathological role in many inflammatory diseases [7].
In periodontitis, the host mechanism is complex, as it involves both protective and destructive elements, which are characterized by immuneinflammatory response to the pathogen and subsequent destruction in the teeth-supporting structure by autoimmune response [8,9].Several studies have associated Th1 and Th2 with the etiology of periodontitis [10][11][12][13][14]. Th1 and Th2 are the important components of the immune response which are associated with inflammatory disease and allergic reaction, respectively.Th1 cells and its cytokines are associated with early periodontal lesions, while Th2 and its cytokines are associated with periodontitis progression [10][11][12][13][14].
The number of anti-cytokine drugs targeting inflammatory cytokines against IL-1b, IL-12p40, IL-6, IL-23p19 and IL-17 has increased significantly [15].The first three cytokine targets (IL-1b, IL-12p40 and IL-60) are the most frequently drug-targeted cytokines, while IL-23p19 and IL-17 are relatively new targets.The objective of the present study was to use Computer-Aided Drug Design (CADD) to study the effectiveness of rocaglamide, a known antimicrobial and anti-inflammatory agent [16], as an anti-periodontitis agent.For this purpose, CADD, which has become a promising tool in new drug discovery [17], was used to target IL-23p19 and IL-17 with rocaglamide.
The crystallographic structure of IL-23p19 protein which contains 189 amino acids bearing PDB ID: 4GRW, was retrieved and modified accordingly by removal of unwanted atoms and molecules to suit the molecular docking experiment.The IL-17 protein contains 155 amino acids and its crystallographic structure PDB ID: 2VXS [18] was downloaded for the study.

Compound selection
Rocaglamide, a natural compound was retrieved from PubChem compound database.The SDF format of the compound was retrieved bearing CID 331783, and it was converted to PDF prior to further studies, using Discovery Studio.

Molecular docking analysis
Molecular docking analysis of the IL-23p19 and IL-17 proteins with rocaglamide was done using Auto Dock4.2tool[19].The Auto Dock4.2toolcalculates energy values by classification of energies as internal energy and torsional free energy as in Eq 1.
where ΔG represents the overall binding energy, while ΔG H-bond , ΔG elec and ΔG tor represent hydrogen bonding, electrostatic bonding and translation/rotation energies, respectively.The term ΔG desolv indicates the desolvation energy on binding and hydrophobic effect.Lamarckian genetic algorithm (GA) default parameters were used for calculating ∆G of each shortlisted compound.Grid box (60 × 60 × 60 A°) was built around the active site.Five top interactions out of ten were studied for each protein.All the visualizations were generated by Discovery studio.

RESULTS
The pathway interaction profile of IL-17A and IL-23A (IL-23p19) derived using Gene Mania database (web-based integrative software) generated the network architecture of the connections of the two cytokines and other important proteins, as shown in Figure 1.The generated protein network were proteins commonly involved in auto-immune diseases such as rheumatoid arthritis.This confirms the importance of IL-23p19 and IL1-7 as drug targets in periodontitis.The 3D structure of the protein retrieved from protein database was trimmed to suit the experimental design using Discovery studio.The modification of both proteins are shown Figure 2a (IL-23p19) and Figure 2b (IL-17).The trimmed structures were used for molecular docking, and blind docking approach was used to look into the possibility of successful docking of rocaglamide onto the two selected cytokines.Table 1 shows the Gibbs free energy of the top two poses of each complex.The best poses of both protein-drug complexes are shown in Figure 3. Table 2 shows the top two interactions of the drug with each protein.The protein-drug interaction (covalent or noncovalent), and the number of hydrogen bonds formed by both proteins with the drug are displayed Figure 4, where 4(a) represents IL-23p14, and 4(b) represents IL-17.
The only interaction formed by rocaglamide and IL-23p14 was the pi-pi interaction as shown in Figure 5 (a), the binding pocket in this pose was TRP26, LEU31, ARG148, GLN151, ALA152, ALA155, VAL156 and ARG159.THR35, PRO37, LYS38, SER40, SER41, TYR44, TRP51, VAL119,GLY120 pi-pi interaction withTRP51 One Hydrogen bond with TRP51 One Hydrogen bond with SER40 Various conceptual models spanning over five decades have been proposed to describe the etiology of periodontitis.Two factors, microbial infection and inflammation, are thought to play pivotal roles in the shift from the healthy state to periodontitis.Thus, the development of strategies is based on these two factors.Compounds of natural origin are invaluable in novel drug discovery, and many natural compounds have been reported to have dual functions [17,20].The present study was focused on a compound with antimicrobial and anti-inflammatory properties.This was based on knowledge from Traditional Chinese Medicine.Rocaglamide, a flavagline of natural origin, is a known traditional Chinese medicinal compound with antimicrobial and antiinflammatory activities.However, the mechanism of action of rocaglamide is yet to be ascertained.It is well established that CADD is an important route for investigating the possible mode of action of drugs.The present study has explored possible targets in the inflammatory pathway that rocaglamide can act on.The data generated here will be useful in developing more robust methods for targeting the pathogenesis of periodontal disease.

CONCLUSION
The results obtained in this study suggest that rocaglamide may have potent anti-periodontitis effects.Being an anti-inflammatory drug with known anti-microbial activity, these findings make it potentially suitable for the development of a new drug for treating periodontitis, a disease in which microbial infection and inflammation are common challenges.

Figure 1 :
Figure 1: Protein sub-networks of IL-17A and IL-23A (queried proteins) with IL-6, NFKB1A and some important proteins involved in arthritis (only significant sub-networks are shown)

Figure 2 :
Figure 2: The original crystallographic structure of the protein available and the trimmed protein used for the study.(a) The trimmed structure represents IL-23p14 protein from amino acid 8-168 (b) The second structure represents amino acids 35-128 of IL-17 Figure 5 (b) represents the second best pose of IL23p14rocaglamide docking.

Table 1 :
Gibbs free energy of top two poses of rocaglamide with IL-23p14 and IL-17

Table 2 :
Auto dock analysis of the top two poses of protein-rocaglamide complexes.