Molecular docking analysis of a virulence factor protein dentilisin from Treponema denticola with oxazole piperazine derivatives

Dentilisin is a surface protease synthesized by the cell wall of Treponema denticola. This protein aids in the invasion of the periodontal tissue by causing infection. To identify drug molecules that have better results, homology modeling of the dentilisin protein was constructed, and molecular docking was performed with the oxazole compounds (1-6) taken from previous studies that are not yet clinically used. Data shows that compounds 1, 2, 3 show better inhibiting properties.


Abstract:
Dentilisin is a surface protease synthesized by the cell wall of Treponema denticola.This protein aids in the invasion of the periodontal tissue by causing infection.To identify drug molecules that have better results, homology modeling of the dentilisin protein was constructed, and molecular docking was performed with the oxazole compounds (1)(2)(3)(4)(5)(6) taken from previous studies that are not yet clinically used.Data shows that compounds 1, 2, 3 show better inhibiting properties.

Background:
Treponema denticola is a helical-shaped, oral spirochete, periodontal pathogen that inflicts damage on host tissue in conjunction with other members of an intricate polymicrobial oral biofilm.These members primarily reside in the subgingival and periodontal pockets, which increases gingival apoptotic cell death and results in hemolysis of human RBC [1, 2].In animal models, it can induce soft tissue degradation and loss of alveolar bone, and its levels rise in the subgingival microbiome of individuals with periodontal disease when compared to healthy people.An operon comprising the genes prcB, prcA, and prtP encodes dentilisin [3, 4, and 5].Among these, prtP encodes for a 72 kDa protease domain that disintegrates extracellular matrix elements like fibronectin, activates C3, and contributes in fibrinogen attachment [6, 7].Additionally, dentilisin functions in immunomodulation, adhesion to fibronectin, as well as other processes.An active protease complex is synthesized on the surface of T. denticola by post-translational modification and interactions between both the expressed proteins [3,8].Dentilisin, also known as acylated chymotrypsin-like protease complex (CTLP), is a substantial virulence factor that triggers a spectrum of cytopathic effects that are consistent with the pathophysiology of periodontal disease [9, 10].An oxygen-containing heterocyclic molecule called oxazole is crucial in the development of several biologically efficient drug molecule, [12,13] including medications that are anti-inflammatory, anti-cancer, anti-depressant, antimicrobial, anti-obesity, anti-diabetic, and analgesic.Similarly, for several years, studies into the anti-microbial characteristics of pyrrole derivatives have driven the synthesis and analysis of a variety of novel compounds, including monodeoxypyoluteorin and derivatives of 2-(2'-hydroxybenzoyl) pyrrole bromine [14,15].Therefore, it is of interest to document the molecular docking analysis data of a virulence factor protein dentilisin from Treponema denticola with oxazole piperazine derivatives for consideration in drug discovery.

Materials and methods: Preparation of ligands:
The 2D mol structures of the selected oxazole piperazine derivatives (1-6) were drawn, and energy minimized using ChemDraw (Figure 1) and Chem3D software respectively (ChemOffice suite 16.0).During the optimization method, all parameters were selected in order to achieve a stable structure with the least amount of energy.Each molecule's 3D coordinates (PDB) were determined using optimized structure [16].

Homology modeling of the dentilisin protein:
The fasta sequence of the protein dentilisin was derived from the Uniprot database (Uniprot ID: P96091).The query sequence is then submitted to the Swiss Model online server and the structure is built using the template with maximum sequence identity.Based on the structure assessment and Ramachandran favored region, the built model was further analyzed for protein preparation

Preparation of protein macromolecule:
The homology modeled structure (Figure 2) of the protein dentilisin of Treponema denticola, was prepared as per standard protocol practiced globally using the software Biovia Discovery tudio.Water molecules, other hetero atoms were chosen for elimination.The previously connected ligands were removed, and the protein was prepared by adding polar hydrogens and kollman charges with Auto Prep.

Auto dock Vina analysis:
The graphical user interface Auto Dock vina was used for Ligand-Protein docking interactions (Figure 3 & Figure 4).Auto Dock Tools (ADT), a free visual user interface (GUI) for the AutoDock Vina software, was used for the molecular docking research.For each ligand, nine alternative conformations were created and ranked based on their binding energies utilizing Auto Dock Vina algorithms.
Drug-likeness and toxicity predictions: SwissADME and PROTOX-II online servers were used.This prediction points users in the direction of drug efficiency, gives information on whether or not the examined ligands have features consistent with becoming an orally active medication.This prediction is based on Lipinski et al's previously established idea known as Lipinski's rule of five [20].
Statistical Analysis: One way ANOVA was used for statistical analysis.The clinically proven drugs are used as control and the results are compared.The significance of the results was found to be p < 0.05.

Results: Molecular docking interaction against protein dentilisin:
All compounds with the protein have binding energy in the range of -6.9 to -8.5 Kcal/mol (Table 1).The compounds show an H binding interaction similar to that of sulfamethoxazole (-5.6).Clinically proven drugs shown in lead binds to the binding site of protein.Azithromycin binds to the Gln-145, Glu-441, Thr-442, Lys-430 binding site.Sulfanilamide binds to the Asp-79, Asp-76, Thr-74 binding sites.Sulfamethoxazole binds to the Ser-291, Arg-299 binding site of the protein.All compounds show similar binding affinity as the lead molecules within the binding site.

ADME & Lipinski rule of five:
The chosen compounds show a logKp value within the range of -6 to -5.12 cm/surface (Table 2).It is inferred that, more the negative value, the higher will be the permeation of skin.All the compounds show lesser negative values of log Kp than the lead compounds.The GI absorption of the compounds is high and there will be no involvement of carrier molecules to perform their function.This is similar to the control compound Sulfamethoxazole and Sulfanilamide.The compounds 4, 5 ,6 and the control group do not show any blood brain barrier permeability.However, compounds 1, 2, and 3 shows blood brain barrier permeation.The compound 4, 5, 6 and the control group taken doses not inhibit the CYP.Nonetheless, compounds 1, 2, and 3 are the inhibitors for CYP.Therefore, the compounds 1, 2, and 3 can be used as potentially lead drug molecules.The compounds 1, 2, and 3 obey the Lipinski rule of five and are similar to the control groups Sulfamethoxazole and Sulfanilamide (Table 3).Additionally, the control group molecule Azithromycin and compounds 4, 5, and 6 shows violation in the molecular weight (748.9, 519.98, 543.87, 533.02 respectively).Also, Azithromycin and compound 4 violates TPSA score (180.08,107.43).In spite of these exceptions, this can still be used as a drug as these violations don't directly affect its function.Compared to this, the oxazole compounds don't have any violations and therefore can be used as lead molecules.

Toxicity profile:
The compounds show class 4 toxicity and have inactive Immunotoxicity, cytotoxicity and hepatotoxicity (Table 4).The lethal dose parameters of the chosen molecules are lesser than the control group and thus can be used as drugs.

Discussion:
The selected compounds (Oxazole piperazine derivatives) 5 (-8.5 kcal/mol) and 6(-8.4 kcal/mol) show better interaction compared to the clinically proven drugs (-6.9 kcal/mol & -7.9 kcal/mol).The compounds 1, 2, 3 obey Lipinski's rule of 5 similar to sulfanilamide and Sulfamethoxazole.Compounds 4, 5, 6 do not obey Lipinski's rule of 5 which is similar to Azithromycin.The selected compounds show better skin permeability (log Kp -6.28 to -5.12 (cm/s)).Compounds 1, 2, 3 & 5 have no toxicity.The reported compounds have stable H bonding, weak hydrophobic and van der Waals interactions within the binding pocket in targeted protein.The SwissADME prediction parameters showed that all the compounds (1-6) have high gastrointestinal (GI) absorption, blood-brain barrier (BBB) permeation and all compounds are substrates of permeability glycoprotein (P-gp) except 1.The CYP's interaction result showed that all the compounds are inhibitors for CYP2C19, CYP2C9, and CYP2D6.Compounds 4, 5 and 6 were found to be potential inhibitors for CYP1A2.For CYP3A4, compound Azithromycin, sulfanilamide and sulfamethoxazole were found to be non-inhibitors whereas compounds 1-6 were found to be potential inhibitors [21].

Table 1 :
Molecular docking scores and residual amino acid interactions of oxazole compounds (1-6) along with control groups against Dentilisin of Treponema denticola (Homology modeled).