In SILICO Molecular Docking Analysis of Pyrazole Derivatives as Potential Anti-Inflammatory Agents Targeting Protein Code IEQG

Pyrazole is a five-membered heterocyclic ring having three carbon atoms and two nitrogen atoms at N1 and N2 position with molecular formula C 3 H 4 N 2 .Pyrazole derivatives have attracted immense interest due to their wide applications in pharmaceutical and agrochemical industries. They are observed to have pharmacological activities such as antibacterial, antifungal, anticancer, antidepressant, anti-inflammatory, anti-tuberculosis and antioxidant effects. Pyrazole is also a significant scaffold in drug discovery and synthesis of organic compounds. Many methods have been reported for the synthesis of pyrazole derivatives. One of the most commonly reported methods is the reaction of 1,3-diketones with hydrazine derivatives.

SOFTWARES

1. ChemDraw

ChemDraw is a molecular editor and chemical structure drawing software that allows you to edit and create chemical structures. It provides chemical structure to name and name to structure conversion functionalities, NMR and mass spectrum simulation, structure cleaning and ligand structure drawing.

2. ChemAxon

ChemAxon is a cheminformatics and bioinformatics software platform for molecular visualization, chemical structure drawing, database searching, molecular property calculation, and drug discovery applications. It offers a range of tools for the manipulation and processing of chemical information.

3. Marvin Sketch

Marvin Sketch is a powerful chemical editor for drawing chemical structures, reactions and queries. It supports 2D and 3D visualization of compounds and reactions . It is useful in chemical structure preparation and drug discovery studies .

4. Swiss-PDB Viewer

Swiss-PDB Viewer is a molecular visualization and protein analysis application for the study of protein structures and structural alignments. Proteins can be overlaid and their structures and active sites compared. It is also helpful for preparing and analyzing protein structures before molecular docking.

5. Studio Discovery

Discovery Studio is a computational software suite for studying small molecules and macromolecules. It offers tools for ligand design, pharmacophore modeling, structure-based drug design, QSAR, ADME studies, macromolecular analysis and predictive toxicity.

6. PyRx Virtual Screening

PyRx is a virtual screening software that can be used to screen libraries of compounds against potential drug targets in computer-aided drug discovery. It provides a user-friendly user interface to tools for ligand and protein preparation, molecular docking, virtual screening and analysis of docking results.

7. PyMOL software

PyMOL is a molecular visualization system for the display and analysis of three-dimensional structures of proteins and small molecules. It is especially useful for visualizing protein-ligand interactions, binding poses, molecular frameworks, and publication quality 3D images

8. PubChem Compound

PubChem is a chemical database maintained by the National Center for Biotechnology Information (NCBI) It contains information about chemical structures, molecular properties, biological activities and related chemical data. Compound information and chemical structure are useful for computational studies.

9. Molinspiration.com

Molinspiration is a cheminformatics tool for molecular property calculation and structure analysis. It supports the manipulation of molecules, conversion of SMILES and SD files, visualization of molecules and calculation of various molecular properties. It is often used to assess properties and drug-likeness.

10. RCSB Protein Data Bank (RCSB-PDB)

RCSB Protein Data Bank (PDB) is a database for the three-dimensional structural data of large biological molecules, such as proteins and nucleic acids. It is widely used to get protein structures for structural analysis and molecular docking studies

MATERIAL AND METHODS:

1. Preparing the Ligands

The novel substituted pyrazole derivatives were designed and drawn by chemical drawing software ( ChemDraw, Marvin sketch). The molecular frameworks were optimized and the canonical SMILES and IUPAC names were recorded for further analysis. Ligands were evaluated for compliance with Lipinski’s Rule of Five using Molinspiration to verify drug-likeness. Discussed in (Table 1).

Results: All the compounds for docking studies showed acceptable drug-like properties. Compound 1 had no Lipinski violations, whereas compounds 2, 3 and 4 each had one violation based on molecular weight or lipophilicity exceeding ideal limits.

2. Protein Preparation

The target proteins of the inflammatory proteins, Prostaglandin H2 synthase (PDB ID: 1EQG) and Prostaglandin G/H synthase (PDB ID: 3LN1) were obtained from RCSB Protein Data Bank. The protein structures were prepared for docking studies using Swiss-PDB Viewer . This involved removing water molecules and other heteroatoms, adding missing residues and hydrogens, and optimizing side chains. Discussed in (Table 2).

Results: The refined protein structures were validated and active binding sites were identified for further docking studies.

3. ADME Prediction

The synthesized ligands were screened for Absorption, Distribution, Metabolism and Excretion (ADME) properties using the SwissADME web tool to predict the pharmacokinetic profiles like gastrointestinal absorption, blood brain barrier permeability and inhibition potential of cytochrome P450 enzymes. Discussed in (Table 3).

Results: SwissADME was used to predict the ADME properties of four substituted pyrazole derivatives and ibuprofen.Compound 1 was predicted to have high gastrointestinal absorption and blood brain barrier permeability. Conversely, Compounds 2, 3 and 4 were predicted to have low gastrointestinal absorption and were not predicted to permeate the blood-brain barrier.The predicted bioavailability score of all the four pyrazole derivatives was 0.55 whereas ibuprofen showed a bioavailability score of 0.85.

4. Molecular Docking

Molecular docking studies were performed using PyRx virtual screening tool which uses AutoDock Vina as docking engine. Protein macromolecules and ligand molecules were loaded in PyRx and grid box parameters were calculated to cover the expected active sites. Docking studies were performed to determine the binding mode and affinity of each ligand to target proteins. The binding strength was assessed by recording the docking scores in kcal/mol.Docking score Discussed in(Table 4)

Results: Compound 1 showed a binding affinity of −8.3 kcal/mol versus −7.6 kcal/mol for ibuprofen.Compound 2 had a docking score of −9.8 kcal/mol whereas Compounds 3 and 4 had docking scores of −10.1 and −10.2 kcal/mol respectively.The lowest docking score (most negative) was obtained for compound 4, suggesting the highest predicted binding affinity for this compound among the tested compounds under the applied docking conditions.

The order of docking score was:Compound 4: −10.2 > Compound 3: −10.1 > Compound 2: −9.8 > Compound 1: −8.3 > Ibuprofen: −7.6 kcal/mol

5. Analysis of Protein-Ligand Interactions

The interactions like hydrogen bonding, hydrophobic contacts and pi-stacking were analyzed by visualization of the docked protein-ligand complexes using PyMOL molecular viewer . This step helped in identifying important amino acid residues involved in ligand binding in the protein active site.Discussed in (Image 1,2,3,4,5)

Results: PyMOL was used to successfully visualize the docked complexes to study the binding orientation of the ligands in the active site region. The interaction analysis was used to compare the predicted binding affinities of the pyrazole derivatives with ibuprofen.

6. Physicochemical Properties and Drug-Likeness

Physicochemical properties such as molecular weight, lipophilicity (logP), topological polar surface area (TPSA), number of hydrogen bond donors and acceptors, and rotatable bonds were calculated by using Molinspiration and SwissADME. These data were useful for the evaluation of the pharmacological efficacy and bioavailability of the compounds.Discussed in (Table 3)

Results: Compound 1 did not violate the Lipinski rule of five and showed a miLogP value of 2.58, a molecular weight of 331.37 g/mol and a TPSA of 64.86 Å2 .Compound 2 had one Lipinski violation with a molecular weight of 431.49 g/mol and a miLogP value of 4.33.Compounds 3 and 4 had one Lipinski violation each. They had molecular weights of 475.31 and 487.35 g/mol, and miLogP values of 4.68 and 4.60, respectively.Compound 1 showed the best drug-likeness profile among the pyrazole derivatives tested according to the Lipinski parameters reported. Compounds 2, 3 and 4 showed one violation each, predominantly because of their higher molecular weight and/or lipophilicity.

TABLE:1 (PREPARATION OF LIGANDS)

SR. NO	NAMES	Canonical SMILES/ IUPACNAME	STRUCTURE
1	Compound 1	Cc2c(C) n(S (=O) (=O) c1ccc (F) cc1) nc2c3ccccn3
2	Compound 2	Cc4c(c1c[nH]c2 cccc12)nn(S(=O)( =O)c3ccc(F)cc3)c 4c5ccccc5
3	Compound 3	[H]c3c(c1cccc(Br)c1F)nn(S(=O)(= O)c2ccc(F)cc2)c3 c4ccccc4
4	Compound 4	[H]c3c(c1cc(OC)ccc1Br)nn (S(=O)(= O)c2ccc(F)cc2)c3 c4ccccc4

TABLE:2 (PREPARATION OF PROTEIN)

SR. NO	ENZYME NAME	PDB CODE	STRUCTURE OF ENZYME
1	Prostaglandin H2 synthase	1EQG
2	Prostaglandin G/H synthase	3LN1

DRUG LIKENESS OF IBUPROFEN

Molinspiration property engine   IBUPROFEN miLogP 3.46 TPSA 37.30 natoms 15 MW 206.28 nON 2 nOHNH 1 nviolations 0 nrotb 4 volume 211.19

COMPOUND 1

MiSMILES: Cc2c(C) n(S (=O) (=O) c1ccc (F) cc1) nc2c3ccccn3

Molinspiration property engine   miLogP 2.58 TPSA 64.86 Natoms 23 MW 331.37  nON 5 nOHNH 0 nviolations 0 nrotb 3 Volume 273.40

COMPOUND 2

MiSMILES: Cc4c(c1c[nH]c2ccccc12)nn(S(=O)(=O)c3ccc(F)cc3)c4c5ccccc5

Molinspiration property engine v2022.08   miLogP 4.33 TPSA 67.76 natoms 31 MW 431.49 nON 5 nOHNH 1 nviolations 1 nrotb 4 volume 361.38

COMPOUND 3

MiSMILES: [H]c3c(c1cccc(Br)c1F)nn(S(=O)(=O)c2ccc(F)cc2)c3c4ccccc4

Molinspiration property engine v2022.08 miLogP 4.68 TPSA 51.97 natoms 29 MW 475.31 nON 4 nOHNH 0 nviolations 1 nrotb 4 volume 338.66

COMPOUND 4

MiSMILES: c3c(c1cc(OC)ccc1Br)nn(S(=O)(=O)c2ccc(F)cc2)c3c4ccccc4

Molinspiration property engine v2022.08   miLogP 4.60 TPSA 61.20 natoms 30 MW 487.35 nON 5 nOHNH 0 nviolations 1 nrotb 5 volume 359.27

ADME PROPERTIES (SWISS ADME):

TABLE:3 PHYSIOCHEMICAL PROPERTIES

PHARMACOKINETICS

WATER SOLUBILITY

DRUGLIKENESS

LIPOPHILICITY

PROTEIN LIGAND INTERACTIONS

IMAGE : 1

IBUPROFEN (STANDARD):

• Pyrazole (standard) binding in the active site of human 1EQG protein complex.
• This was assessed by docking studies and the docking score generated was –7.6 kcal/mol.

COMPOUND 1

IMAGE :2

• Compound 1 binding into the active site of human 1EQG protein complex.
• This was evaluated by docking studies and the score obtained is -8.3 kcal/mol, which was compared with the standard score -7.6 kcal/mol of the drug ibuprofen

COMPOUND 2

IMAGE :3

• Binding of compound 2 in the active site of human 1EQG protein complex.
• This was checked by docking studies and obtained score -9.8 kcal/mol was compared with standard score -7.6 kcal/mol of drug ibuprofen.

COMPOUND 3:

IMAGE :4

• Compound 3 binding in the active site of human 3pty protein complex.
• Docking studies were performed to evaluate this and the generated score of -10.1 kcal/mol was compared with standard score -7.6 kcal/mol of drug ibuprofen

COMPOUND 4:

IMAGE :5

• Compound 4 binds to the active site of the human 3pty protein complex.
• Which was evaluated by docking studies and generated score -10.2 kcal/mol was compared with standard score -7.6 kcal/mol of drug ibuprofen.

TABLE:4

DOCKING RESULTS