In silico molecular docking against C-KIT Tyrosine Kinase and ADME studies of 3-Ethyl-2-(2,3,4-trifluoro-phenylimino)-thiazolidin-4-one derivatives

 

Shreyash D. Kadam¹*, Denni Mammen¹, Deepak S. Kadam², Sudhakar G. Patil²

¹School of Science, Navrachana University, Vasna-Bhayli Main Rd, Bhayli, Vadodara-391410, Gujarat, India

²Organic Chemistry Research Laboratory, Maharashtra Udaygiri Mahavidyalay, Udgir-413517,  Maharashtra, India

*Corresponding Author E-mail: kadamshreyash28@gmail.com

 

 

1. INTRODUCTION

Computational docking methods are used to screen a variety of possible compounds, searching for new compounds with specific binding properties or testing a range of modifications of an existing compound. Due to the rapid rise in the amount of molecular biological data available, the computer-aided analysis of molecular interactions becomes more realistic in addition to which as of now the computer prediction of the interaction between proteins and small molecules has advanced to the point that it allows accurate prediction of bound conformations and affinity.

 

Also binding of small molecule majorly organic compounds which are ligands to large protein targets is significant to both understanding biological processes and designing drugs¹. As many proteins regulate biological functions by interacting with small molecules, these receptor proteins are often prime targets for therapeutic agents. A detailed understanding of interactions between small molecules and proteins may therefore form the basis for a rational drug-design strategy which is attractive in drug development concept due to two reasons: it may facilitate the development of more selective therapeutic agents with fewer undesirable side effects and will offer some hope for reduction of the enormous costs and time required in traditional random screening protocols for drug discovery. So, by assuming the receptor structure is available in the PDB database, a major challenge in lead discovery and optimization is to predict both ligand orientation as well as binding affinity which could often be referred to as “molecular docking”².

Molecular docking has become an increasingly important tool for drug discovery and is the most widely employed technique whose goal is to predict the position and orientation of a ligand (a small molecule) when it is bound to a protein receptor or enzyme. The completion of the human genome project has resulted in broadening the scope of new therapeutic targets in drug design and discovery. With this, the advancement in strategies such as excessive high-throughput protein purification, crystallography and nuclear magnetic resonance (NMR) spectroscopy has been providing structural information of protein–ligand and protein complexes. This leads to advancement which resulted in the development of computer-aided drug design, also known as molecular docking³. Molecular docking is a key tool in structural molecular biology and computer-assisted drug design which tries to predict the structure of the intermolecular complex formed between two or more constituent molecules, also trying to predict the position and orientation of a ligand when it is bound to a protein to know the predominant binding modes of a ligand with a protein of known three-dimensional structure. Simply this can be said that docking is a method that predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. Usually, these binding partners are biological macromolecules (e.g., protein, DNA/RNA, peptide) or small molecules (e.g., endogenous ligands, drugs) and their preparations for the docking is just as important as the docking itself ^4,5. Computational approaches are currently being used for screening large databases of compounds to identify potential lead drug molecules. So it can be said that its main application lies in structure-based virtual screening for the identification of new active compounds towards a particular target protein⁶. It can also be said that for a selected set of structures of a protein and a ligand, the ultimate goal of all docking methods is to predict the structure of the resulting complex and to predict the biological activity of a given ligand.

 

In this study, molecular docking is performed between receptor i.e. protein molecule and ligand i.e. novel thiazolidin-4-one derivatives⁷ which were already synthesized by the authors. They are the oxo derivatives of thiazolidine which belong to an important group of heterocyclic compounds containing sulfur and nitrogen as well as carbonyl group in the 4^th position in a five-member ring^8,9. They are an important class of bioactive molecules having diverse biological activities so it’s often called “wonder nucleus”. Thiazolidinone also gives out various derivatives which attracted great attention due to the diversity of their biological effects¹⁰ such as antidiarrheal¹¹, antimicrobial^12-14, antifungal¹⁵, antihyperglycemic¹⁶, antibacterial^17-19, antidiabetic^20,21, antihistaminics²², anticancer^23-26, anti-HIV²⁷, Ca²⁺ channel blocker²⁸, cardioprotective²⁹, anti-ischemic³⁰, cycloxygenases inhibitory³¹ and anti-platelet activating factor³², antimalarial³³, antioxidant^34,35.

 

1.1. Experimental Section:

1.1.1 Molecular Docking:

Docking studies of all the synthesized analogues through Discovery studio docking suite has been performed on target protein (PDB ID 1T46) (figure 2) against novel synthesized ligands (figure 1).

1.1.1.1. Ligand preparation:

The structures of the novel synthesized ligands (figure 1) were drawn in Chemdraw Ultra 7.0 and saved in PDB format using Biovia Discovery Studio. Later prepared ligand by applying charges using MGL Tools and saved in (.pdbqt) format for docking study.

 

 

Figure 1 – Synthesized novel thiazolidin – 4 - derivatives

1.1.1.2. Protein selection and Preparation:

The X-ray crystallographic structure of kinase (PDB ID 1T46) (figure 2) was obtained from pdb data-base and saved as pdb format for further studies. Selected protein macromolecule was prepared using Biovia Discovery Studio and charges were applied using MGL Tools.

 

 

Figure 2 - Proteins used for Molecular Docking - 1T46, C-KIT Tyrosine Kinase target protein

 

1.1.1.3. Docking Studies:

In this study, the affinity and binding modes of the examined molecules against the target protein were determined. At first, the water molecules were removed from the crystal structures of target proteins, retaining only main-chain amino acids which are essential for binding. The co-crystallized ligands were used as reference ligands to predict the binding pockets (figure 3) and later ligand was removed. Then, the polar hydrogen atoms were added to protein structures to protonate them. The structures of the examined compounds were drawn using ChemDraw Ultra 7.0 and BIOVIA Discovery Studio Visualizer 2021 which were later saved using PDB formats. Then, the saved files were opened using MGL AutoDock Tools software where preparation of protein was done and selected as macromolecule then saved in PDBQT format. The configuration file was created which contained receptor name, ligand name, output file name, X, Y, Z coordinates of the grid box and also the size X, Y, Z of the grid box. Then the ligand was prepared and any rotatable bonds if available were added. Next, the Command Prompt was opened and AutoDock Vina software was used for running the docking process for each target receptor by ligand by entering necessary codes or commands. In each case, 9 docked structural poses, affinity and RMSD data were generated using the algorithm. The output from the Vina split software was further analyzed and visualized using BIOVIA Discovery Studio Visualizer 2021.

 

The (figure 2) depicts the structure of the protein 1T46 on which molecular docking of all 18 earlier synthesized compounds have been performed.

 

1.1.2. ADME Study:

ADME study had been performed via using the Swiss ADME site (SwissADME). Primarily the structures were created with the help of ChemDraw Ultra 7.0 software and later uploaded on the Swiss ADME website in order to generate Smiles. These Smiles were used to generate ADME analysis data represented as boiled egg in (figure 5) and data are represented in (table 3, 4, 5, 6, 7 and 8).

 

2. RESULTS AND DISCUSSION:

2.1. Molecular Docking:

Molecular docking study helps to analyze and obtained the information about appropriate orientation of synthesized compound (ligand) (figure 1) inside the active site of the macromolecule (protein) (figure 2). Here the active sites were selected on the basis of previously bound inhibitor in the downloaded crystal strucutre of respective proteins. The best compound was selected as per few criteria such as binding modes, good molecular interactions with the active site components of protein and docking energy at 0.00 RMSD value. The compounds were docked with C-KIT Tyrosine Kinase protein (figure 2). Docking scores and interacting amino acids were depicted in the (table 1) below. Substituted thiazolidin-4-one derivatives (1-18) had shown various H-Bonding, Pi-Sigma, halogen interactions towards 1T46 protein molecule. It was observed that compounds were showing good affinity value within the range of -6.6 to -10.7 kcal/mol with favorable binding poses. The amino acid LYS623 from 1T46 protein showing hydrogen bonding with most of the compounds such as  3, 4, 5, 6, 7, 8, 9, 10, 14, 17 and 18. Apart from this amino acids THR670, GLY676, SER639 and ASP810 were also involved in H bonding with various compounds. Among these compounds 4 and 5 exhibit strong interaction with active site amino acids with binding energy of -10.7 and -10.5 kcal/mol respectively towards 1T46 Kinase protein.

 

Table 1 - Docking scores and interactions

 

 

 

To visualize the interactions their 2D diagram and 3D interactions (colour of bond interaction is justified in the table) of compound 4 are displayed which is having lowest affinity value -10.7 (Figure 4 and 5) and interactions are shown in Table 2.

 

2.1.1. 3-Ethyl-5-(4-trifluoromethyl-benzylidene)-2-(2,3,4-trifluoro-phenylimino)-thiazolidin-4-one docking results against 1T46

 

Figure 3 - Molecular Docking 3D interaction output of 3-Ethyl-5-(4-trifluoromethyl-benzylidene)-2-(2, 4, 5-trifluoro-phenylimino)-thiazolidin-4-one, 4 against 1T46

 

Figure 4 - Molecular Docking 2D interaction output of 3-Ethyl-5-(4-trifluoromethyl-benzylidene)-2-(2, 4, 5-trifluoro-phenylimino)-thiazolidin-4-one, 4 against 1T46

 

 

Table 2 - Total Number of Favourable Interactions: 17

 

In order to perform molecular docking analysis, the structure of 3-Ethyl-5-(4-trifluoromethyl-benzylidene)-2-(2, 3, 4-trifluoro-phenylimino)-thiazolidin-4-one has been selected as a ligand which is docked against protein molecule 1T46 (Figure 2) acting as a receptor. During the analysis 4 different poses were observed, by refining which the pose of which pose having lowest affinity (kcal/mol) was selected as the best docking pose and was considered for the ligand interaction. At this time, 17 favorable interactions were observed in the selected pose where the ligand has bonded at the chosen pocket site in the selected pose. The above table provides information regarding bonding interaction between the ligand and amino acid chains which contains bond distance, types of bonds, from where the bond is forming and their types. The bonding interactions observed in this moiety while docking were -: one hydrogen bonds [conventional hydrogen bond with fluorine], four halogen bonds [with fluorine], one electrostatic bond [pi-cation] and eleven hydrophobic interactions [i.e. five pi-sigma, one pi-pi T-shaped, one alkyl and four pi-alkyl].

 

2.2. ADME:

 

Figure 5 – ADME Boiled egg Diagram

The results of ADME studies of the 18 compounds have been illustrated (Fig. 4). The molecules that appear in white portion of the boiled egg diagram molecules have the capability of being absorbed through the intestine while the molecules in the yellow portion are the absorbed molecules which have the potential to cross the blood brain barrier. According to the analysis it is stated that molecules 1, 2, 3, 8, 9, 10, 11, 12, 13, 16, 17 and 18 are capable of being absorbed through the human intestine (HIA). Among these 12 molecules just molecule 1 which has no substituents on the phenyl ring attached to the thiazolidin-4-one ring shows good permeability and is observed to be capable of crossing the BBB barrier. In addition to this molecule 1 has shown to follow all five rules of Lipinski’s Rule, without any violations of the rules. Owing to the presence of two methyl groups attached to nitrogen of the amine on the phenyl ring molecule 8 shows good potential to be able to cross the BBB barrier. Molecules 16 and 18 are close to the potential molecules that could cross the BBB barrier due to the existence of heterocycles furan and pyrrole attached to the thiazolidin-4-one ring, while the existence of thiophene in molecule 17 decreases its potential to do so, probably due to bigger size of sulphur as compared to oxygen and nitrogen. The moieties with various methoxy and hydroxyl groups on the phenyl ring are good candidates that could be absorbed by the intestine represented by molecules 9, 10, 11, 12, and 13 whereas molecule 2 is close to crossing of BBB barrier due to presence of single methoxy group.

 

Shockingly, functionalities possessing –CF₃ on the phenyl ring i.e. molecules 4, 5, and 6, while possessing phenoxy group and fluoro groups on the phenyl ring attached to the thiazolidin-4-one ring i.e molecule 14, show no potential for absorption through the intestinal lining. Because of the presence of halogen atoms –Br and –Cl is shown to hamper its potential for intestinal absorption as observed in molecules 7 and 15.  However, if only the –F group is present on the ring, the intestinal absorption improves and shows more potential to cross the BBB barrier as observed in the case of molecule 3.Shown below are some other parameters that were analysed during this analysis as represented in Table 3, 4, 5, 6, 7 and 8.

 

Table 3 - Physicochemical Properties

 

Table 4 - Lipophilicity

 

Table 5 - Water Solubility

 

Table 6 - Pharmacokinetics

 

Table 7 - Drug likeness

 

 

Table 8 - Medicinal Chemistry

 

 

3. CONCLUSION:

Docking of the novel thiazolidin-4-one derivatives were performed through Discovery studio, MGL tools and Auto Dock Vina to acquire the active conformations of the derivatives that showed good interactions with the C-KIT Tyrosine Kinase (1T46) target protein. ADME analysis give an idea about groups like fluoro, methyl, hydroxyl and methoxy illustrate good human intestinal absorption, but it is opposite in case of moieties having functionalities chloro, bromo and trifluoromethyl groups attached to the heterocyclic ring. This type of in-silico methods can indicate which functional groups in the molecules could aid in absorption in the body which could give a proper direction to researchers for synthesizing biologically active derivatives of thiazolidin-4-one.

 

4. CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

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Received on 11.08.2022                    Modified on 25.10.2022

Accepted on 03.12.2022                   ©AJRC All right reserved