Inhibition of γ-H2AX Protein by Molecular Modeling in Radiation-Resistant Cancer Cells

Research Article

J Drug Discov Develop and Deliv. 2020; 6(1): 1034.

Inhibition of γ-H2AX Protein by Molecular Modeling in Radiation-Resistant Cancer Cells

Dali-Sahi M1*, Nafuye G1, Dennouni-Medjati N1, Merad M2 and Harek Y1

¹Department of Biology, University of Tlemcen, Algeria

²Department of Chemistry, University of Tlemcen, Algeria

*Corresponding author: Dali-Sahi M, Department of Biology, University of Tlemcen, Laboratory of Analytical Chemistry and Electrochemistry, Algeria BP119 13000, Tlemcen, Algeria

Received: March 03, 2020; Accepted: April 28, 2020; Published: May 05, 2020

Abstract

The objective of this study is to propose therapeutic targets to inhibit in silico the activity of γ-H2AX with MDC1 responsible for recruiting DNA repair proteins to make cancer cells radiosensitive.

The protein complex to be studied was retrieved from a protein database (PDB ID - 2AZM) and the constraints were removed using Biova Discovery Studio Visualizer. The docking ligands were selected from the PubChem database and modifications were made using ChemDraw ultra 12.0 and molecular docking was performed with Autodock 4.2. After docking, the ADME analysis and toxicity were performed against possible inhibitors using the admetSAR web server.

The molecular docking results indicated that ligand 6 (C20H14N2O3S2) and R6 (C19H14N2O3S2) had a minimal binding energy (-6.7Kcal/mol) and a positive ADMET analysis prediction profile. After modification of ligand 6, the results also showed that R6 had the minimum binding energy (-7.3Kcal/mol) and a convincing ADMET prediction profile.

We therefore conclude that the ligands used in this study, in particular ligand 6 and its modified derivatives R1 (C21H16N2O3S2), R2 (C21H16N2O2S2), R3 (C20H16N2OS2), and R6 (C19H14N2O3S2) are considered as potential radiosensitizers to improve the effectiveness of radiotherapy and can also be used for further studies.

Keywords: DNA repair; γ-H2AX; ADMET; Molecular docking; Radiosensitizer

Introduction

Since the discovery of ionizing radiation in 1895, radiation therapy has become the treatment of choice for many types of cancer and has been applied as a first-line treatment for many malignant tumors in humans [1].

However, many cancer cells have a standard resistance to radiotherapy, and in many cases, resistance to radiotherapy is an adaptive response to the hyperactive repair mechanisms of Double- Strand Breaks (DSB) [2].

Phosphorylated H2AX, called gamma-H2AX (γ-H2AX), is one of the first proteins involved in DNA damage response pathways (DDRs). It is necessary for amplification of DNA damage signal and subsequent accumulation of many DDR proteins at DSB sites to form ionizing radiation-induced foci (IRIFs) [3-6].

In response to DSB, the conserved C-terminal tail of H2AX rapidly becomes phosphorylated on the serine-139 by Phosphoinositide Kinase 3-kinase (PI3-K) kinases, including Ataxia Telangiectasia Mutated protein kinase (ATM), Ataxia Telangiectasia and Rad3- related protein (ATR) and a DNA-dependent protein kinase, catalytic sub unit (DNA-PKcs).

ATMs and DNA-PKcs show functional redundancy in H2AX phosphorylation after ionizing irradiation, whereas ATRs are more important for phosphorylation of H2AX in response to DNA damage that would slow or block replication [7].

The mediator of DNA damage check point protein 1 (MDC1) works closely with γH2AX in DDR, as it is necessary for almost all foci formation events induced by ionizing radiation dependent on γ-H2AX as a result of DNA damage. In response to DSB, MDC1 binds directly to γ-H2AX through its C-terminal BRCT protein domains [8,9].

The objective of this study is to propose a therapeutic target, use insilico methods to inhibit γ-H2AX activity with the MDC1 responsible for the recruitment of DNA repair proteins to make cancer cells radiosensitive.

Materiel and Methods

Software

Discovery Studio v17.2.0.16349 [10], AutoDock tools and vina 4.2 [11] and ChemDraw Ultra 12.0 were used for three dimension structure preparation, binding site defining, molecular docking and derivatives generating.

Protein structure and ligand presentation

The crystal structure of the BRCT domain of MDC1--H2AX complex was downloaded from the Protein Data Bank (PDB code: 2AZM). According to the residues of the BRCT domain of MDC1 revealing its hydrogen bond interaction with γ-H2AX, the involved residues were defined as its binding site.

We screened a chemical library (PubChem database) to find potential inhibitors of the MDC1-H2AX interaction. The search was based on the chemical similarity of the functional groups of the phosphoserine of -H2AX. Six potential inhibitors (Lig 1, Lig 2, Lig 3, Lig 4, Lig 4 and Lig 6) were identified.

Molecular docking

Here we used Autodock 4.2 for molecular docking. Molecular docking fits two molecules in favorable configuration using their topographical features. Practically molecular docking has been an important technique for the modeling protein-ligand interactions and has been used in studies of the structural basis of biological functions. Essential parameters like hydrogen atoms, and kollman charges were added to the modeled protein structure using Autodock tool. Grid box was then generated using Autogrid program so that it cover entire protein binding sites and make ligand to move freely in that site. For the inhibitor, charges of the Gasteiger type were assigned using Autodock Tool. Other docking parameters were set to the software’s default values. After docking completion the docked model was ranked according to their docked energy as implemented in the AutoDock program.

Molecular docking of ligands at the MDC1 binding site was performed using Autodock Vina software. The docking tests were carried out with a radius of 0.375Åwith the coordinates x: 47.35, y: 77.28 and z: 85.487. The best ranked docking pose of each ligand in complex with MDC1 was obtained based on the scores and the binding energy value. The docked complex was then analyzed using BIOVIA Discovery Studio Visualizer to show the type of interactions between the ligands and MDC1, to determine the distance of the ligands from the binding site on MDC1 and to generate the 2D structures of the complexes.

The ADMET Analysis

The Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties of drug candidates or environmental chemicals play a key role in drug discovery and environmental risk assessment. The ADMET structure-activity relations server, called admetSAR, is a comprehensive tool for predicting the ADMET properties of drug candidates and environmental chemicals [12]. This web server allowed us to calculate the penetration of the Blood-Brain Barrier (BBB), Human Intestinal Absorption (HIA), permeability of human colon adenocarcinoma cell lines (Caco2), plasma glycoprotein binding substrate and inhibition, CYP inhibitory promiscuity, human ether-a-go-go gene inhibition (hERG), AMES toxicity and carcinogenicity. Pre-ADMET is useful for high throughput screening and combinatorial chemistry library design considering the Lipinski’s rule or lead-like rule, drug absorption and water solubility.

Results and Discussion

Molecular docking allowed us to evaluate the interaction energies of the complexes; first between the BRCT domain of the MDC1 protein (NFBD1) and the γ-H2AX tail (Ref) and then between the BRCT domain of the MDC1 protein and the different prospective inhibitors (ligands) that were downloaded from the PubChem databases. Table 1 includes the results of calculations made in the search for the best possible conformation.

Table 1: Molecular docking results of -H2AX and inhibitors with MDC1.



TABLECREATED







Table 1: Molecular docking results of -H2AX and inhibitors with MDC1.

The results in Table 1 show that the energy of interaction obtained after docking between the ligands and MDC1. From these results, we can attest that ligand 6 presents the minimum energy of interaction (-6.7Kcal/mol) and ligand 4 (C16H16BrNO3S2) presents the maximum energy of interaction (-5.3Kcal/mol). As it is in molecular docking, the smaller the energy of interaction the more stable the complex formed between the ligand and the receptor.

Analysis of the MDC1 BRCT-H2AX co-crystal structure revealed that 3 residues of BRCT1 engage in direct hydrogen-bond interactions with γ-H2AX: Thr1898 and Lys1936 contact the phosphoserine, and Arg1933 contacts both the peptide backbone and the C-terminal carboxylate group [9]. However this almost corroborated in our results as Thr1898 engaged in direct hydrogen bond with the phosphoserine, whereas Lys 1936 engaged in salt bridge interaction with an attractive charge with the phosphoserine (Figure 1&2). Meanwhile Arg1933 contacts both the peptide backbone and the C-terminal carboxylate group as documented elsewhere [9].

Figure 1: 3D structure of the interaction between the BRCT domain of MDC1 protein and the γ-H2AX tail.

    

    
    

    

    Figure 1: 3D structure of the interaction between the BRCT domain of MDC1
protein and the γ-H2AX tail.

Figure 2: 2D structure of the interaction between the BRCT domain of MDC1 protein and the γ-H2AX tail.

    

    
    

    

    Figure 2: 2D structure of the interaction between the BRCT domain of MDC1
protein and the γ-H2AX tail.

More to that, from the docking results all the ligands engaged in a direct interaction with at least one of the three functional amino acids of the binding site on the BRCT 1 domain of MDC1. Lig 1, Lig 2, Lig 4 and Lig 6 (Figure 3,4 &5) engaged in direct hydrogenbond interaction with Lys 1936, whereas ligand 3 engaged in a pication interaction with Lys 1936 and ligand 5 engaged in a pi-alkyl interaction with Lys1936 (results not shown).

Figure 3: 3D structure of the interaction between the BRCT domain of MDC1 protein and ligand 6.

    

    
    

    

    Figure 3: 3D structure of the interaction between the BRCT domain of MDC1
protein and ligand 6.

Figure 4: 3D structure of the interaction between the BRCT domain of MDC1 protein and ligand 6.

    

    
    

    

    Figure 4: 3D structure of the interaction between the BRCT domain of MDC1
protein and ligand 6.

Figure 5: Structure of ligand 6 and R6 with their respective binding energy.

    

    
    

    

    Figure 5: Structure of ligand 6 and R6 with their respective binding energy.

The Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) analysis results and their Probabilities (Prob) are summarized in table 2. These Results (Res) are categorical for instance, blood brain barrier penetration (BBB+/BBB-), human intestinal absorption (HIA+/-), human adenocarcinoma cell lines permeability (Caco2-/Caco2+), p-glycoprotein substrate and inhibitor (yes/no), CYP inhibitory promiscuity (low/high), human Ether-a-gogo- Related Gene inhibition (yes/no), AMES toxicity (toxic/no) and carcinogenicity (carcinogen/no) Table 2.

Table 1: Results of the ADMET predicted profile with admetSAR.



TABLECREATED







Table 1: Results of the ADMET predicted profile with admetSAR.

Using Chemdraw Ultra 12.0, we drew and modified Lig 6 by substituting the carboxylic group on the benzene ring with different chemical groups (CH3C00- to form R1, CH3CO- to form R2, CH3 to form R3, (CH3)2CH2 to form R4, OH to form R5and (OH)2 to form R6) and Chem3D Pro were used to change their structures from twodimension to three-dimension. The purpose of these modifications was to see if there could be variation in the energy of interaction. We determined the physicochemical properties of this ligand and docked them into the binding site of the BRCT domain of MDC1 to determine their energy of interaction Table 3. These modifications further decreased the energy of interaction with ligands R1, R2, R3, and R6 showing improvement Table 3.

Table 3: Summary of the physicochemical properties and interaction energy between the modified ligands and MDC1.



TABLECREATED







Table 3: Summary of the physicochemical properties and interaction energy between the modified ligands and MDC1.

The Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) predicted profile also showed improved results Table 4, there was improvement in the blood-brain barrier penetration, human intestinal absorption, decreased hERG inhibition, non-AMES toxic, and non-carcinogenic. However, the predictions showed that there could be inhibition of the plasma glycoproteins and also a high CYP inhibitory promiscuity as compared to the ADMET predicted profile of ligand 6.

Table 1: Results of the ADMET predicted profile with admetSAR.



TABLECREATED







Table 1: Results of the ADMET predicted profile with admetSAR.

In other studies conducted elsewhere in search for radiosensitizing agents, success has been registered. An antimetabolite designed by Taiho Pharmaceuticals is currently used in conjunction with radiotherapy in Japan [13] and it’s under phase III trials in Europe and USA [14]. Another radio-sensitizing agent AZ0156 which targets ATM kinase has demonstrated potential to hypersensitize cancer cells to ionizing radiation [15] and is currently in phase one trials. Another radio-sensitizing agent veliparib which targets Poly(ADP-ribose) Polymerase (PARP) has shown promising results in sensitizing Melanoma, pancreatic cancer, glioma, non-small cell lung cancer, breast cancer to ionizing radiation and is currently under phase III/clinical trials [16]. Though most radio-sensitizers are of chemical nature, few natural compounds have also been identified to sensitize cancer cells to ionizing radiations. These include curcumin [16-18], genistein [19,20] and quercetin [21].

Conclusion

Ionizing Radiation (IR) as the basis of radiotherapy is one of the three standard treatment modalities used against cancer and is indicated for approximately 60% of cancer patients [20]. Certain cancers such as glioblastoma, cancer of the bladder, breast cancer, advanced non-small cell lung cancer, soft tissue carcinoma show high survival rates after treatment with radiotherapy due to radio-resistance. Targeting pathways such as the DNA Damage Repair (DDR) which induce radio-resistance could improve on the effectiveness of radiotherapy.

In our study we analyzed the protein-protein interaction between the gamma-H2AX and the BRCT domain of MDC1 using molecular docking tools and further anticipated inhibitors which could prevent this interaction. As the interaction between these two proteins leads to the recruitment of DNA Damage Repair (DDR) proteins and thus enhances radio-resistance in cancerous cells [9].

The results obtained after molecular docking of the BRCT domain of MDC1 and various ligands showed that ligand 6(C20H14N2O3S2) presented the minimum energy of interaction (-6.7Kcal/mol) and a positive ADMET predicted profile. Modification of ligand 6 by substitution of its carboxylic group with several chemical groups again showed better results with the modified ligand R6(C19H14N2O3S2) presenting the minimum energy of interaction (-7.3Kcal/mol) and a positive ADMET predicted profile.

Virtual screening methods are regularly used for the cost and time of new drug discovery. It has been clearly demonstrated that the approach used in this study proves that the new inhibitors to be modified (R1, R2, R3, and R6) have shown a high binding energy affinity with a score of (-7.0, -7.2, -6.8 and -7.3) Kcal/mol, respectively. According to Lipinski’s rules, all compounds could be good candidates for the development and could improve on the effectiveness of radiotherapy.

To conclude, given the results obtained in this work, which consists in elucidating the inhibition of the gamma-H2AX protein by molecular modeling methods, it seems that R6 probably has a better contribution to inhibition for prevent recurrence after treatment. The modification of ligand 6 by addition of the radical probably increased the stability of the complex formed. Subsequently the synthesis of compound is proposed as well as the study of the biological activity.

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Citation: Dali-Sahi M, Nafuye G, Dennouni-Medjati N, Merad M and Harek Y. Inhibition of γ-H2AX Protein by Molecular Modeling in Radiation-Resistant Cancer Cells. J Drug Discov Develop and Deliv. 2020; 6(1): 1034.