Introduction

Cancer is one of the major chronic diseases worldwide, and every year 12.7 million new incidences are recorded. This number is expected to reach 26 million by 2030¹. The emerging targeted therapies provide a lot of hope, whereby a single target protein or enzyme is blocked using an inhibitor. The accumulating evidence established nuclear factor kappa B (NF-κB) as an excellent cancer therapeutic target^{2, 3}. The NF-κB, which acts as a transcription factor, exists in the form of homo or heterodimeric complex. These complexes are made up of Rel-like domain including proteins RelA (p65), RelB, p105 (NFκB1), (p50) NFκB1, Rel and p52 (NFκB2), and the complex from two different subunits p65 and p50 seems to be the major one. NF-κB’s linkage with cancer is shown through the constitutive activation of NF-κB in several types of cancer like breast, lung, liver, pancreatic, prostate and various kinds of lymphoma^4-9. The most important role of NF-κB is its ability to promote cell survival through the transcription of target pro-survival genes, resulting in protein expression that inhibits the programmed cell death (PCD) mechanism in both types of malignant and normal cells^{10, 11}. Due to the broad significance of NF-κB, it has been challenging to target NF-κB in cancer cells¹². Last few years’ studies have unveiled that NF-κB is activated by carcinogens, chemotherapeutic agents, tumour promoters, and by inflammatory cytokines¹³. Studies on preclinical models have shown that several chemotherapy drugs like taxanes, anthracyclines and platinum-based agents induce the activation of NF-κB pathway¹⁴, while there are a majority of drugs identified to be the inhibitor of NF-κB signaling, with potencies as low as 20nM. Some of these drugs like narasin, sunitinib malate, lestaurtinib, tribromsalan, bithionol, fluorosalan and emetine inhibit NF-κB signaling via inhibition of IkBa phosphorylation¹⁵.

The natural compounds have increasingly been exploited by the scientific community for anticancer potential^16-19. In this view, green tea catechins were extensively studied for anticancer mechanisms controlling cell proliferation, cell death in tumour cells and vascular angiogenesis in solid tumours^{20, 21}. Recent studies have suggested that natural compounds catechins found in green tea have inhibitory activity against the NF-κB²². There are four major components in green tea found as catechins; 1) (-)-epicatechin (EC), 2) (-)-epigallocatechin (EGC) 3) (-)-epicatechin-3-gallate (ECG) and 4) (-)-epigallocatechin-3-gallate (EGCG) (Fig.1). EGCG and EGC are the most abundant in green tea^{23, 24}. Current findings have unveiled that green tea catechins particularly interact with cell surface receptors, including trans-membrane receptor 67L, lipid rafts, receptor tyrosine kinases (RTKs) and endoplasmic reticulum (ER)²⁵. These catechins modulate gene expression through a direct effect on transcription factors or indirectly by epigenetic mechanism, and also interfere with intracellular proteostasis at various levels²⁶. Recent developments in the advancement of knowledge in this field have shown that EGCG shows an anticancer effect by inhibiting the activity of NF-κB and several other receptors^{22, 27}. In recent years, the computational studies are increasingly being used for exploring the inhibitory mechanism of active compounds and in drug designing^28-34. Despite the knowledge of anticancer potential and NF-κB inhibitory activity, the exact binding poses and molecular interactions of these catechins for NF-κB were not explored yet. The current study is aimed to explore the inhibitory mechanism of catechin derivatives against NF-κB DNA binding activity through computational methods.

Materials and Methods

Data Retrieval

The 3-D structure of human transcription factor NF-κB p50 subunit in complex with DNA was retrieved from protein data bank with PDB ID, 1svc. The molecular structures of (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG) were obtained from PubChem with compound IDs, CID: 72276, 72277, 107905, and 65064 respectively.

Molecular Docking

Dock v.6.5³⁵ was used for docking simulation of all four catechin derivatives into the DNA binding site of NF-κB. The binding site was considered as the residues around 5 Å vicinity of bound DNA. Chimera v.1.6.2³⁶ was used in initial structure preparation of protein and ligands.

Analyses of Docked Protein-Ligand Complex

Pymol v.1.3³⁷ was used to generate illustrations and to analyze the protein-ligand complexes. LigPlot v.1.4.3 program³⁸ was used to generate protein-ligand interaction plots and to analyze the polar and non-bonding interactions between the protein and the ligands. X-Score v.1.2.11³⁹ was used for binding energy and dissociation constant calculation prediction from the protein-ligand complex.

Result and Discussion

Molecular Docking  of EC to NF-κB

Epicatechin (EC) was found to bound deep in the binding site of NF-κB and interacted with eight residues namely Tyr-60, Lys-147, Asp-209, Leu-210, Ser-211, Lys-244, Pro-246 and Asn-247 (Fig. 2). The EC forms 28 non-bonding interactions and two hydrogen bonds (through Leu-210 and Asn-247) with the binding site stabilizing the protein-ligand complex (Table 1). Of eight interacting residues, Lys-244 was proposed to be the key interacting residue as it was involved in maximum number (10) of non-bonding interactions. The high absolute values for the dock score (-32.01), binding energy (-7.14 kcal/mol), and dissociation constant (pKd, 5.23) also suggested towards quality binding of EC to NF-κB protein and therefore it may serve as a good NF-κB inhibitor for DNA binding activity (Table 2).

Figure 1: Two dimensional schematic representations of four catechin derivatives EC, EGC, ECG, and EGCG. Click here to view figure

Figure 2: Molecular docking of EC to DNA binding site of NF-κB. Click here to view figure

Table 1: The transcription factor NF-κB residues interacting with EC are listed with the number of non-bonding contacts and hydrogen bonds.

Molecular Docking of EGC to NF-κB

The molecular docking results showed that EGC bound well to the NF-κB binding site and interacted with seven residues namely Tyr-60, Asp-209, Leu-210, Ser-211, Lys-244, Pro-246 and Asn-247 (Fig. 3). The seven interacting residues formed 24 non-bonding interactions and two hydrogen bonds (through Leu-210 and Asn-247) with EGC stabilizing the protein-ligand complex (Table 3). Of seven interacting residues, Lys-244 was proposed as the key interacting residue as it was involved in maximum (9) non-bonding interactions (Table 3). In addition to 24 non-bonding interactions and two hydrogen bonds, the high absolute values of dock score (-33.12), binding energy (-7.08 kcal/mol), and dissociation constant (pK_d, 5.19) also indicated towards quality docking and thus EGC may also serve as potential inhibitor for DNA binding activity of NF-κB (Table 2).

Figure 3: Molecular docking of EGC to DNA binding site of NF-κB. Click here to view figure

Table 2: The values for the binding strength scores (dock score, binding energy, and K_d, dissociation constants) are provided for the docked catechin derivatives.

Table 3: The transcription factor NF-κB residues interacting with EGC are listed with the number of non-bonding contacts and hydrogen bonds.

Molecular Docking of ECG to NF-κB

The molecular docking of ECG to NF-κB revealed that ECG bound well in the binding site and interacted with 10 residues including Tyr-60, His-144, Thr-146, Lys-147, Asp-209, Leu-210, Ser-211, Lys-244, Ala-245 and Pro-246 (Fig. 4). These 10 residues formed 46 non-bonding interactions and four hydrogen bonds making the protein-ligand complex stable (Table 4). Of 10 interacting residues, Lys-244 was found to be the key residue as it was involved in the maximum number (10) of non-bonding interactions and one hydrogen bond (Table 4). The absolute values of binding strength scores: dock score (-43.72), binding energy (-7.84 kcal/mol), and dissociation constant (pK_d, 5.75) indicated towards quality binding and thus ECG may serve as a potential NF-κB inhibitor for DNA binding activity (Table 2).

Figure 4: Molecular docking of ECG to DNA binding site of NF-κB. Click here to view figure

Table 4: The transcription factor NF-κB residues interacting with ECG are listed with the number of non-bonding contacts and hydrogen bonds.

Molecular Docking of EGCG to NF-κB

The molecular docking results showed that EGCG bound deep in the NF-κB binding site and formed interaction with 10 residues namely Tyr-60, His-144, Thr-146, Lys-147, Asp-209, Leu-210, Ser-211, Lys-244, Ala-245 and Pro-246 (Fig. 5). The ten interacting residues exerted 47 non-bonding interactions and three hydrogen bonds with EGCG and made the protein-ligand complex stable (Table 5). Of 10 interacting residues, Lys-244 was found to be the key residue as it was involved in maximum non-bonding interactions (11) and one hydrogen bond (Table 5). Further, the high absolute values of dock score (-42.70), binding energy (-7.78 kcal/mol), and dissociation constant (pK_d, 5.70) also suggested that EGCG bound tightly to NF-κB and thus may act as a potential inhibitor for DNA binding activity.

Figure 5: Molecular docking of EGCG to DNA binding site of NF-κB. Click here to view figure

Table 5: The transcription factor NF-κB residues interacting with EGCG are listed with the number of non-bonding contacts and hydrogen bonds.

Comparative Binding Analyses of Catechin Derivatives

It is noteworthy that all the interacting residues of EC and EGC were common except for one residue of EC, Lys-147 (Table 2) and the binding scores were comparable i.e., dock score (EC, -32.01; EGC, -33.12), binding energy (EC, -7.14 Kcal; EGC, -7.08 Kcal) and pK_d (EC, 5.23; EGC, 5.19) indicating towards similarity in binding pose and strength (Table 6). This finding is in agreement with what is expected as the structure of EC and EGC are similar (Fig. 1). Similarly, the binding pattern of ECG and EGCG were found similar as both have same set of ten interacting residues (Table 6) and have comparable binding scores i.e., dock score (ECG, -43.72; EGCG, -42.70), binding energy (ECG, -7.84 Kcal; EGCG, -7.78 Kcal) and pK_d (ECG, 5.75; EGCG, 5.70) as shown in Table 2. This property of similarity in binding pattern is also attributed to similarity in structures of ECG and EGCG (Fig. 1). However, the binding affinity order is ‘ECG ≈ EGCG > EGC ≈ EC’ based on the binding scores. Collectively, all the four catechin derivatives have six common interacting residues (Tyr-60, Asp-209, Leu-210, Ser-211, Lys-244 and Pro-246) and interestingly the residue Lys-244 was identified as the key residues in all cases as it was involved in the maximum number of non-bonding interactions. These findings indicated towards overall similarity in the binding of catechin derivatives in the DNA binding site of NF-κB.

Table 6: Comparative binding analyses of catechin derivatives in the binding site of NF-κB. Each column contains the interacting residues with the catechin derivative mentioned on the top of the column. The common interacting residues for different derivatives are placed in the same row.

Conclusion

The molecular docking results suggest that the green tea catechin derivatives, EC, ECG, EGC, and EGCG showed substantial binding to NF-κB and may inhibit DNA binding activity of NF-κB, which may result in inhibition of NF-κB mediated transcription of target genes. The anti-cancer properties of these compounds are well established, and NF-κB is a known therapeutic target. Therefore, this study proposes the catechin derivatives as a potential inhibitor of NF-κB and hence downstream pathways, which may prevent or minimize the onset and progression of various cancer types. The order of their binding affinity in decreasing order based on the dock scores, binding energy and dissociation constant values are as follows: ECG ≈ EGCG > EGC ≈ EC. In general, all these four catechin derivatives have high binding strength and may act as a potential inhibitor of NF-κB mediated carcinogenesis. However, the ECG (the top scorer) and EGCG (2nd rank), having a similar structure, specifically showed high comparative values for the binding affinities. Thus these may be proposed as potentially effective inhibitors of NF-κB for DNA binding activity.

Acknowledgements

The authors are thankful to School of Life & Allied Health Sciences, The Glocal University, Saharanpur, Uttar Pradesh, India for the facilities provided.

Conflict of Interest

The authors confirm that they do not have any conflict of interest.

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