Epigallocatechin Gallate (EGCG) – A Novel Covalent NF- kB Inhibitor: Structural and Molecular Characterization

Research Article

J Cardiovasc Disord. 2021; 7(1): 1041.

Epigallocatechin Gallate (EGCG) – A Novel Covalent NF- κB Inhibitor: Structural and Molecular Characterization

Reddy AT*, Lakshmi SP, Varadacharyulu N.Ch and Kodidhela LD

Department of Biochemistry, Sri Krishnadevaraya University, Anantapur, Andhra Pradesh, India

*Corresponding author: Aravind T Reddy, Department of Biochemistry, Sri Krishnadevaraya University, Anantapur, AP, India

Received: May 08, 2021; Accepted:May 31, 2021; Published: June 07, 2021


Tea contains antioxidant catechins thought to exert health-promoting protective effects against conditions involving chronic inflammation, such as cardiovascular diseases. The most abundant catechin in tea is Epigallocatechin Gallate (EGCG), thought to be a key contributor to tea’s health-promoting actions. EGCG exerts protective cardiovascular effects via its antioxidant, antiinflammatory, hypolipidemic, anti-thrombogenic, and anti-hypertensive actions. Because EGCG inhibits the strong proinflammatory gene-inducing transcription factor NF-κB, we analyzed the chemical and molecular details of the mechanism by which EGCG mediates NF-κB inhibition. We quantified and mapped key parameters of its chemical reactivity including its electrophilic Fukui ƒ+ function, in silico covalent binding, and identified its frontier Molecular Orbitals (MOs) and nucleophilic susceptibility. These physical and chemical reactivity parameters revealed that the bond-forming MOs are distributed on the B ring of the EGCG oxidized state with nucleophilic susceptibility, and that this B ring has properties that favor participating in a Cys-alkylating 1,4-addition reaction. Molecular modeling and docking analysis further revealed that EGCG bonds covalently with Cys-38 of NF-κB-p65, and thereby inhibits its DNA binding ability. We also generated a model pharmacophore based on the EGCG-NF-κB complex. We conclude that EGCG covalently binds to NF-κB-p65 and inhibits it by abolishing its DNA binding, by chemical mechanisms that may inform design of EGCG derivatives as novel anti-inflammatory agents.

Keywords: Epigallocatechin gallate; NF-κB; Covalent bond; 1,4 addition; Cys-alkylation; Molecular modeling; Pharmacophore; Tea


EGCG: Epigallocatechin Gallate; NF-κB: Nuclear Factor- κB; RHD: Rel Homology Domain; IκBs: Inhibitory κB Proteins; Cys: Cysteine Residue(s); CVDs: Cardiovascular Diseases; MOs: Molecular Orbitals; CΒ: Β-Carbon; DFT: Density Functional Theory; LDA: Local Density Approximation; ADF: Amsterdam Density Functional; QTAIM: Quantum Theory of Atoms in Molecules; PM6: Parameterization Method 6; HOMO: Highest Occupied Molecular orbital; LUMO: Lowest Unoccupied Molecular Orbital; CHARMM: Chemistry at HARvard Macromolecular Mechanics; NP Dock: Nucleic Acid-Protein Docking; QZ4P: Quadruple Zeta with 4 Polarization Functions; H: Hydrogen; HBA: H Bond Acceptors; HBD: H Bond Donors; HY: Hydrophobic; CADD: Computer-Aided Drug Design


Cardiovascular Diseases (CVDs) are the leading cause of death worldwide, and will give rise to a predicted increase in annual deaths from 17.5 million in 2012 to 22.2 million by 2030 if current trends persist [1]. CVDs including congestive heart failure, stroke, ischemic and coronary heart disease, coronary artery disease, and peripheral vascular disease [2] inflict high societal costs, and >75% of deaths in countries of low and middle incomes. Worldwide, millions of people strive to control CVD risk factors, while others are unaware of the risks [3]. Inflammation and apoptosis are major pathogenic contributors to these conditions, and induction of many of the pathways involved is heavily mediated by activation of the transcription factor nuclear factor-κB (NF-κB) [4]. CVDs in which NF-κB activation plays an essential pathogenic role include myocardial infarction [5], ischemia/ reperfusion injury [6], transplant rejection [7], angina pectoris [8], autoimmune myocarditis [9], congestive heart failure [10], and cardiomyocyte hypertrophy [11]. Therefore, modulators of NF-κB activity can influence these conditions.

NF-κB consists of a group of structurally-related transcription factors including NF-κB1 (p50/p105), NF-κB2 (p52/p100), RelA (p65), RelB, and C-Rel, each characterized by a highly conserved Rel homology domain (RHD), the domain which regulates its interaction with inhibitory κB proteins (IκBs), dimerization, and DNA binding to evoke changes in target gene expression [12]. Interactions among NF-κB family members lead to formation of homodimers or heterodimers, among which the most abundant and wellcharacterized is the p50/p65 heterodimer [13]. The p50/p65 dimer interacts with consensus DNA sequences known as κB motifs, which are located in promoter or enhancer regions of target genes, and consist of 5'-GGGRNNYYCC-3', where R is an unspecified purine, Y is an unspecified pyrimidine, and N is any nucleotide [14]. As a result of its activation by cytokines, pathogens, and other stressful conditions, NF-κB induces production of numerous inflammatory mediators including cytokines, chemokines, adhesion molecules, inducible enzymes, and growth factors [15,16]. A highly conserved cysteine residue (Cys-38 in human NF-κB-p65 RHD) is required for its interaction with κB DNA [17]. Several natural and synthetic antioxidant compounds that contain functional electrophilic carbons can inhibit NF-κB DNA-binding activity, by alkylating Cys-38 [18] via a 1,4-addition (S-alkylation) reaction [19].

Antioxidant polyphenolic catechins are present in many nutrientrich foods, such as fruits, berries, and leaves (especially tea), and their health benefits via such antioxidant properties have been wellestablished by in vivo and in vitro studies (reviewed in [20]). Many such beneficial antioxidant activities are attributed to flavonoids that contain dihydroxy or trihydroxy groups, and their antioxidant activity further increases with increasing content of these groups [21-23]. Among the catechins in tea, the most abundant is epigallocatechin gallate (EGCG; C22H18O11) is [24], which can covalently modify proteins and alter their functions [25-27]. Such chemical activities of EGCG reside in its two adjacent trihydroxy structures, the B (gallyl) and D (gallate) rings. These can readily undergo auto-oxidation to form a semiquinone that then rearranges to an electrondeficient and electrophilic Β-carbon (CΒ)- containing O-quinone, which is susceptible to nucleophilic attack by thiols. Such electrophilicnucleophilic attack forms EGCG-S-cysteinyl protein adducts.

EGCG exerts cardiovascular protection via its antioxidant, anti-inflammatory, hypolipidemic, anti-thrombogenic, and antihypertensive actions [28]. Antioxidant properties of catechins include free radical scavenging [29], metal ion chelation [30], inhibition of redox responses, and induction of antioxidant enzymes [31]. EGCG-mediated inhibition of NF-κB via multiple mechanisms [20] contributes to its anti-inflammatory activities. Catechins in tea also improve blood lipid profiles, regulate vascular tone, and impede progression of atherosclerotic lesions, by inhibiting cytokine production, inflammatory cell transmigration, platelet adhesion, and vascular smooth muscle cell proliferation [reviewed in [28]]. Experimental and clinical studies identified protective roles of EGCG in CVDs [32], attracting attention toward developing novel therapeutic strategies targeting Nrf2 activation and NF-kB inhibition [33].

We recently found that EGCG selectively and covalently binds to cysteinyl thiol of NF-κB via 1, 4-addition reaction and effectively suppresses its activation. The cysteine found as the reactive sulfhydryl moiety as S-carboxymethylation blocked the 1, 4-addition reaction between EGCG and NF-κB [34]. Based on our previous findings and EGCG’s biochemical properties, we hypothesized that EGCG covalently binds to NF-κB and inhibits NF-κB-p65’s DNA binding ability. To test this idea we analyzed the operant mechanisms and explored the potential for targeting the relevant sites pharmacologically for therapeutic benefits, by characterizing EGCG’s chemical reactivity and electrophilicity. We found that its oxidized B ring contains proton donating O-quinones and CΒs, which therefore readily undergo chemical reactions. Herein we describe further analyses of the frontier Molecular Orbitals (MOs), nucleophilic susceptibilities, molecular modeling and docking, and identified a new putative pharmacophore based on the EGCG-NF- κB complex. These findings will inform further in silico and in vitro research to enable design of novel EGCG derivatives as potential NF-κB inhibitors.

Materials and Methods

Computational methods

Structures of EGCG in the reduced state (EGCG-RS; C22H18O11) and oxidized state (EGCG-OS; C22H16O11) were generated in ChemOffice (v 17.0, CambridgeSoft, Cambridge, MA, USA). Geometric optimizations and all electronic structure calculations were performed as we described [35], using Density Functional Theory (DFT) by Local Density Approximation (LDA) exchangecorrelation and the QZ4P base set with Amsterdam Density Functional (ADF) Modelling Suite [36]. The critical points, bond paths, atomic properties and energies, and reactivity indices were analyzed via the Quantum Theory of Atoms in Molecules (QTAIM) proposed by Bader, as implemented in ADF. For semi-empirical quantum chemical calculations we used using Parameterization Method 6 (PM6) as implemented by SCiGRESS (v 2.8.1, Fujitsu Ltd., Tokyo, Japan). The resulting parameters, Energies of Highest Occupied Molecular Orbital (EHOMO), and Lowest Unoccupied Molecular Orbital (ELUMO) values were used in standard equations, to determine global chemical reactivity descriptors including hardness (η), chemical softness (σ), chemical potential (μ), electrophilicity (ω), nucleophilicity (ω-), and local reactivity descriptors including the Fukui functions (ƒ+ and ƒ-), Koopmans DD, and philicities.

Molecular modeling

We selected the X-ray structure 1vkx [14] from the RCSB Protein Data Bank to build docking receptors. NF-κB-p65/p50 heterodimer complexed with κB DNA, its C38S mutant (the cysteine at the residue 38 was substituted with a serine), p65 wild-type subunit, and p65 C38S mutant subunit with BIOVIA Discovery Studio (BIOVIA, San Diego, CA, USA). Energy minimization (constraining the heavy atoms) analysis was performed using Chemistry at HARvard Macromolecular Mechanics (CHARMM) force fields [37].

Molecular docking

Molecular docking studies were carried out as we described [38] with Discovery Studio. Briefly, the geometry-optimized EGCG structure, generated based on DFT was used as a ligand. The energyminimized three-dimensional structures and complexes (as described above) were used as receptor molecules to model covalent docking. We further characterized the lowest energy pose of the covalent EGCG-p65 wild-type complex to determine ring conformation changes and generate a pharmacophore model using the receptorligand complex based-common features mapping model.

In vitro electrophilic addition reaction

To determine the EGCG covalent adduction of NF-κB and the involvement of Cys residue an in vitro electrophilic adduction was performed as we described previously [34]. Briefly, unmodified NF- κB-p65 recombinant protein or S-carboxymethylated NF-κB-p65 protein was incubated with various concentrations of biotin taged EGCG. After incubation the formation of the covalent adduction was determined by Western blotting as we previously reported [38].

Nucleic acid–protein docking

We analyzed DNA-protein docking studies as we described [39] to identify interactions between EGCG-bound NF-κB and κB (5′-TGGGGACTTTCC-3′) in the nucleic acid-protein docking (NP Dock) server as described [40], with default docking parameters. We set the RMSD threshold 5 Å for clustering, and used the best-scored decoy in the clusters with the highest probability to identify DNA– protein interactions using Discovery Studio Visualizer.


Calculated chemical properties of EGCG

We determined electrophilicity of EGCG-RS (Figure 1A) and EGCG-OS (Figure 1B) by quantum chemical calculations. We analyzed chemical properties including chemical hardness (η), chemical softness (s), chemical potential (μ), electrophilicity (ω), and nucleophilicity or reactivity (ω-) indices (Tables 1 and 2). The data in Table 1 indicate that EGCG-OS is softer (s: 0.278 eV-1), more reactive (μ: -5.900 eV), and more highly electrophilic (ω: 4.835 eV) than EGCG-G.