Targeting NOX2:

An In-Silico approach with Phytoconstituents to Alleviate Oxidative Stress

Payal Shah^1,2, Gaurang Shah²*

¹Research Scholar, Gujarat Technological University, Ahmedabad.

²Department of Pharmacology, L. M. College of Pharmacy,

Navrangpura, Ahmedabad 380009, Gujarat, India.

*Corresponding Author E-mail: gaurang.shah@lmcp.ac.in

ABSTRACT:

The NADPH oxidase (NOX) enzyme family is essential for producing reactive oxygen species (ROS), which act as signaling molecules in numerous physiological functions but can also lead to diseases related to oxidative stress when improperly regulated. NOX2, a crucial isoform mainly found in phagocytic cells, aids in microbial destruction during the respiratory burst through the generation of superoxide and hydrogen peroxide. Nonetheless, its excessive activation has been linked to cardiovascular illnesses, neurodegenerative conditions, and cancer. Utilizing the structural insights from the P22Phox-P47Phox complex (PDB ID: 1WLP), Cytochrome b558 (PDB ID: 1NG2), and NOX2 regulatory domains (PDB ID: 3A1F), this research explores the potential of phytochemicals to act as NOX2 inhibitors. Utilizing in-silico examinations, significant phytochemicals were discovered that exhibited high binding affinity against NOX2. These results establish a basis for creating specific treatments for disorders associated with oxidative stress and inflammation.

KEYWORDS: NOX2, NADPH oxidase, ROS, Molecular docking, Oxidative stress, Phytochemicals.

1. INTRODUCTION:

Among the various enzymes that produce Reactive Oxygen Species (ROS), the NADPH oxidase (NOX) family stands out for generating ROS as its main function. The NADPH oxidase (NOX) enzyme family features seven isoforms—NOX1 through NOX5, along with DUOX1 and DUOX2—that vary in their tissue locations, regulatory processes, and ROS generation¹. NOX enzymes facilitate the conversion of molecular oxygen to produce superoxide (O₂⁻) and hydrogen peroxide (H₂O₂), important ROS compounds that play a role in cellular signalling, immune reactions, and host defence mechanisms^2-4.

NOX2, the isoform that has been studied the most, is mainly found in phagocytic cells like neutrophils and macrophages. During the respiratory burst, NOX2 produces ROS in phagosomes to eradicate microbial pathogens⁵.

The NOX2 core complex, previously referred to as cytochrome b558, is made up of the catalytic gp91phox subunit and the supporting p22phox subunit. When activated, regulatory subunits (p47phox, p67phox, and p40phox) along with small GTPases such as Rac combine with the NOX2-p22phox heterodimer to create an enzymatically functional complex^6-9.

NOX2, referred to as gp91phox, is a crucial isoform primarily located in phagocytic cells, where it is essential for innate immunity by producing superoxide radicals during the respiratory burst. Impairment of NOX2 function leads to oxidative stress associated with different diseases. In cardiovascular conditions like atherosclerosis and hypertension, oxidative stress mediated by NOX2 worsens endothelial dysfunction and inflammation^10,11. In a similar manner, in neurodegenerative disorders such as Alzheimer’s and Parkinson’s, excessive NOX2 activation results in neuronal injury^12,13. Additionally, NOX2 is linked to tumour advancement and metastasis via ROS-driven signalling¹⁴.

The structural clarification of NOX2 and its regulatory complexes has enhanced our comprehension of its role. The crystal structures of the P22Phox-P47Phox complex (PDB ID: 1WLP), Cytochrome b558 (PDB ID: 1NG2), and the regulatory domain (PDB ID: 3A1F), offer essential knowledge about NOX2 activation and its subunit interactions. These frameworks act as important resources for the systematic creation of selective NOX2 blockers^15,16.

This research investigates the capability of phytochemicals as specific NOX2 inhibitors via in-silico, focusing on finding candidates that reduce NOX2-related oxidative harm while preserving immune functions.

2. METHODOLOGY:

2.1 Data assortment and processing:

Total 140 phytochemicals with known anti-oxidants activity were identified through extensive literature survey and their 3-dimensional structures were obtained from PubChem. The ligands were optimized and transformed into PDBQT format with OpenBabel software for further molecular modelling¹⁷.

The 3D crystal structures of the P22Phox-P47Phox complex (PDB ID: 1WLP), Cytochrome b558 (PDB ID: 1NG2), and the regulatory domain (PDB ID: 3A1F) were obtained from RCSB protein data bank. The proteins were prepared using AutoDock Vina for further process^18,19. To balance the protein's polarity and charges, all water molecules were removed and polar hydrogen atoms were introduced together with Kollman charges. Receptor grid box was generated having x, y and z co-ordinates 60 × 60 × 60 A˚ (1WLP), 126 × 126 × 126A˚ (1NG2), and 60 × 50 × 66 A˚ (3A1F), respectively. Co-ordinates for size and centres were set 3.40 × 8.57 × 3.10 A˚(1WLP), 17.246 × 49.571 × 10.014 A˚(1NG2) and 15.597 × -13.324 × 10.201 A˚(3A1F) for x, y, and z dimensions, respectively. Proteins were saved in PDBQT format to use it in molecular docking.

2.2 Molecular Docking:

The ligand molecules were positioned on the selected domains of NOX2 proteins with AutoDock Vina for assessing the binding strength and interactions, resulting in ten poses for each ligand. The protein-ligand interactions were visualized and analysed using Biovia Discovery studio 2020²⁰ and hits were chosen having binding energy below -6.0 kCal/Mol. Being a known and previously reported NOX inhibitor, Apocynin was selected as a control to compare binding affinity of other compounds^6,21,2.

3. RESULTS AND DISCUSSION:

Docking was performed on the active site at which grid boxes were generated on each protein. Compounds having better binding affinity than apocynin were observed and described in the Table along with residues involved in the polar interactions. Apocynin as a reported and known NOX inhibitor, observed having binding energy of between -4.6 to -5.1Kcal/mol.

Table 1 presents the molecular docking results of selected phytoconstituents against three structural domains of NOX2—namely 3A1F (regulatory domain), 1NG2 (cytochrome b558), and 1WLP (P22Phox-P47Phox complex). The table lists compounds that exhibited binding energies lower than -6.0kcal/mol, indicating potential inhibitory activity stronger than the reference compound Apocynin, which showed binding energies ranging from -4.6 to -5.1kcal/mol.

Each compound is accompanied by its calculated binding affinities for the three NOX2 protein structures, along with the specific amino acid residues involved in polar interactions such as hydrogen bonds, Pi-stacking, Pi-cation, Pi-anion, and Pi-sulfur interactions. Among the top-performing compounds were Gedunin, Theaflavin, Procyanidin, and Rutaecarpine, all of which displayed binding energies significantly better than Apocynin across all targets. This suggests a promising role for these phytochemicals as selective NOX2 inhibitors, warranting further investigation.

Table 1. Compounds having binding energy less than -6.0 Kcal/mol against any of the protein.

Phytoconstituents	Binding Affinity/Energy (kcal/mol)			Residues Involved in Polar Bonds
Phytoconstituents	3A1F	1NG2	1WLP	3A1F	1NG2	1WLP
Gedunin	-8.2	-8.8	-8.0	LYS137, VAL177	ILE164, ALA165, MET175	GLN281
Theaflavin	-7.5	-9.0	-8.2	GLU18, LYS53, HIS143 Pi-Cation - LYS54	GLU218, LYS317, ARG318, LEU319, TYR324 Pi-Cation - ASP221	ASP166, GLU244
Procyanidin	-7.5	-8.2	-8.1	ASN169, GLY176, VAL177 Pi-Cation - ARG175	ALA207, TRP264, ILE295, ARG296 Pi-Sulfur-MET278	GLU211, GLU220, TYR 274 Pi- Anion ASP261
Celastrol	-7.4	-8.5	-8.2	LYS137, ASN169	LEU280 Pi-Sigma- MET278	Pi-Cation- HIS257
Ginkgolide B	-7.4	-8.4	-8.3	LYS137, ASN169, VAL177	ILE164, CYS196	SER208, ASP261
Scutellarin	-7.1	-7.5	-8.7	ASN169, ARG175, Pi- Sigma – LYS137, ALA140	MET278, LEU280, ARG296	LEU210, GLU211, LEU260, ASP261
Ruteacarpine	-7.0	-9.3	-8.8	GLU154 Pi-Sulfur – CYS153	GLU192, Pi-Pi-stacked TRP194	ASP261, Pi-Sigma- SER208, Pi-Pi-T-shaped TRP194
Tanshinone II A	-6.7	-8.4	-7.8	LYS137, ASN169	Pi-Sigma TRP194	Pi-Sigma THR219
Apigenin	-6.9	-8.7	-7.8	GLU184, PHE186, Pi-cation LYS183	GLU262, TRP264, MET278, LEU280	Pi-Alkyl PRO16, ARG21
Hesperidin	-6.9	-9.0	-8.5	ASN169 Pi-Sigma LYS137	ALA165, MET175, CYS196 Pi-Pi stacked TRP194	SER208, LEU210, GLU211, GLU220, TYR274
Morindone	-6.4	-8.1	-7.1	LYS137, SER168	TRP194	Pi- Sigma THR219
Ellagic acid	-6.3	-7.6	-7.4	LYS137, ASN169, VAL 177	TRP194	GLN7, GLU174, Pi-Pi-T shaped PHE195, TRP204
Emodin	-6.5	-8.1	-7.0	ASN169, ARG175	TRP 194	THR219
Quercetin	-6.8	-7.4	-7.2	LYS137, ASN169, GLU171	SER173, MET175	PRO24, THR219, ASP261 Pi-Anion GLU211
Curcuminoid	-6.1	-7.9	-7.0	SER34, TRP69	TRP264, ARG296 Pi-Sul fur MET278	HIS257, Pi-Sigma THR219 Pi-Anion ASP261
Naringenin	-6.5	-7.3	-7.4	LYS137, SER141, SER168, ASN169, GLU171	MET175, Pi-Pi stacked TRP194	Pi-Anion GLU211 Pi- Alkyl ALA20, ALA207
Berberine	-6.7	-8.3	-7.3	LYS137, ASN169, ARG175, Pi-Anion GLU171	MET175, CYS196	Alkyl/Pi- Alkyl PRO16, ALA 20, ARG21, TRP194, PRO212
Epigallocatechin	-6.6	-7.2	-7.0	SER168, ARG175	TYR274, ARG301, ARG302, SER303, Pi-cation ARG316	SER5, LYS6, GLN7, Carbon Hydrogen bond TRP204, Pi-Pi T shaped PHE195
Resveratrol	-5.5	-7.0	-6.5	ILE167	ALA207, ASP261, Pi Sulfur- ARG296, Pi-Cation MET278	ALA20, LYS23, THR219, ASP261, Pi- Anion GLU211
Apocynin	-4.6	-5.1	-5.1	ASN169, ARG175	TRP194	Carbon Hydrogen GLU218, LEU260, Pi-Sigma THR219

Figure 1 describes the interaction of top molecules with 3A1F. Gedunin interacts through hydrogen bonding with GLY176, and LYS137. Along with this, it makes alkyl bonds with ALA140 and PRO144 of 3A1F. Theaflavin makes hydrogen bonds with amino acids HIS143, GLU18 and GLY88. It also interacts with LYS54 with a Pi-cation interactions.

Figure 1. (A) Interactions of Gedunin with protein 3A1F, (B) Interaction of Theaflavin with protein 3A1F.

Figure 2 shows the interaction of top molecules with 1NG2. Rutaecarpin makes hydrogen bond with GLY192, and alkyl bonds with CYS196, MET175 and LEU177 of protein 1NG2. Hesperidine was found to interact through hydrogen bonds with ASP166, ALA165, CYS196 and MET75. It also makes alkyl bond with VAL183 and TRP194.

Figure 2. (A) Interactions of Rutaecarpine with protein 1NG2, (B) Interaction of Hesperidine with protein 1NG2.

Rutaecarpine makes hydrogen bonds with ASP261, alkyl bonds with ALA20, ARG21, Pro16 and ALA207. Pi-Sigma interaction was also observed with SER208. Scutellarin was found having hydrogen bonds with LEU210, GLU211, GLU218, LEU260 and ASP261. It also interacts with alkyl bonds with amino acids ARG21, PRO16 and ALA20 of 1WLP protein describe in the Figure 3.

Figure 3. (A) Interactions of Rutaecarpine with protein 1WLP, (B) Interaction of Scutellarin with protein 1WLP.

4. CONCLUSIONS:

The in-silico analysis identified several phytoconstituents with strong binding affinities toward key NOX2 domains, indicating their potential as effective NOX2 inhibitors. These compounds, as listed in Table 1, demonstrated greater inhibitory interactions than the reference compound Apocynin. Therefore, they represent promising candidates for the development of selective, plant-based NOX2 inhibitors and warrant further validation through in vitro and in vivo studies.

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Received on 01.02.2025 Revised on 22.05.2025

Accepted on 21.08.2025 Published on 01.12.2025

Available online from December 06, 2025

Research J. Pharmacy and Technology. 2025;18(12):5758-5762.

DOI: 10.52711/0974-360X.2025.00830

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