Molecular docking studies of natural compounds of naringin on enzymes involved in the urea cycle pathway in hyperammonemia

Purpose: To investigate the anti-hyperammonemic activity of naringin by molecular docking via in silico studies. Methods: Urea cycle proteins were docked to the natural compound naringin as well as a standard drug, sodium benzoate. Hydrogen bonds and binding energy were obtained using Catalytic Site Atlas and Cast P Finder Software Tool. Results: There were six urea cycle enzymes, including N-acetyl glutamate synthase, carbamoyl phosphate synthase I, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase and arginase I. On evaluating protein interactions with naringin, which is dynamically connected to the urea cycle pathway with hyperammonemia, naringin showed more hydrogen bonds and also produced higher binding energy when compared to the standard drug, sodium benzoate. Conclusion: The results of the molecular docking study show that naringin interacts with urea cycle enzymes with more hydrogen bonds and higher bonding energy than the standard drug, sodium benzoate. This supports the hypothesis that naringin can prevent experimental hyperammonemia.


INTRODUCTION
Naringin is one of the natural flavonoids present in grapes and citrus fruits. The molecular formula of naringin is C27H32O14 and its molecular weight is 580.4 g/Mol. The chemical formula of naringin is 4',5,7,-trihydroxyflavonone-7-rhamnoglucoside. Naturally, it possesses a distinct bitter taste of grapefruit juice and has strong antioxidant properties [1]. Naringin is soluble in water and can be well absorbed from the gut [2]. It has been suggested that naringin has a variety of biological properties, including anti-ulcer [3], antidiabetic [4], renoprotective [5], cardioprotective [6], anti-cancer [7], anti-mutagenic [8], and antiinflammatory effects [9], as well as neuropro-tective activities [10]. Moreover, naringin has no significant side effects identified, as humans have been ingesting grapes and citrus fruits for a long time [11].
Ammonia is a neurotoxin in humans as well as other living organisms. Free ammonia (NH3) is also known to be a non-proteinaceous nitrogen compound which perfuses into the cells. NH3 is converted into urea, which takes place in the liver by six urea cycle enzymes. Later, the synthesized urea is removed via urine by the kidneys [12]. The activities of carbamoyl phosphate synthase I (CPS I), N-acetyl glutamate synthase (NAGS), and ornithine transcarbamylase (OTC) occur in the hepatic mitochondria, whereas argininosuccinate synthase (ASS), argininosuccinatelyase (ASL) and arginase I (ARG) act in the hepatic cytoplasm which regulates the urea cycle pathway [13]. Due to the deficiency of urea cycle enzymes, there is a significant increase in ammonia at the blood brain barrier, which triggers hepatic encephalopathy (HE) [14]. HE exhibits a substandard cerebral edema, such as nitrosative/oxidative stress, as well as consequent dearrangements of the signal transduction pathway, neurotransmission, oscillatory networks, and synaptic plasticity in the brain as the clinical manifestations [15]. It has been suggested that hyperammonemia could lead to the hyperactivation of NMDA receptors, ultimately causing neurotoxicity [16]. Sodium benzoate has been widely used as a preservative agent for food and beverages. In 1979, USA accepted the use of sodium benzoate for medicinal purposes, but the Food and Drug Administration (FDA) stated that its use should not exceed a maximum level of 0.1 %. Sodium benzoate has since been used as a marker for the treatment of HE patients with hyperammonemia, initially in those with urea cycle enzyme deficiencies and later in patients with cirrhosis [17]. Sodium benzoate utilizes the non-urea cycle pathway for ammonia removal in the liver. Clinical medicines which contain sodium benzoate and sodium phenylacetate have been approved by the FDA for patients with urea cycle disorders and hyperammonemia [18,19]. Until now, molecular mechanism triggering anti-hyperammonemic activity by naringin has not been explored. Hence, this study aimed to investigate the antihyperammonemic activity of naringin by identifying the interactions between naringin and the structure of urea cycle enzymes by molecular docking using in silico studies. It has been reported that naringin regulates the urea cycle enzymes in the liver in an ammonium chloride (NH4Cl)-induced hyperammonemic experimental rat model [10]. In the present study, we explored in silico properties such as interacting residues, binding energy, the number of hydrogen bonds and the bond length of urea cycle enzymes compared to the standard drug, sodium benzoate.

Preparation of ligand structures
The naringin and sodium benzoate structures were downloaded from the PubChem database and Drug Bank (<http: //www.drugbank.ca>). The downloaded naringin structure was converted from the .sdf format to .pdb using Openbabel. This method has frequently been used in the discovery and optimization of molecular interactions with target binding energy, interacting residues, number of hydrogen bonds and bond length characterization.

Catalytic site Atlas and Cast, P Finder Software tool
The catalytic site Atlas and Cast, P Finder software tool offers a broad range of predicted active sites. The heteroatoms were removed from the PDB file and energy was minimized using GROMOS96 to stabilize the protein molecule.

Molecular docking
To determine the structure of protein ligand interaction complex in docking, studies were carried out using Auto Dock 4. Hydrogen atoms, charges were added to protein structure and grid coordinates were calculated based upon the active site and high volume surface area of the protein. Grid co-ordinates were set to generate the grid box. The docked structure was analyzed and visualized using the Molegro molecular viewer.

RESULTS
The protein molecule crystal structure was downloaded from Protein Data Bank and visualized by PyMol. The energy was minimized using GROMOS96. Figure 1 A shows that the standard drug sodium benzoate docked with CPS I and the natural drug compound naringin. The level of binding energy of CPS I with the standard drug sodium benzoate was found to be -5.98 KJ/Mole, with three hydrogen bond interactions at SER625, VAL626 and THR627 (Table. 1). The level of binding energy of CPS I with naringin (Figure 1 B) complex was found to be -5.10 KJ/Mole with ten hydrogen bond interactions at SER588, GLU631, THR660, GLU714, ASN716, ARG718, SER720, ARG721 and LYS772.

OTC complex interacts with standard drug sodium benzoate and natural compound naringin
The protein molecule OTC is docked with sodium benzoate and naringin. Figure 2 A shows the molecular docking structure of sodium benzoate. OTC had a binding energy of -3.24 KJ/Mole, with three hydrogen bond interactions at SER90, THR91 and ARG92 (Table 1). In contrast, the structural affinity of naringin with the OTC complex appeared as -6.45 KJ mole binding energy with nine hydrogen bond interactions at ARG89, THR91, ARG92, TYR143, ASN198, ASN199, ASP263 and THR264 (Table 1). Figure  2 B shows that the naringin complex had better structural affinity than the standard drug sodium benzoate; this might be due to its natural origin with greater binding energy and an increased number of hydrogen interactions.

ASS complex interacts with standard drug sodium benzoate and natural compound naringin
When another target molecule, ASS, was docked with the standard drug sodium benzoate and the natural compound naringin, the binding energy between sodium benzoate and ASS was -4.80 KJ/Mole, with two hydrogen bond interactions at TYR11 and ALA36 (Figure 3 A). In contrast, the docked structure of naringin and ASS had binding energy of -12.92 KJ/Mole, with 11 hydrogen bond interactions identified at ALA10, SER12, GLN40, GLU42, TYR87, GLY117, SER180, MET181, GLU183 and TYR282 (Figure  3 B). Comparatively, naringin complex had better docking scores than the sodium benzoate complex, as evidenced by its higher binding energy and more hydrogen bond interactions.  Figure 4 A and B comparatively show that the sodium benzoate had slightly lower binding energy, but naringin had a higher binding energy and increased range of hydrogen bond interactions than sodium benzoate.

DISCUSSION
To estimate the best anti-hyperammonemic agent, the synthetic drug sodium benzoate [20] and natural compound naringin were docked with urea cycle pathway target molecules involved in the pathway to produce hyperammonemia. The drug sodium benzoate and compound naringin were docked with the protein after forming a grid; these complexes were analyzed. The best binding energy score was selected. The same structural binding predictions were followed for all of the protein and the most stable conformation was selected. A deficiency of CPS I could result in the accumulation of elevated levels of glutamine and alanine and reduced levels of citrulline and arginine in the liver tissue. The genetic location of the CPS I enzyme was chromosome 2p 35 [21]. This study was designed for the analysis of the interaction of CPS I with naringin via molecular docking. From this result, it has been suggested that naringin may modify CPS I through binding interactions.
Increased levels of glutamine and alanine and decreased levels of citrulline and arginine were observed in OTC deficiency in liver tissues of rats. The OTC enzyme gene is localized in X linked OTC (Xp21.1) [22,23]. In AL (argininosuccinic aciduria) and AS (citrullinemia) deficiencies, the level of plasma citrulline has been enhanced. If the level of serum citrulline is in the normal range, then disorders including lysinuric protein intolerance, argininemia, and the hyperammonemia-hyperornithinemiahomocitrullinuria syndrome should be measured.
The deficiency of argininosuccinate synthase enzyme is also known as citrullinemia, which refers to higher levels of citrulline and reduced levels of arginine in the liver tissue of rats. The gene sequences responsible for this disorder are present on chromosome 9q34. Affected patients are able to partially incorporate the waste nitrogen into urea-cycle intermediates [24], making the treatment much easier. Our findings support the ASS molecular docking analysis, and indicate that naringin could be rewired back to the ASS enzyme mechanism via binding interactions.
A deficiency of argininosuccinatelyase leads to the elevation of citrulline and argininosuccinic acid and decreased levels of arginine in the liver tissue of rats. The gene for this ASL enzyme is present on chromosome AR-ASL 7cen-q11.2 [22,23]. Our results suggest that the structure of naringin may alter the structure of ASL through the binding interaction. ASL deficiency is also known as argininosuccinic aciduria (ASA); the diagnosis is suspected by monitoring hyperammonemia in the presence of enhanced AL levels (without acidosis) in the plasma as well as the urine. Measuring the activity of AL range in erythrocytes or fibroblasts could verify the diagnosis [25]. Arginase I deficiency, also called hyperargininemia, causes increased levels of arginine in the liver tissue of hyperammonemic rats. The gene for this enzyme is found on chromosome AR-ARG1 6q23 [22,23]. Arginase deficiency may also cause systemic hypertension due to the secondarily reduced synthesis of nitric oxide. Conversely, excess argininosuccinic acid may be culpable for the liver disease observed in some ASL-deficient patients. Our findings suggest that naringin alters ARG through the structural binding affinity [26].

CONCLUSION
Naringin can play an important role in enzyme catalysis which depends on the delicate balance of energy for different reaction pathways while participating in energy-based metabolic reactions. Thus, naringin is the most active natural compound exhibiting more energy bonds and structural interactions with urea cycle enzymes than the standard drug. Hence, it is proposed that naringin can exert a potential chemopreventive effect on urea cycle disorders as well as hyperammonemic diseases. Since hyperammonemia is an important cause of neurotoxicity via the hyperactivation of NMDA receptor (26), it is reasonable to speculate that naringin may also have a neuroprotective effect. Therefore, this study reveals that naringin is a potentially useful compound for the prevention of neurodegenerative diseases.