Tangeretin and its Derivatives: An Integrative Bioinformatic Study of Obesity and Related Immunodeficiency

Obesity is a complex and major public health concern known to exacerbate many diseases. There are increasing evidences stating the obese people due to adiposity are getting more susceptible to immune deficiency disorders. Tangeretin is a key member of flavonoids reported to have many favourable biological activities. In search of novel leads in ameliorating obesity and related immunodeficiency, the present study is aimed at the in silico evaluation of tangeretin derivatives to assess their biological role. Initially tangeretin derivatives are designed by molecular manipulation approach.Drug likeness and bioactivity score prediction was done using Molinspiration web tool. Swiss ADME prediction and toxicological predictions were performed. In silico Molecular Docking studies were performed by employing a flexible ligand docking approach using Schrodinger on the protein targets namely leptin, Fat mass and obesity associated protein (FTO), Pancreatic lipase, Peroxisome proliferated receptor (PPARɣ) and NADH oxidase. Further the electronic parameters were computed for the best fitted ligands by DFT analysis. The evaluation of results was made based on Glide (Schrodinger) dock score. Out of 18 screened compounds, some of them showed the best docking scores with the targets when compared with the standard (Lovastatin). Particularly the two ligands (L-13 and L-8) showed the best binding score with all five targets. Moreover, DFT analysis carried out for the tangeretin and best fitted ligands (L13 and L8) substantiated the other in silico studies. These findings probably provide excellent lead candidates for the development of therapeutic drugs in combating obesity and related immune deficiency. Original Research Article Sruthi et al.; JPRI, 33(50B): 247-266, 2021; Article no.JPRI.77177 248


INTRODUCTION
Obesity is a major public health concern all over the world and its prevalence is increasing day by day. It is a major contributor to the global burden of chronic diseases and complications including cardiovascular disorders, diabetes, renal toxicity, inflammation related disorders etc [1]. Less well known is the fact that there is a link between obesity and impaired immune function. There are increasing evidences stating the obese people due to adiposity are getting more susceptible to infectious diseases. Further as per reports of WHO it was clear that of 2.5 million COVID-19 deaths reported in February 2021, 2.2 million were reported in countries where more than half of the population are classified as overweight [2]. Hence there is a dire need to concentrate on the discovery of drugs to combat obesity and also to enhance the immune responses.
Flavonoids have a potential role in combating many disorders including obesity and also play vital role in enhancing immunity [3]. Among various flavonoids tangeretin is a key member of flavonoids having favourable biological activities, which have a prospect to develop as novel leads in drug discovery [4].Presently in the drug discovery process, in silico methodologies have become a crucial part and playing an everincreasing role. These computational strategies can impact the entire drug development trajectory, identifying and discovering new potential drugs with a significant reduction of cost and time [5]. Hence in view of scope to design new derivatives and assess their biological role, the present study is planned to use computational studies in screening some semisynthetic tangeretin derivatives and predict their probability in developing as novel leads in drug discovery to ameliorate obesity and related immunodeficiency.

Prediction of Molecular Properties
Tangeretin derivatives are designed by molecular manipulation approach and the chemical structures for the proposed compounds were drawn using Chem draw software. Lipinski's rule of five was employed to determine drug likeness and also to estimate whether a chemical substance predicted is possessing with some biological activity consists of properties to be orally active [6]. By using Molinspiration, an online server calculation of essential molecular properties like "molecular weight, log P, hydrogen bond acceptor and donor of selected ligands" for selected ligands was performed.

Calculation of Bioactivity Score
Bioactivity of different selected ligands was determined by determining the activity score of "GPCR ligand, ion channel inhibitor, Kinase inhibitor, nuclear receptor ligand, protease inhibitor, enzyme inhibitor using molinsipartion server".

ADMET Studies
ADME properties of a compound were estimated using Swiss ADME and PreADMET web tools in which, various physicochemical, pharmacokinetic, drug-likeliness, GI absorption, BBB permeability and skin permeability and toxicity can be predicted [7].

In silico molecular docking studies
The tangeretin and its derivatives were docked against five protein targets namely Leptin, Fat mass and obesity associated protein (FTO), Pancreatic lipase, Peroxisome proliferated receptor (PPARɣ) and NADH oxidase (NOX 4) and using Schrodinger Glide software (Version).

Preparation of ligand
The 2D structures of the prepared ligand were downloaded in the SDF format from Pubchem online data base. These molecules were then prepared in Schrodinger Ligprep wizard. In ligand preparation all possible conformations were taken into account. The ligands were then subjected to further predocking preparations where hydrogens were added followed by minimization and optimization of force field and finally in working ligand directory files were created.

Preparation of protein
The protein structure codes for theLeptin (1AX8), Fat mass and obesity associated protein (FTO) (3LFM), Pancreatic lipase (1LPB), Peroxisome proliferated receptor (PPARɣ) (2PRG) and NADH oxidase (NOX 4) (3A1F) were obtained from protein data bank (PDB) online data base (https://www.rcsb.org/).The proteins were prepared using Schrodinger's protein preparation wizard by removal of crystallographic water molecules and addition of hydrogen atoms, followed by minimization and optimization using force field of Schrodinger.

Grid Preparation and Docking
By applying Maestro search in Glide receptor grid was generated by specifying the binding site residues using site map tool. Upon the preparation of the grid for each protein, ligands were docked to each protein using "Extra precision mode (XP)" and the docked conformers are assessed by employing Glide (G) Score [8].

DFT Analysis
Density functional theory (DFT) is a computational quantum mechanical modelling method used to examine the electronic structure and also to investigate the interactions involved between the receptors and the ligands. The electronic and structural properties of the two best ligands along with tangeretin were calculated using the Becke3-Lee-Yang-Parr (B3LYP) method with the 6-31G(d,p) basis set aided by Gaussian 09. The computed parameters include the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) energies, Mulliken charge analysis, and Reactive descriptor values [9].

Prediction of Molecular properties
The proposed tangeretin derivatives with their structures, IUPAC name generated using Chem draw software were mentioned in Table 1. Molecular properties of the selected compounds are read using Molinspiration software to satisfy lipinski's rule of five, which is essential for rational drug design. Most of the selected compounds have number of violations equal to zero. Apart from the standard (L19), L14 and L15 showed high molecular weight and hydrogen bond acceptors. All the other designed compounds obeyed Lipinski's rule of five for drug likeness ( Table 2).

Calculation of Bioactivity Score
The predicted bioactivity scores of the screened compounds against various types of receptors were found be in the range of -5.0 to 5.0 which indicates that the compounds have ability to possess moderate to good activity towards biological targets (Table 3).

ADMET Studies
The ADME predicted are summarized in Table 4 and it could be observed that except L15 and L19 all the compounds have shown promising human intestinal percentage absorption and varied cell permeability with Caco2.
The compounds skin permeability was also found to be within permissible limits. The computed distribution and metabolic parameters showed the compounds have low BBB permeability. Further, all the compounds were shown to exhibit significantdrug interactions through inhibition of CYP and by toxicity parameters it is observed that there is low to medium risk of hERG inhibition.

Molecular Docking Studies
The results were represented in the terms of Glide docking score, Glide energy, H-bonds and nonbonded interactions (van der Waals and Coulombic) and mentioned in Table 5,6,7,8 and 9. The validation of the modelled proteins structures was done using the Ramachandran plot was represented in Fig 1. The ligand interactions are shown in theLigand interaction tool of maestro (Schrodinger) and it was observed that in case of all the targets some of the compounds exhibiting potent score compared with the standard.Most of the compounds showed hydrogen bond interactions and also hydrophobic interactions in the active site of proteins. The more negative values of the glide docking score represent tighter binding to the targets. Among all the compounds L 13 and L8 showed the best G score with all the protein targets[ Fig 2&3].

DFT Analysis
The DFT analysis performed for the tangerertin and selected L13 and L8 compounds showed the energy gap between the LUMO and HOMO ( Fig  4). Further Mulliken charge analysis was represented in Table 10. The computed reactive descriptors include ionization potential, electron affinity, chemical potential, chemical hardness, softness, electronegativity and electrophilicity index were represented in Table-11 clearly supported the ability of the compounds to bind with the receptors and revealed that L 8 and L13 have good chemical reactivity and charge transferability.

DISCUSSION
In the presentscientific world, in silico prediction is a valid alternative to experimental studies and plays a major role in selection of hit molecules from large library in drug discovery process.
Because of the wide applications in evaluating bioactive substances and their physicochemical and pharmacokinetic propertiesin the area of research and development these in silico studies has been gaining immense importance. This helps to predict numerous failure that arise in the process of new drug development [10].  A drug will be potent when it reaches its active target in the body at sufficient amount and produces biological effect in its active form. About 40% of the candidate compounds not being marketed is due to their poor biopharmaceutical properties (drug likeliness) [11].Prediction of drug likeliness properties like Lipinski's rule of five, bioactivity score and ADME properties has been used immensely to filter out undesirable compounds in early phases of drug discovery [12]. In the current study, tangeretin and its derivatives possess all the drug like properties and these findings come in accordance with the earlier scientific reports of bioactive phytoconstituents and it strongly supports that these can be considered as drug candidates for further studies [13].

Caco-2 permeability (Colorectal Adenocarcinoma cells permeability), GI (Gastro intestinal absorption), BBB (Blood Brain Barrier)
Molecular docking was performed to predict the preferred orientation and binding affinity of molecules to receptor/binding site/an enzyme.Glide was used to calculate the docking score and binding free energy of molecules with proteins. Forin silico assessment of the ability of compounds in ameliorating obesity and related immunodeficiency the proposed compounds were docked with the five targets Leptin, Fat mass and obesity associated protein (FTO), Peroxisome proliferated receptor (PPARɣ), NADH oxidase (NOX 4) and Pancreatic lipase.

Fig. 2. Binding interactions (2D) of Ligands with Leptin -1AX8
The leptin receptor is crucial for energy homeostasis and regulation of food uptake. The rational design of leptin agonists/antagonists could be an appealing challenge in the battle against obesity [14]. Many of the ligands like L5, L8, L11and L13showed good Glide score near to that of standard and it was observed that these ligands had made prominent hydrophobic interactions with the Asp 40 and Leu 39 of Leptin. These findings come in accordance with the earlier findings on computational studies of other phytoconstituents which played a potential role in combating obesity [15].
Fat mass and obesity associated protein is mainly associated with energy and body weight regulation. Inhibition of FTO is a main target in controlling obesity and in the current study L8, L9, L13and L16 showed high glide score nearly to that of standard and it was revealed that they show prominent pi-pi stacking with HIS 231 and TYR 108 and hydrogen bonding interactions with ASP 233, GLU 234 and ARG 96. These studies were in harmony with the previous findings on molecular docking studies on FTO protein for flavonoids like quercetin, Kaempferol which proved to be used in attenuating obesity [16].
Pancreatic lipase is an essential enzyme recognized for the digestion and absorption of lipids and reported to be a promising drug target towards the future development of antiobesity therapeutics in the cure of obesity disorders [17].In present investigation many of the compounds showed better Glide score than that of standard. L13 and L8 showed the best glide score by exhibiting pi-pi staking with HIS 263 and TYR 114 and hydrogen bonding and hydrophobic interactions with ASP79 and PHE77. These findings are in line with earlier scientific reports on various bioactive phytoconstituents which binds to the pancreatic lipase enzyme and interrupt the conformational changes required for the fat hydrolysis [18].
PPARγ plays an important role in regulating lipid metabolism, insulin sensitivity, and glucose homeostasis and their agonist are used in treating hyperlipidemia. Interestingly PPARγ is prominently involved in maturation and function of various immune system-related cell types [19]. In the present study, the molecular docking studies result in high glide score for most of the proposed compounds mainly L13, L18 and L8 exhibited prominent binding interactions with the receptor by forming hydrophobic interactions with ARG 288, GLU343, TYR 327 sites. These studies are in accordance with the earlier reports which revealed that some phytoconstituents like flavonoids and other phenolic derivatives possess both anti obesity and immunity enhancing capability [20].
NADH oxidase (NOX4) is an enzyme that exhibits vital role in free radical scavenging which inturn leads to the importance in controlling adipogenesis and regulating immune deficiency disorders [21]. The docking studies in the current screening revealed that L8 had showed the best glide score followed by L18, L11 and L13 by forming strong hydrophobic interactions with GLU 156 and GLH 104. These observations come in harmony with various previous scientific reports on phytocompounds possessing both antioxidant and antihyperlipidemic potential [22].
Upon Molecular docking analysis of all the proposed compounds with different targets it is clearly evident that L8 and L13 exhibited efficient docking score with all proteins. This may be due to the modification of OCH3 group at the fifth position of tangeretinwith polar groups OH and COOH groups in L8 and L13 respectively.

Fig. 4. Energies for the HOMO and LUMO of Selected compounds
Further DFT analysis carried out for the selected potent Ligands and tangeretin substantiated the molecular docking and other in silico predictions. The higher HOMO value states that molecule with a good electron donor, whereas a lower value indicates a weak electron acceptor. Moreover, a smaller energy gap between the LUMO and HOMO energies has a considerable influence on the intermolecular charge transfer and bioactivity of molecules [23]. In the current study the tangeretin and its derivatives L8 and L13 were found to possess less energy gap which supports their ability to binding with receptors. The reactive descriptors like chemical potential (μ) indicate negative values for all the compounds, which implies good stability, and the formation of a stable complex with the receptor. The other reactive descriptors also clearly indicated that the analysed compounds possess better bioactivity and chemical reactivity with considerable intra-molecular charge transfer between electron-donor to electron-acceptor groups which comes in accordance with previous in silico studies on biologically active phytocompounds [24].

CONCLUSION
In the current computational study, it is evident that proposed tangeretinderivatives have marked binding ability with obesity and associated immune related targets. Hence, the present bioinformatic findings probably provide excellent lead candidates for the development of therapeutic drugs in combating the obesity and related immune deficiency.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.