Int J Pharm Pharm Sci, Vol 6, Issue 10, ??-??Original Article

MOLECULAR DOCKING STUDIES ON FLAVONOID COMPOUNDS: AN INSIGHT INTO AROMATASE INHIBITORS

INDU A. G¹, PUNNAGAI MUNUSAMI²*, C. S. VASAVI², DIVYA G²

¹Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum 695012, Kerala, India, ²Bioinformatics Division, School of Bio Sciences and Technology, VIT University, Vellore 632014, Tamil Nadu, India.
Email: punnagai.munusami@vit.ac.in

Received: 14 Aug 2014 Revised and Accepted: 15 Sep 2014

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ABSTRACT

Objective: Aromatase is a key enzyme that plays a crucial role in the synthesis of estrogen and has a major effect in pathogenesis of estrogen‑dependent disease, including breast cancer, endometrial cancer, and endometriosis. The abnormal over expression of aromatase can be inhibited by aromatase inhibitors. The objective of the present study is to evaluate the binding interaction of flavonoid compounds with cytochrome P450 enzyme aromatase, which is involved in the metabolism of estrogens and considered as a powerful target for treatment of estrogen-dependent breast cancers.

Methods: To understand the mechanisms involved in the binding of flavonoid compounds and their interactions with the binding site of aromatase, molecular docking studies were carried using Autodock 4.2.

Results: The docking results revealed that, benzoflavanones showed higher binding affinity compared to other class of compounds. The presence of hydrogen bond interaction and cation–π interaction contributed to their higher binding affinity. The flavonoid compounds with unsubstituted or less substituted rings showed higher binding affinity than those with substituted rings. The hydrogen bonding interactions were predominant in all the classes of compounds considered for the study and were found to be important for inhibition. The docking studies showed that the binding energies mainly depend on aromatic properties like cation–π and π–π interactions. These properties play a key role in determining the biological activity of flavonoid compounds.

Conclusion: The present findings provided valuable information on the binding process of flavonoid compounds to the binding site of aromatase and revealed the structural requirement needed for binding.

Keywords: Breast cancer, Estrogen, Aromatase, Aromatase inhibitors, Flavonoid compounds, Docking studies.

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INTRODUCTION

Breast cancer is the most frequently diagnosed cancer and leading cause of death in females, worldwide. Many breast cancers are sensitive to the hormone estrogen, which causes breast cancer tumor to grow. The cancer of this type is called as HER2 positive breast cancer. HER2 refers to a gene that helps cells to grow, divide, and repair themselves. Mutation or damage in this gene or in the cell signalling pathway, leads to cancer [1]. Since 2008, breast cancer incidence has increased by more than 20%, while mortality has increased by 14%. It is also estimated that breast cancer is the most prevailing cancer worldwide [2]. Other than radiotherapy, chemotherapy many other surgical approaches are used for the treatment [3]. Estrogen plays a crucial role by binding to estrogen receptors, in promoting the proliferation of both the normal and neoplastic breast epithelium by inducing intracellular signalling cascades [4,5].

Aromatase is the enzyme involved in estrogen biosynthesis, which can be inhibited for limiting the growth and development of estrogen dependent tumors. Human aromatase is the product of the CYP19A1 gene. This enzyme is located in the endoplasmic reticulum of the cell and its activity is regulated by tissue specific promoters that are in turn controlled by hormones, cytokines, and other factors. Inhibition of aromatase is an important approach for reducing growth-stimulatory effects of estrogens in estrogen-dependent breast cancer. Two primary approaches have been developed to reduce the growth stimulatory effects of estrogens in breast cancer like interfering with the ability of estrogen to bind to its receptor, and decreasing circulating levels of estrogen. Anti-estrogens (Tamoxifen) compete for binding to the estrogen receptors and reduce the number of receptors available for binding to endogenous estrogen [6]. Both steroidal and non-steroidal aromatase inhibitors have also shown clinical efficacy in the treatment of breast cancer. The potent and selective third- generation aromatase inhibitors, like anastrozole, letrozole, and exemestane were used in postmenopausal patients failing anti-estrogen therapy alone or multiple hormonal therapies. Use of an aromatase inhibitor as initial therapy or after treatment with tamoxifen is now recommended as adjuvant hormonal therapy for a postmenopausal woman with hormone-dependent breast cancer [7].

As like anti-estrogen, steroidal and non-steroidal aromatase inhibitors, flavonoids also play a major role in aromatase inhibition. Flavonoids are a group of phytochemicals produced by various plants in high qualities. Based on their skeleton, flavonoids classified to flavans, flavones, flavanones, isoflavones, isoflavanones, anthocyanidines, chalcones, flavanoligans and flavonols, each possessing the benzopyranone ring system as the common chemical scaffold. Flavonoids exert anti-inflammatory, analgesic, anti-allergic effects, apoptotic, estrogenic and anti-estrogenic properties. Flavonoids interacts with CYP450 enzymes in atleast three way: by inducing biosynthesis of several CYPs, by modulating the enzymatic biosynthesis of CYPs (inhibited or stimulated) and the last is the metabolism of flavonoids by several CYPs in turn. They poses both induction of CYP mediated carcinogenesis as well as inhibition of the same by scavenging free radicals formed by CYP catalysed reactions. As aromatase inhibitors, flavonoids has structure resembling the estrogen skeletons, they bind to the estrogen receptor in turn modulates its activity [8]. Flavones have been demonstrated to be competitive inhibitors of aromatase with respect to the androgen substrate, with K_i values at low micromolar concentrations. The binding characteristics and the structural requirement necessary for the inhibition of human aromatase by flavone and isoflavone phytoestrogens were obtained using computer modelling and confirmed by site–directed mutagenesis. It was found that these compounds bind to the active site of aromatase in an orientation in which their A-ring and C-ring mimic rings D and C of the androgen substrate, respectively [9]. Initial attempts to study the aromatase and its inhibitors interaction have been relied on homology derived models. At present there are crystal structures available for human placental aromatase. Thus the availability of the structural details of aromatase active sites help to understand the binding nature of aromatase inhibitors and also to evaluate key residues needed for binding. Molecular docking is one of the foremost approaches to understand the binding interactions that govern the enzyme inhibition [3]. In this study, docking calculations were performed to elucidate the binding modes of all flavonoid compounds with aromatase. The crystal structure of the human placental aromatase (PDB ID: 3EQM) solved by Gosh et al, 2009 [10] was considered for docking studies. The tertiary structure consists of 12 major helices and 10 strands distributed into one major and 3 minor sheet, and follow the characteristic CYP450 fold.

A striking feature of the tertiary aromatase structure is that long loops interconnect well–defined secondary structure elements, was observed with other P450 structures. The active site of aromatase is the distal cavity of the heme–binding pocket and the Heme Fe is the reaction centre of the enzyme. The amino acid Asp309 is proposed to be a catalytically important residue. The residue Asp309 appears to be involved not only in catalysis but also in substrate binding. Several flavonoid compounds were synthesized and tested for aromatase inhibitor [11-16]. These structural features play an important role in determining the binding mode of several drugs including aromatase inhibitors [17].

MATERIALS AND METHODS

Preparation of the protein and Ligand Structures

The three dimensional X–ray structure of aromatase complexed with androstenedione (ASD), was obtained from Brookhaven Protein Data Bank (PDB ID: 3EQM). The crystal structure was refined by removing water molecules and the cofactor, phosphate ion. The heme was retained in the binding site. Hydrogen atoms were added and electronic charges were assigned to the protein atoms using kollman united atoms force field by using AutoDockTools–1.5.6 [18].

To understand the binding nature of the ASD, the ASD was removed and redocked into the binding site of aromatase. The docking calculations were carried out using both the Genetic Algorithm and Lamarckian Genetic Algorithm.

The docked conformations obtained using LGA could able to reproduce the binding modes reported in the crystal structure solved by Ghosh et al. In this paper, the Lamarckian Genetic Algorithm (LGA) was used to explore the binding conformational landscape of flavonoid compounds. The flavonoid compounds considered for the present study are given in the Scheme 1 [11-16]. The compounds were geometrically optimized using AM1 Hamiltonian in MOPAC software package [19]. The gasteiger partial atomic charges were added and all possible flexible torsion angles of the ligand was defined by using AUTOTORS. The structures were saved in a PDBQT format for AutoDock calculations.

Docking Studies

Docking calculations were performed to predict the binding affinity between the aromatase and flavonoid compounds using AutoDock 4.2 [20]. AutoDock requires pre–calculated grid maps, one for each atom type present in the ligand being docked. These maps were calculated by using the auxiliary program AutoGrid. The compounds treated as flexible molecules were allowed to move in the six spatial degrees of freedom for orientation and torsional degrees of freedom within the grid box. Lennard–Jones parameters 12–10 and 12–6, implemented with the program, were used for modeling H–bonds and van der Waals interactions, respectively.

AutoDock uses a semi empirical free energy force field to evaluate conformations during docking simulations. The energy functions define what contributes to the energy of the molecule, like bond stretching, bending etc., so that energy of the molecule can be calculated for any particular conformation. The force field evaluates binding in two steps. In the first step, the intramolecular energetics is estimated for the transition from unbound states to the conformation of the ligand and protein in the bound state. The second step then evaluates the intermolecular energetics of combining the ligand and protein in their bound conformation.

ΔG = ΔG_vdw + ΔG_hbond + ΔG_elec +ΔG_tor+ ΔG_desolv

First three terms are for van der Waal’s, hydrogen bonding, electrostatics respectively. The term ΔG_tor is for rotation and translation and ΔG_desolv is for desolvation upon binding and the hydrophobic effect [20].

BF1	BF2	BF3	BF4
BF5	TF1	TF2	TF3
IF1	IF2	PF1	PF2
PF3	PF4	TIF1	TIF2
TIF3	TIF4	TIF5	TIF6
TIF7	TIF8	TIF9	TIF10
TIF11	TIF12	TIF13	TIF14

Scheme 1: The analogs of flavonoid compounds are shown.

HF1	HF2	HF3	HF4
HF5	HF6	HF7	HF8
HF9	HF10	HF11	HF12
HF13	HF14	HF15	HF16
HF17	HF18	HF19	HF20
HF21	HF22	HF23	HF24
HF25	HF26

Scheme 1 (continued): The analogs of flavonoid compounds are shown.

RESULTS AND DISCUSSIONS

The binding energy and their corresponding intermolecular energy, electrostatic energy, internal energy and torsional energy for each class of flavonoids with aromatase are given in Table 1. All the energies are reported in kcal/mol. Figure 1 shows the lowest binding energy conformation of each flavonoid class. Their interacting residues in the binding site of aromatase are represented in elemental coloured ball and stick model. The maroon colour dashed lines between the atoms represent the hydrogen bonds.

Table 1: Molecular docking energies of flavonoids with aromatase are shown.

Compounds	∆GBE	∆Gintermol	∆Gelect	∆Ginternal	∆Gtor
BF1	–7.24	–7.54	–0.05	–0.32	0.30
BF2	–7.56	–7.86	–0.19	–0.34	0.30
BF3	–7.16	–7.46	–0.02	–0.33	0.30
BF4	–7.40	–7.99	–0.16	–0.26	0.60
BF5	–6.93	–7.52	–0.24	–0.27	0.60
TF1	–5.99	–6.89	–0.16	–0.63	0.89
TF2	–6.53	–7.43	–0.31	–0.42	0.89
TF3	–6.64	–7.54	–0.17	–0.60	0.89
IF1	–5.97	–6.86	–0.23	–0.22	0.89
IF2	–6.13	–7.02	–0.21	–0.46	0.89
PF1	–7.02	–7.62	–0.14	–0.72	0.60
PF2	–5.45	–6.34	0.02	–0.82	0.89
PF3	–6.97	–7.57	–0.07	–0.61	0.60
PF4	–7.04	–7.94	–0.12	–0.72	0.89
HF1	–5.63	–6.82	–0.21	–0.25	1.19
HF2	–5.88	–7.37	–0.24	–1.20	1.49
HF3	–6.13	–7.62	–0.28	–1.21	1.49
HF4	–5.70	–7.78	–0.78	–1.42	2.09
HF5	–5.30	–6.50	–0.50	–0.31	1.19
HF6	–6.75	–7.94	–0.51	–0.80	1.19
HF7	–6.48	–7.98	–0.78	–0.94	1.49
HF8	–5.96	–6.86	–0.24	–0.66	0.89

Docking analysis of Benzoflavanones

The benzoflavanones (BF) [11] binding energy were in the range ~ –7.0 kcal/mol. They possessed lowest binding energy compared to all other class of compounds considered for study. The common binding site residues interacting with BF were Met374, Ser478, Ala306, Asp309, Thr310 and Arg115. Interestingly BF1, BF3 and BF5 showed cation–π interactions between Arg115 and the benzo ring of the flavanone.

Even though the above compounds exhibited cation–π interactions, comparing to BF3 and BF5 the binding energy was low for BF1 compound, which may be due to the unsubstituted benzo group in BF1. In BF2 and BF4 there are hydroxy (–OH) and cyano (–CN) groups present at the meta (C₃^׳) positions of the B-ring.

Presence of electron-withdrawing groups like cyano and electron-donating groups such as the hydroxyl on B-ring, increased the inhibitory effect can be seen in BF2 and BF4. Among all the five compounds only the BF4 hydroxyl group which is in the meta position in B-ring showed hydrogen bond with Met374 (Met374 NH···OH). In benzoflavanones, ∆G_elect, ∆G_internal interactions contributed very less to the binding energy and the significant contribution to the binding energy was due to ∆G_intermol energy, which is the sum of vdW, hydrogen bonding and desolvation, and torsional energies.

The torsional energy was almost the same for BF1–3 which was ~0.3 kcal/mol and BF4–5 showed torsional energy of ~0.6 kcal/mol. Torsion angles within a molecule are defined by four atoms connected by three bonds. BF1–3 had one rotatable bond and BF4–5 had two rotatable bonds, therefore, higher torsional energy than BF1–3.

Docking analysis of Triazolylflavans

Binding energy of Triazolylflavan (TF) [12] was in the range ~–6.0 kcal/mol and among them the lowest binding energy was shown by TF3. The common residues in the binding site interacting with TF were Ser478, Thr310, Asp309, Trp224, Leu477, Met374 and Ile133. The oxygen of oxymethyl groups in TF2 and TF3 showed hydrogen bond with the hydrogen ion of hydroxyl group in Ser478 (Ser478-OH···OCH₃) residue at a distance of 1.745 Å and 1.778 Å respectively. Though TF2 showed a stronger hydrogen bond than TF3, its binding affinity was less than TF3. TF1 exhibited a π–π interaction between the C-ring of flavan and imidazo group of tryptophan (Trp224) residue but its binding energy was very high compared to TF2 and TF3.

BF2	TF3
IF2	PF4
HF14

Fig. 1: Flavonoid compounds having lowest binding energy with aromatase are shown.

Table 1 (Continued): Molecular docking energies of flavonoids with aromatase are shown.

Compounds	∆GBE	∆Gintermol	∆Gelect	∆Ginternal	∆Gtor
HF9	–5.85	–7.35	–0.46	–0.85	1.49
HF10	–6.77	–7.07	–0.18	–0.25	0.30
HF11	–7.03	–7.63	–0.10	–0.26	0.60
HF12	–7.02	–7.62	–0.25	–0.27	0.60
HF13	–6.80	–7.10	–0.07	–0.27	0.30
HF14	–7.15	–7.45	–0.05	–0.30	0.30
HF15	–6.55	–7.15	–0.18	–0.21	0.60
HF16	–6.53	–7.43	–0.12	–0.81	0.89
HF17	–6.43	–7.33	–0.31	–0.18	0.89
HF18	–6.47	–7.66	–0.48	–0.78	1.19
HF19	–6.32	–6.92	–0.09	–0.30	0.60
HF20	–6.21	–7.10	–0.31	–0.81	0.89
HF21	–6.06	–7.26	–0.16	–1.29	1.19
HF22	–6.16	–7.95	–0.90	–1.64	1.79
HF23	–6.30	–6.90	–0.13	–0.21	0.60
HF24	–6.17	–7.06	–0.36	–0.17	0.89
HF25	–6.47	–7.07	–0.22	–0.82	0.60
HF26	–5.27	–6.47	–0.37	–0.88	1.19

As in benzoflavonones, the contribution of ∆G_elect, ∆G_internal interactions were very less to binding energy in the triazolylflavans, and energy contribution was due to the ∆G_intermol energy. The torsional energy for TF1–3 was same of ~0.9 kcal/mol as they had three rotatable bonds.

Docking analysis of imidazolylflavans

The imidazolylflavans (IF) [13] showed binding energy in the range ~–6.0 kcal/mol. The lowest energy was exhibited by IF2 which has a 7-oxymethyl substitution. The most common residues in the binding site that interact with IF were Ser478, Thr310, Ala306, Asp309, Phe221, Leu477, Met374 and Ile133. In IF2, the oxygen atom of the 7–oxymethyl group showed hydrogen bond with hydroxyl hydrogen of Ser478 (Ser478 OH···OCH₃) at a distance of 1.725Å whereas the hydroxyl group of IF1 showed no hydrogen bond with the nearby residues.

This could be one of the reasons why IF1 had higher binding energy or lower binding affinity than IF2. In imidazolyl flavans, ∆G_elect, ∆G_internal interactions contributed less to the binding energy and the significant contribution to the binding energy was due to ∆G_intermol energy. The compounds IF1 and IF2 had three rotatable bonds and a same torsional energy of ~0.9 kcal/mol.

Docking analysis of pyridinyl-substituted flavanones

The Pyridinyl-substituted flavanones (PF) [14] showed binding energies between –7.0 and –6.0 kcal/mol and of which the lowest energy was shown by PF4. The binding site residues that commonly interacted with PF were Ser478, Thr310, Ala306, Trp224, Leu477, Met374 and Ile133. The hydrogen bond with the hydroxyl group of Ser478 (Ser478 OH···N) were with the nitrogen of the pyridinyl ring in PF3 and PF4 at a distance of 2.097Å and 2.024Å respectively.

The oxygen of 7-oxymethyl group of PF4 also made hydrogen bond with the amino hydrogen of Met374 (Met374 HN···OCH₃) at a distance of 1.974Å. This could be one of the reasons for the lowest binding energy or highest binding affinity of PF4 compared to PF1–3 as the 7-methoxy group of PF4 acts as hydrogen bond acceptor and interacts well with amino acid residues of aromatase.

The benzo group of all the compounds from PF1­–4 showed cation–π interactions with the Fe of heme, and pyridinyl ring of PF2 showed cation–π interaction with Arg115. From the docking results obtained for PF1–4, Z-isomer of oxymethyl pyridinyl substituted flavanones and E-isomer of unsubstituted pyridinyl flavanones showed lower binding energy or higher binding affinity as the restrained configuration of E–isomer could bring the pyridine nitrogen closer to heme Fe for a possible interaction.

In pyridinyl-substituted flavanones, the contribution of ∆G_elect, ∆G_internal interactions was less to the binding energy and the significant contribution to the binding energy was due to ∆G_intermol energy. PF1 and PF3 had two rotatable bonds and the same torsional energy of ~0.5 kcal/mol. PF2 and PF4 had three rotatable bonds with a torsional energy of ~0.9kcal/mol.

Docking analysis of hydroxy flavones

Hydroxyflavones (HF) [15] showed binding energies between –7.0 and –5.0 kcal/mol and the lowest binding energy was shown by HF14. The residues of binding site interacting with the HF were Ser478, Thr310, Ala306, Phe221, Leu372, Arg115, Trp224, Met374 and Ile133. HF14, which has the lowest binding energy among all the hydroxyflavones, showed a cation–π interaction between the B-ring and Arg115. HF14 was the only unsubstituted flavone in that class and studies have shown that unsubstituted flavones showed higher aromatase inhibiting activity as they can correctly fit in the binding site. Out of the 26 hydroxyflavones taken for the study, 20 of them showed cation–π interactions with Fe of heme. HF11 and HF12 followed HF14 in having low binding energies, where, HF11 showed a cation–π interaction between B-ring and Fe of heme and Arg115, and hydroxyl oxygen of its B–ring showed a hydrogen bond with amine hydrogen of Met374 (Met374 NH···OH) at a distance of 1.797Å. HF12 also showed cation–π interaction with Arg115 and a hydrogen bond between hydroxyl oxygen of 7,8-benzo ring and amine hydrogen of Met374 (Met374 NH···OH) at a distance of 1.634Å. In addition, studies have shown that presence of hydroxyl group at the C₇ position increases inhibitory potency as they serve as hydrogen bond acceptor to interact with the nearby residues, as seen in HF11 and HF12.

As mentioned in the literature isoflavones showed less binding affinity and less inhibition of aromatase than flavanones and flavones. HF12 formed a stronger hydrogen bond than the rest of the hydroxyflavones and therefore, is lower binding energy. HF26 is an isoflavone and showed the highest binding energy –5.27 kcal/mol. It also showed a hydrogen bond between C₇ hydroxyl hydrogen and hydroxyl oxygen of Thr310 (Thr310 HO···HO) at a distance of 2.102Å, which is a weak interaction, compared to that seen in HF12 and HF14. Hydroxyl substituted derivatives showed lower binding energies than methoxy derivatives as the hydroxyl groups showed the ability to form hydrogen bonds. In hydroxyflavones, ∆G_elect, ∆G_internal interactions contributed less to the binding energy and the significant contribution to the binding energy was due to ∆G_intermol energy. The torsional energy ranges from 0.30–2.09 kcal/mol. HF4 had eight rotatable bonds, therefore, showed the highest torsional energy 2.09 kcal/mol.

Docking analysis of pyridylmethyl thio isoflavones

The binding energies obtained for TIF [16] ranged between –6.0 and 2.0 kcal/mol. The lowest energy was exhibited by TIF1. These compounds were found to bind near a binding site other than the experimentally identified binding site. Therefore, a search was conducted in the Q-site finder [21] to identify the possible binding sites in the aromatase. Q-site finder showed ten binding sites in aromatase and the thio compounds were found to bind near site 4, which is located away from the experimentally identified binding site. The common residues that interact with thio compounds were Tyr424, Phe427, Phe430, Lys 448, Met444 and Val 445. Though these compounds were bound at a different site, they displayed significant hydrogen bond and cation–π interactions and high torsional energies. The torsional energies obtained were 2.09 and 2.39 kcal/mol. The compound with the lowest binding energy (TIF1) showed there hydrogen bond interactions, two π–π interaction between and one cation–π interactions. A hydrogen bond was formed between amine hydrogen of Gln428 and nitrogen of the pyridine ring of TIF1 (Gln428 NH···N) at a distance of 1.962Å. The other hydrogen bonds were formed between hydroxyl hydrogen of Tyr361 and carbonyl oxygen of C-ring of flavone (Tyr361 OH···OC) at a distance of 2.233Å and amine hydrogen of Lys448 and oxygen of the nitrobenzene ring of TIF1 (Lys448 NH₂···O₂N) at a distance of 1.809Å. This could be one of the reasons why TIF1 showed higher activity than the other compounds of the same class.

CONCLUSION

In the present study, the binding and interactions of flavonoid compounds (flavans, flavanones, isoflavones and flavones) with aromatase have been studied using molecular docking calculations. Most of the compounds have shown significant binding interactions with the aromatase. It was observed that the benzo ring is an important but not the only structural requirement to evoke aromatase inhibition. The hydrogen bond interactions and cation–π interactions also contributed to the strong binding of these compounds to the binding site of aromatase. An increase in binding energy was observed when there is substitution at C₇ and C₈ position of A-ring of the flavanone. Compounds with unsubstituted or less substituted rings showed higher activity than substituted rings.

This is possibly because the binding cavity was not large enough to accommodate bulky substituted compounds without steric clashes. Hydrogen bonding interactions were predominant in all the classes of compounds taken for study and were found to be important for inhibition. Among the compounds considered for the study, BF4, TF2, TF3, IF2, PF3, PF4, HF1–9, HF11–12 and HF15–26 showed hydrogen bonding with some of the residues in the binding site of aromatase. BF1, BF3, BF5, PF1–4, HF1–5, HF7, HF8, HF10–17, HF19 and HF21–26 showed cation–π interaction. Amino acids Thr310, Met374 and Ser478 are the common residues that interact with the flavonoid compounds BF, TF, PF, IF and HF. The amino acid Asp309 which is involved in ASD substrate binding also plays a key role in binding of flavonoid compounds BF, TF and IF. Interestingly the thio isoflavones, which had an extended hydrocarbon chain showed decreased activity and those compounds, were found to bind at a different binding site in the enzyme rather than the experimentally identified binding site. The decrease in activity compared to the other class of compounds may be due to unfavourable interactions with the binding site residues. This study provides a detailed insight into binding of the flavonoid compounds, to the aromatase.

ACKNOWLEDGEMENT

We would like to thank the management of VIT University for supporting this study.

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