In silico analysis of Adenosine deaminase inhibitory activity of some flavones and N-arylazetidin-1-yl-2-Flavonyl oxy acetamide Derivatives

Durbhaka Sai Padmini; Dugasani Swarnalatha; Prasad S. V. U. M

In silico analysis of Adenosine deaminase inhibitory activity of some flavones and N-arylazetidin-1-yl-2-Flavonyl oxy acetamide Derivatives

Durbhaka Sai Padmini ✉ ¹ , Dugasani Swarnalatha ² , Prasad S. V. U. M ³

1 Department of Pharmaceutical Chemistry, Creative Educational Society's College of Pharmacy, Chinnatekuru, Kurnool-518218, Andhra Pradesh, India, 9885355377

2 Department of Pharmacognosy and Phytochemistry, Annamacharya College of Pharmacy, Newboinapalli, Rajampeta -516126, Andhra Pradesh, India

3 School of Pharmaceutical Sciences, Jawaharlal Nehru Technological University, Kakinada-53303, Andhra Pradesh, India

Abstract

Adenosine deaminase(ADA) is a vital enzyme distributed in both intra and extra-cellular locations in humans. Its primary function is purine metabolism and also plays an essential role in maintaining immunological response. Altered regulation of ADA is related to many malignancies. Over-expression and increased serum ADA activity are reported in breast cancer. Flavonoids are potential sources of protective effects against chronic diseases. In this study, we investigated the insilico adenosine deaminase inhibitory activity of some selected plant flavones, synthetic 2-azetidino flavones [SLP II 1-2 and SLP 61(a-d)-2(a-d)] and ADA inhibitor drugs EHNA and pentostatin. Molecular docking studies have been performed by using Autodock tools. The obtained results indicate that all the selected ligands have a potent inhibitory effect on ADA in the range of -5.19 kcal/mol to -10.56 kcal/mol binding energy. Among the plant flavones, Baicalein is shown good binding energy of -7.83 kcal/mol with the highest conventional H- bonds and in synthetic ligands, SLP VI 2c has -10.56 kcal/mol. The synthetic compounds SLP II 1-2 and SLP 61(a-d)-2(a-d) exhibit greater binding energies than the selective ADA inhibitors and plant flavones. Therefore the present study highlights the use of these synthetic ligands in drug development against specific malignancies like breast cancer that may show high ADA activity.

Keywords

Adenosine deaminase, Flavone, 2- azetidinone, breast cancer, molecular docking

Introduction

The intracellular and intercellular adenosine regulates multiple diverse physiological processes by acting as a messenger in cardiovascular, neurological and immunological systems. Upon injury, it potently restricts immune responses and promotes wound healing. The extracellular adenosine regulates vasodilation, renal reabsorption of water, pain perception and other physiological processes (Vigano et al., 2019). ADA is ubiquitously expressed in various cells and tissues. It is a key enzyme in regulating adenosine levels by irreversible catalysis. In the lymphatic system, Adenosine deaminase (ADA) is released by monocyte/

https://typeset-prod-media-server.s3.amazonaws.com/article_uploads/8eefe1d8-668a-4732-8409-931350525a3f/image/4cb3fb8d-fb4f-42b4-9f1c-685560cb9257-upicture1.png — **Figure 1: 2D structure of plant flavone derivatives**

https://typeset-prod-media-server.s3.amazonaws.com/article_uploads/8eefe1d8-668a-4732-8409-931350525a3f/image/3fc628a2-8ef7-4281-80f6-93f09771068d-upicture2.png — **Figure 2: 2D structure of Adenosine deaminase inhibitors**

https://typeset-prod-media-server.s3.amazonaws.com/article_uploads/8eefe1d8-668a-4732-8409-931350525a3f/image/989bd298-af40-4b3b-b887-2257bbb7f514-upicture3.png — **Figure 3: 2D structure of 2-azetidinyl flavone derivatives [SLP II1-2 and VI 1(a-d)-2(a-d)]**

macrophages, which potentiate the proliferation and differentiation of T lymphocytes. Monocyte/macrophage activation by intracellular infectious microorganisms and inflammatory diseases leads to elevated levels of ADA in serum (Conde, S.R.Marinho, & Kritsk, 2002).

Oxidative stress is effective in many pathologic conditions, including cancer, cardiovascular diseases, rheumatoid arthritis, ischemia/reperfusion and ageing. A close relationship has been demonstrated between inflammatory reactions and FORs formation in chronic diseases. Adenosine deaminase (ADA) enzyme directly or indirectly contributes to FORs development by the catabolism of anti-inflammatory adenosine (Arun, Sharanya, Abhithaj, & Sadasivan, 2019). Altered Serum ADA activity is used to evaluate the clinical status of diseases related to cell-mediated immune responses, tuberculosis, rheumatoid arthritis, systemic lupus erythematosus, liver diseases, schizophrenia etc. (Sasidharan et al., 2017). ADA inhibitors are being considered to develop promising candidates for various diseases (Arun, Sharanya, Abhithaj, & Sadasivan, 2020; Sharanya, Arun, Abhithaj, & Sadasivan, 2020).

India exhibits heterogeneity in cancer and the number of patients with cancer in India is around 13,000 per year. Breast cancer is among the common sites of cancer and trends in cancer incidence rates showed an increase in all sites of cancer in both sexes. The ADA has significance in breast cancer development by regulating the pool of adenosine (Mahajan, Tiwari, Sharma, Kaur, & Singh, 2013). Extracellular purines mediate host cell response or regulate growth and cytotoxicity on tumour cells. Elevated levels of ADA were observed in tumour angiogenesis from actively proliferating endothelial cells. Inhibition of adenosine deaminase activity has beneficial effects like decreased aggressiveness of tumour cells (Ryszard & Smolenski, 2018).

The antiproliferative properties of intracellular ADA inhibition could be due to the accumulation of nucleotides that suppress DNA synthesis. The risk of cardiovascular disease is a major complication in cancer therapy. There is an association between serum adenosine deaminase and atherosclerosis and coronary artery calcification (Gloria-Bottini, Safranow, & Banci, 2013). Experimental investigations indicate that adenosine has a cardioprotective effect through its coronary vasodilation and antioxidant effect. Chao Xuan et al. demonstrated that serum concentrations of adenosine metabolites were associated with endothelial dysfunction and the severity of Cardiovascular diseases (Xuan, Tian, & Zhang, 2019). Since ADA involve in irreversible catabolism of adenosine, its relationship to cardiovascular disease remains a concern. The present study is aimed to determine the inhibitory activities of a set of natural flavones and experimentally synthesized 2-azetidino flavone compounds on adenosine deaminase.

https://typeset-prod-media-server.s3.amazonaws.com/article_uploads/8eefe1d8-668a-4732-8409-931350525a3f/image/edfd2b1b-8fb1-43cd-af5b-81580becdf09-upicture4.png — **Figure 4: 3D Secondary Structure of target enzyme adenosine deaminase with a resolution of 1.52 A^{^⸰}**

Experimental

According to Padmini et al., a set of novel 2-azetidino flavones were synthesized from 7-hydroxy flavones through the intermediate Schiff bases. These compounds were evaluated for in-vitro anticancer activity against MCF-7 cells. The reported novel 2-azetidino flavones were used in docking studies against the target enzyme Adenosine deaminase (PDB ID: 3IAR) along with a set of plant flavones and selective ADA inhibitor drugs like EHNA and pentostatin. Figure 1 and Figure 2 represent structures of selected natural flavones and selective ADA inhibitors. Figure 3 represents structures of synthetic flavones derivatives as ligands named as compounds SLP II1-2 and SLP VI 1(a-d)-2(a-d).

https://typeset-prod-media-server.s3.amazonaws.com/article_uploads/8eefe1d8-668a-4732-8409-931350525a3f/image/b55d1c7d-87bb-4faf-8ea5-af6376667777-upicture5.png — **Figure 5: Binding mode of some natural flavones and selective ADA inhibitors with 3IAR**

https://typeset-prod-media-server.s3.amazonaws.com/article_uploads/8eefe1d8-668a-4732-8409-931350525a3f/image/9a6a442b-fadd-405a-a1ad-e646af69e308-upicture6.png — **Figure 6: 2D interactions of (A) Baicalein (B)EHNA (C) SLP VI 2c with residues of ADA**

https://typeset-prod-media-server.s3.amazonaws.com/article_uploads/8eefe1d8-668a-4732-8409-931350525a3f/image/44ca9844-2260-40f6-b31e-f73c78c1fb69-upicture7.png — Figure 7: 3D illustrations of interactions of SLP VI 2a(a) receptor interaction (b) interactions with pocket atoms of corresponding amino acid residues of ADA (c) hydrogen bonding surface (d) 2D illustration of interactions. Autodock 4.2 and Biovia discovery studio2019 Viewer were used to generate and visualize the docked structures, respectively

https://typeset-prod-media-server.s3.amazonaws.com/article_uploads/8eefe1d8-668a-4732-8409-931350525a3f/image/38a7886a-b647-4609-b809-5bc37d181948-upicture8.png — Figure 8: 3D illustrations of interactions of SLP VI1c (A)receptor interaction (B) interactions with pocket atoms of corresponding amino acid residues of ADA (C) hydrogen bonding surface (d) 2D illustrations of interactions. Autodock 4.2 and Biovia discovery studio2019 Viewer were used to generate and visualize the docked structures, respectively

Table 1: Docked energy valuesof ligands with target protein 3IAR

Compound	Binding energy(kcal/mol)	Predicted Inhibitory constant (Ki) (µm)	No interactions and bonds	No of H-bonds
Chrysin	-7.27	4.72	8	3
Baicalein	-7.83	1.83	12	5
Apigenin	-7.09	6.34	8	3
Acacetin	-7.18	5.44	6	2
Luteolin	-7.08	6.51	6	3
Diosmetin	-5.19	155.87	8	4
Tricetin	-6.98	7.60	8	5
Wogonin	-7.02	7.18	8	1
Tangeritin	-6.45	18.79	11	3
Nobiletin	-6.38	21.21	6	1
SLP II 1	-6.71	12.11	5	1
SLP II 2	-7.16	5.66	6	2
SLP VI 1a	-8.45	635 nm	7	1
SLP VI 1b	-7.48	3.31	7	3
SLP VI 1c	-8.58	514 nm	12	3
SLP VI 1d	-8.21	964 nm	7	2
SLP VI 2a	-8.66	446nm	9	3
SLP VI 2b	-7.80	1.92	7	0
SLP VI 2c	-10.56	18.22 nm	11	0
SLP VI 2d	-8.67	445 nm	7	1
EHNA	-6.03	37.85	10	4
Pentostatin	-6.13	32.16	8	5

Table 2: Interaction of the amino acid residues of adenosine deaminase to natural flavones

Residues $*$ H- bonding	Chry sin	Baica lein	Apige nin	Acac etin	Lute olin	Diosm etin	Tric etin	Wog onin	Tanger itin	Nobil etin
His17	✔ $*$	✔ $*$	✔ $*$
Ser21				✔ $*$
Phe61	✔	✔	✔
Leu62	✔	✔	✔			✔ $*$
Phe65	✔	✔				✔
Met89				✔
Glu93				✔ $*$
Leu106	✔	✔	✔
Pro114						✔ $*$
Pro116						✔
Trp117						✔ $*$
Met155	✔	✔	✔
His157						✔ $*$
Ala183		✔	✔
Arg211										✔ $*$
Glu217	✔ $*$	✔ $* *$	✔ $*$
Lys232					✔ $*$
Thr233										✔
Glu234					✔					✔
His238		✔ $*$
Leu243							✔ $*$	✔	✔
Gln246							✔ $*$
Glu255					✔ $*$					✔
Asn256					✔
Met257					✔
Ala271							✔ $*$	✔ $*$	✔
Lys273									✔ $*$
Ala279							✔ $*$	✔	✔ $*$
Arg282							✔ $*$	✔	✔ $*$
Asp296	✔ $*$	✔ $*$	✔ $*$
Ser332					✔ $*$					✔

Table 3: Interaction of the residues of adenosine deaminase with the synthetic derivatives

Residues $*$ H- bonding	$E H N A$	$\begin{array}{l} P e n t o \\ s t a t i n \end{array}$	SLP II 1	SLP II 2	SLP VI 1a	SLP VI 1b	SLP VI 1c	SLP VI 1d	SLP VI 2a	SLP VI 2b	SLP VI 2c	SLP VI 2d
His17	$✓ *$										✔
Leu62											✔
Phe86						✔	✔
Met89							✔			✔
Leu106	$✓$										✔
Trp117	$✓$										✔
Asn118											✔
Arg149					✔
Cys169		$✓$
Gln173		$✓ *$
Gln174		$✓ *$			✔ $*$
Thr176					✔
Val177		$✓ *$
Val178		$✓ *$			✔
Ala183	$✓$										✔
Gly184	$✓ *$										✔
Gly208		$✓$
His210		$✓ *$
His 214	$✓$										✔
His238	$✓$
Tyr240								✔	✔			✔
His241			✔	✔				✔ $*$	✔			✔
Leu243			✔	✔					✔			✔
Ala271			✔ $*$	✔ $*$
Lys273								✔ $*$	✔ $* *$			✔ $*$
Thr276									✔ $*$
Ala279			✔	✔ $*$				✔	✔			✔
Arg282			✔	✔					✔			✔
Asp295	$✓ *$										✔
Asp296	$✓ *$
Pro297						✔	✔
Lys301						✔	✔ $*$			✔
Thr303						✔ $*$	✔ $*$
Asp305						✔ $*$				✔
Thr306						✔ $*$	✔ $*$

The crystal structure of Adenosine deaminase (PDB ID: 3IAR, 1.52 Å X-ray diffraction resolution) complexed with 3D1, NI, NO3 and GOL was obtained from the RCSB Protein Database (PDB). The three-dimensional structure of protein Adenosine deaminase (PDB ID: 3IAR) is known experimentally and is shown in Figure 4.

Molecular Docking

Automated dockings were performed using various AutoDock 4.2 for determining the orientation of natural flavones, designed ligands ( 2 azetidino flavones) and selective ADA inhibitor drugs to the binding site of ADA(PDB ID: 3IAR). Solvent, hetero atoms (Ligands) and ions were removed from the original PDB files. Polar hydrogens were added and Kollmann charges were incorporated into the receptor. Gasteiger partial charges were allotted. The structures of ligands were relaxed by energy minimization; rotations and torsions were enabled for the bonds present in all the ligands. Grid maps were optimized by using Autogrid carefully. Docked conformation having the lowest binding energy was identified for every selected ligand.

Results and Discussion

Docking studies were performed with the target protein adenosine deaminase. All the ligands have good binding energies. The binding energy values of the selected ligands are represented in Table 1. The binding energy values of natural flavones were found to be between -5.19 to -7.83kcal / mol, and synthetic 2-azetidino flavones were found to be between -7.48 to -10.56 kcal/mol. The ADA inhibitor drugs EHNA and pentostatin showed the binding energy of -6.03 and -6.13 kcal/mol, respectively.

Among the selected natural flavones, Baicalein has good binding energy of -7.83kcal/mol, and Diosmetin has the least energy of -5.19 cal/mol. In the synthetic derivatives, the 7-OH flavones (SLP II1-2) have a binding energy of -6.71kcal/mol and -7.16kcal/mol, correlated with the natural flavones binding capacity. Among the 2-azetidinoflavone derivatives, SLP VI 2(a-d) has a greater binding capacity than SLP VI 1(a-d) compounds. 4-(N-dimethyl)amino substitution on the Ring B of flavones and aryl ring of 2-azetidino structure influence the hydrophobicity and binding capacity.

The SLP VI 1c and SLP VI 2c showed high binding energy values of -8.58kcal/mol and -10.56kcal/mol, respectively, supporting that the electron donating group favours the interaction with residues of the target protein.

The 3-OH,4-OCH₃ substitution in ring B of Diosmetin and 4-OH, 3-OCH₃ substitution on aryl ring of 2-azetidino structure in SLP VI 1b and SLP VI 2b may be responsible for low affinity towards the active site of adenosine deaminase supported by energy values of -5.19kcal/mol, -7.48 kcal/mol and -7.80 kcal/mol respectively.

The predicted inhibitory constant (k_i) value of natural flavones is 1.83 µm - 21.21 µm and synthetic derivatives are 18.22 nm -3.31 µm. These values are substantially low compared to selective ADA inhibitors, EHNA and pentostatin in the 32-38 µm range except diosmetin.

Binding mode

The entire ligand sets selected bound efficiently with binding sites of the target protein, 3IAR. The binding mode of selected natural flavones and selective ADA inhibitors with 3IAR is shown in Figure 5.

Table 2 and Table 3 represent the interaction of the amino acid residues of adenosine deaminase to the selected ligands of plant flavones and synthetic azetidinoflavones, respectively.

These interactions were studied further in detail. It clearly shows that all compounds have good binding interactions with residues of the target protein by conventional Hydrogen bond, Pi- alkyl, Pi-Pi staked, Pi-lone pair, Pi-sigma, Pi-Pi T shaped and vanderwaal interactions.

The flavones, Chrycin, Baicalein and Acaetin have shown binding with residues of HIS17, PHE61, LEU62, LEU106, GLY184, GLU217, and ASP296. They form H-bond with HIS17, GLU217 and ASP296 and Baicalein has the highest H- bond interactions among all the selected ligands.

The selective ADA inhibitor EHNA and SLP VI2c has the same binding residues as that of Chrycin, Baicalein, Epigenin and Acacetin. SLP VI2c has no conventional H-bonds, but its similarity in binding mode with that of EHNA persuades the highest binding energy of -10.56 among all the selected ligands (Figure 6).

The similarity in binding mode has been observed in pentostatin and SLP VI1a to the residues of GLN174, VAL177, VAL178, and THR176 with conventional hydrogen bonds, Pi-alkyl and amide-pi stake interactions. Tricetin, Wogonin, Tangeritin and synthetic 7-OH flavones SLP II 1-2 and 2-azetidino flavones derivatives SLP VI 1d, 2a, 2d have a similar binding mode with target protein residues of TYR240, HIS241, LEU243, GLN246, GLU255, ASN256, MET257, ALA271, LYS273, THR276, HIS278, ALA279 and ARG282 with the possible number of conventional hydrogen bonds (Figure 7).

The compounds SLP VI1b and SLP VI1c have entirely different residual binging interactions with PRO297, LYS301, THR303 and ASP305. The above interactions indicate that all the ligandes have different binding affinities at various pockets of target protein based on the hydrophobic substitutions and position of substitution on the ligand (Figure 8). The active site of ADA contains a zinc ion cofactor coordinated by five atoms from His15, His17, His214, Asp295, and the substrate. The inhibitory potency of selected ligands may be due to the efficient binding to the amino acid residues of active sites HIS17, ASP295 and HIS 214.

Conclusion

This study highlights the insilico binding efficiency of various flavone derivatives to adenosine deaminase enzyme. Low binding energy values of synthetic azetidinoflavone derivatives[SLP VI 1(a-d)-2(a-d)] compared to natural flavones and selective ADA inhibitors revealed that these compounds are active scaffolds and can be considered in the design of new drugs against diseases related to high Adenosine deaminase activity. Adenosine deaminase inhibition has a beneficiary effect on reducing breast cancer development. This molecular docking study supports the previously reported in-vitro anti-breast cancer evaluation on synthetic azetidinoflavone derivatives. The synthetic derivatives can be further explored as anti-breast cancer agents as they have strong inhibitory binding potencies with active site residues of ADA.

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