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ORIGINAL ARTICLE
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Antiproliferative, apoptosis-inducing activity and molecular docking studies of sydnones compounds


1 Department of Biotechnology, Sir M Visvesvaraya Institute of Technology, Bengaluru, India
2 Department of Chemistry, St. Joseph's College (Autonomous), Devagiri, Calicut, Kerala, India
3 Centre for Nano and Soft Matter Sciences, Bengaluru, India
4 Department of Biotechnology, S.E.A. College, Bengaluru, India

Date of Submission26-Nov-2020
Date of Decision19-Jan-2021
Date of Acceptance23-Feb-2021
Date of Web Publication25-Oct-2021

Correspondence Address:
C Rajendra Singh,
Department of Biotechnology, Sir M Visvesvarya Institute of Technology, Via, Yelahanka, International Airport Road, Bengaluru - 562 157, Karnataka
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_1614_20

PMID: 34708812

 > Abstract 


Objective: To evaluate the antiproliferative and apoptosis inducing activity of different sydnones on cancer cell lines and their interaction with cancer proteins by molecular docking studies.
Material and Methods: Antiproliferative activity was carried out by MTT assay and apoptosis inducing activity was performed by DAPI and Annexin V and propidium iodide staining. Molecular docking studies were performed using AutoDock Tools 1.5.6. Pharmacokinetics properties like ADME and toxicity were analysed by pkCSM web server.
Result: In this study, four new sydnone compounds 3-(4-nonylbiphenyl-4'-yl) sydnone (MC-182), 3-(4-propylbiphenyl-4'-yl) sydnone (MC-454), 3-(4-hexylbiphenyl-4'-yl) sydnone (MC-433), and 3-(4-methylbiphenyl-4'-yl) sydnone (MC-431) were screened for antiproliferative and apoptotic effect against BT-474 (human breast cancer), HeLa (human cervical cancer) and Jurkat (human myeloid leukemia) Mostly, all the sydnone compounds exhibited decent antiproliferative effectiveness, but compound MC-431, MC-433, and MC-454 showed more antiproliferative activity (IC50 1.71, 10.09 and 2.87 μM against BT-474, Hela and Jurkat cell line, respectively). The changes of morphological characteristics of cancer cells determined by staining techniques indicate the apoptotic cell death. The molecular docking and interaction studies were carried out between sydnones with cancer proteins (epidermal growth factor domain receptor tyrosine kinase [EGF-TK], tumor necrosis factor-alpha [TNF-α] and Caspase3. Among all four sydnone molecules, two compounds MC-454 and MC-431 showed good binding energy with targeted proteins. Drug-like property was predicted by ADME toxicity study.
Conclusion: The results indicate sydnone compounds were found to exhibit anticancer activity by inducing apoptosis. The molecular docking study of sydnones with cancer proteins showed a decent interaction affinity. The results of absorption, distribution, metabolism, excretion and toxicity studies by the Insilco approach also proved that MC-454 sydnone showed better In-Vivo administration. Thus, the current research work indicates that these sydnone compounds would be prospective in developing anticancer medicines.

Keywords: Sydnones, apoptosis, cancer proteins, Binding energy, toxicity



How to cite this URL:
Hossain SL, Mathews M, Bhyranalyar Nagarajappa VS, Kumar B K, Veerappa Yelamaggad CV, Singh C R. Antiproliferative, apoptosis-inducing activity and molecular docking studies of sydnones compounds. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 5]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=329189




 > Introduction Top


Cancer is known as an important cause of death in humans and according to the WHO more than 70% of all cancer deaths happen in developing countries.[1] Recently, there has been an increase in deaths from different types of cancers globally, with a prediction of 12 million deaths in 2030.[2] In spite of the improvement in the understanding of bioorganic procedures linked with carcinogenic potential, significant challenges are still there for effective treatment of cancer because of the general toxicity of conventional cancer chemotherapeutic agents. Thus, the search for a new medication to treat cancer is still an essential and demanding job for researcher.

Recently, many scientists have shown a path in drug discovery based on the biological properties of liquid crystal pharmaceutical (LCP). The LCP molecule Tolecine was identified for antitumor, antibacterial and antiviral properties.[3] Sydnone derivatives have liquid crystalline properties that flow like liquids but maintain some of the ordered structure of its molecules.[4] Sydnones are novel mesoionic compounds due to their versatile applications in various fields.

Mesoionic compounds have attractive structural traits since they have regions of complementary opposite charges connected with a polyheteroatomic structure with exclusive variation in electron density in the ring.[5] All these features suggest a great possibility of persistent interactions with biomolecules like DNA or proteins. Even though mesoionic compounds are neutral generally, they are internally charged, thus, they can cross biological membranes.[6] Sydnones, sydnonimines, iso-sydnones, and 1, 3, 4-thiadiazoles are the various classes of mesoionic compounds that have been widely studied because of their exclusive structures, biological potency, and probable pharmaceutical use.[7] These are anti-inflammatory, analgesic, antibacterial, antifungal, and antitumor activities.[8]

Apoptosis is an induced program of cell death to treat cancer.[9] The changes in normal cell cycle regulation and apoptosis-related genes can lead to cancer and are linked to cancer progression.[10] Certain drugs have proved to be cytotoxic by inducing an apoptotic pathway that could effectively treat cancers.[11] Around 20 million compounds were documented, of half were heterocyclic compounds.[12] It is reported that sydnone was found to have a significant cytotoxic against sarcoma 180, Ehrlich carcinoma, and B10MCII fibrous histiocytoma.[13]

Sydnone compounds have very interesting structural traits they can cross the biological membrane and interact with biomolecules which could be the target site for cancer research.

Epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase family; it has been associated with a large number of epithelial tumors such as breast, lung, brain, prostate, and liver cancer. Overexpression, deletion, and mutation of the EGFR gene lead to impact on carcinogenesis.[14],[15] Caspase-3 is the cysteine-aspartic acid protease (caspase) family and has an important function in the induction of apoptosis. Research has shown the connection between caspase-3 expression and breast cancer.[16],[17] Tumor necrosis factor (TNF) is a cell-signaling protein (cytokine) that has a vital role in various cellular processes. TNF is secreted by cells that cause inflammation, which could be associated with inflammation-related carcinogenesis.[18]

Sydnone compounds were docked with three above mentioned proteins (Caspase3, EGF-TK, and TNF-α) by molecular docking study to understand the binding interaction between sydnone ligands and the receptor proteins.

Pharmacokinetics or absorption, distribution, metabolism, excretion (ADME) toxicity properties of a compound is a very essential factor to get drug-like property. These properties are evaluated routinely at an early stage of drug development to reduce the failure rate in drug discovery processes because most of the clinical trial failures have been reported due to ADME and toxicology (ADMET) issues.[19],[20] Another important drug property is oral absorption, which is analyzed by Lipinski's Rule of Five.[21]

We report herein the evaluation of four sydnone compounds (MC-182, MC-454, MC-433 and MC-431) as possible anticancer agents by MTT Assay, fluorescence staining, docking, and ADME toxicology studies.


 > Materials and Methods Top


Sydnone derivatives

Four sydnone compounds were generously provided by the Department of Nano and Soft Matter Science (CENS), Bengaluru, India. The structural details of sydnone compounds are given in [Table 1]. The stock solutions of sydnone (20 mM) were prepared with 1% dimethyl sulfoxide.
Table 1: Chemical structure of sydnone derivatives

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3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide cell proliferation assay

3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) was used to assess the cell division rate and cell viability reduction. The water-insoluble blue color formazan crystals are formed by reducing the yellow compound MTT by mitochondrial dehydrogenase, depending on the cell viability.

BT 474 breast cancer, the HeLa human cervical carcinoma cell, and Jurkat cell (2 × 104 cells/mL) were seeded in 96-well plates and treated with different concentrations (100, 33.3, 11.11, 3.70, 1.23, 0.41, 0.14, 0.05 μM) of sydnones for a period of 24 h. After incubating with sydnones, MTT reagent was added and kept in the dark for 3–4 h at 37°C; dark purple formazan crystals were produced. After solubilization of these crystals with isopropanol, absorbance was taken at 595 nm spectrophotometrically and IC50 of all the compounds was evaluated in triplicate.[22]

4′,6-diamidino-2-phenylindole (DAPI) Staining

Cell nuclear morphology was assessed by fluorescence microscopy, followed by DAPI staining. Human cervical cancer (HeLa) and BT 474 breast cancer cell lines cells were treated with the sydnone compounds (IC50 values) for 24 h. The cells were set with 70% ethanol (ice-cold) after washing with phosphate-buffered saline (PBS) (pH 7.4). After that, DAPI stain was added and kept for 15 min at 37°C. The cells were observed under Nikon Eclipse Fluorescence microscope (Nikon Instruments Inc., Tokyo, Japan) after washing them with PBS.[23]

Apoptosis assay (Annexin V and propidium iodide staining) by fluorescent microscope and flow cytometry

The two important cell deaths, necrosis, and apoptosis were distinguished by the Annexin V-FITC and propidium iodide (PI) kit (Sigma Aldrich) using fluorescent microscopy for Hela and BT 474 cell lines. For Jurkat cell lines, two major cell deaths, necrosis and apoptosis were distinguished by the Annexin V-FITC and PI kit (Sigma Aldrich) using flow cytometry. After washing with PBS as mentioned in DAPI staining, the annexin V and PI stains were added to the cells according to the instructions of the kit used (BD-Bioscience-Catalogue no. 556547). The stained cells were viewed and pictures were taken under an Eclipse 50i Nikon fluorescent microscope (Nikon, Tokyo, Japan). As Jurkat cells are suspension lymphoblast, it was analyzed through flow cytometry.[24]

Ligand, protein preparation, and active site prediction

The synthesized sydnones compound's structure was drawn by ChemDraw software and converted to PDB format by smile converter. The target proteins Caspase 3 (PDB ID-5I9B), EGFR domain receptor tyrosine kinase (EGF-TK, PDB ID-1M17), and TNF-α (PDB ID-1TNF) were retrieved from Protein Data Bank (www.rcsb.org) and the water molecules were removed from the protein. Furthermore, a commercially available antibreast cancer drug molecule, Anastrozole (PubChem CID: 2187) was accessed from PubChem Database. CASTp server was used to predict the active site for ligand binding.[25]

Molecular docking analysis and optimization of drug likeliness by Lipinski rule

The compound structures were drawn by ChemDraw and assessed for their drug likeliness property using “Lipinski Rule of Five”[26] by chemine tools open babel software library.[27] Molecular docking is a computational approach to analyze binding between ligand and target protein receptor. Thus, molecular docking analysis has a major role in drug design. Docking was carried out between the crystal structure of the proteins (Caspase3, EGF-TK, and TNF-α) and the four sydnone compounds (ligands). It was performed using AutoDock Tools 1.5.6 The protein and ligand files were changed to “pdbqt” format, and grid variables were given as per the result produced by CASTp. Docking parameters were default selected according to Autodock4.[28] The docked results were viewed by Molegro Molecular Viewer (www.molegro.com) and PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC) as well the protein-ligand interactions were analyzed by ProteinsPlus[29] and protein-ligand interaction profiler.[30],[31]

Absorption, distribution, metabolism, excretion toxicity prediction

All four sydnone compounds were subjected to predict pharmacokinetics properties like ADMET with an online pkCSM webserver with the canonical smiles of sydnone compounds. Approximately 11 important pharmacological descriptions and properties of the compounds were evaluated by the pkCSM webserver.


 > Results and Discussion Top


3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide assay

All four sydnone compounds (MC-182, MC-454, MC-433 and MC-431) were screened for their antiproliferative activity on the BT 474 breast cancer cell line, HeLa human cervical carcinoma cell line, and Jurkat cell lines. This screening was based on the determination of IC50 values by MTT assay. Each cell line (BT 474 breast cancer cell line, HeLa human cervical carcinoma cell line, and Jurkat cell lines) showed different cytotoxicity to the sydnone compounds (MC-182, MC-454, MC-433 and MC-431), as demonstrated by IC50 values shown in [Table 2]. All the compounds showed dose-dependent activity and were found to possess IC50 values lower than 50 μM against the HeLa, BT 474, and Jurkat cell lines. The sydnone compound MC-431showed remarkable antitumor activity against BT 474 cell line with an IC50 value of 1.71 μM. MC-433 was found to be cytotoxic on the HeLa human cervical carcinoma cell line with an IC50 value of 10.09 μM. MC-454 showed very good antitumor activity with an IC50 value of 2.87 μM against the Jurkat cell line.
Table 2: IC50 values

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Cell division is a vital mechanism for the progression, development, and regeneration of eukaryotic organisms. The changes in the mechanism of cell division lead to uncontrollable growth and cause cancer.

All sydnone compounds were found to possess IC50 values lower than 50 μM against the HeLa, BT 474 cell lines, and Jurkat cell lines. The cytotoxic activity of sydnone could be because of the heterocyclic structure of sydnone compounds, which has different regions of positive and negative charges. These charges are connected with a poly-heteroatomic system, this makes sydnone molecule cross cellular membranes and may have strong interaction with DNA or proteins.[6] A similar cytotoxic effect was reported on 1, 3, 4 thiadiazolium mesoionic compound on human melanoma[32] and Sydnone 1:[33] A mesoionic compound has antitumor effect on Walker-256 carcinosarcoma and reduced tumor growth by 54%. The anticancer activity of 4-[1-oxo-(substituted aryl)-2-propenyl]-3-phenylsydnones also was reported.[34]

Determination of apoptosis and necrosis using fluorescence microscopy (BT 474 and the HeLa human cervical carcinoma cell line)

Apoptosis assays were conducted to clarify the basic antiproliferative mechanisms of sydnone compounds at a concentration according to their IC50 value against both BT 474 breast cancer and the HeLa cell lines. Assessment of apoptosis is vital to distinguish it from necrosis. Apoptotic cell death includes cellular morphological changes like membrane blebbing, formation of apoptotic bodies, contracted and emarginated nuclei in contrast to the normal cell nucleus, which provides the evidence for apoptotic activity of compounds.[35] DAPI (a fluorescent DNA-binding agent), annexin V, and PI staining techniques were used to find the qualitative identification of apoptotic and necrotic death against both BT 474 breast cancer and the HeLa cell lines. In identifying apoptosis, Annexin V conjugated to a green fluorescent dye and PI is a red fluorescent dye that stains DNA of both necrotic and late apoptotic cells with damaged membranes. Fluorescence microscope was used to identify both apoptotic and necrotic death qualitative and quantitatively.

Anticancer screening results indicated that all compounds were active in the MTT assay. All the sydnone compounds at their IC50 concentrations were incubated with HeLa and BT 474 cells and performed apoptosis assay and observed under a fluorescence microscope. If the cells are pink colored with PI, the cells are necrotic. If the cells are colored green with annexin V, they are found to be apoptotic cells. Live cells will not have any color (Annexin V_/PI_), apoptotic cells and late apoptotic (Annexin V+/PI_) are green and necrotic cells (AnnexinV_/PI+) are pink. The results indicated that all the compounds showed maximum apoptotic and late apoptotic activity with both Hela and BT 474 cell lines as shown in [Figure 1] and [Figure 2], except MC-433. The percentage of apoptotic and necrotic cells determined for BT 474 breast cancer and Hela cell lines as shown in [Table 3]. All the sydnone compounds showed apoptosis effect, except MC-433 which had 43% of necrotic cells compared to other sydnone compounds.
Figure 1: Apoptosis and necrosis in BT-474 cells. (a) Control-BT 474 breast cancer cell with DAPI staining, (b) MC-182, (c) MC-454, (d) MC-433, (e) MC-431 represent different compounds were treated with BT 474 breast cancer cells in the IC50 value (red arrows indicate apoptotic cells and yellow arrows indicate necrotic cell)

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Figure 2: Apoptosis and necrosis in HeLa cell lines. (a) Control-HeLa with DAPI staining, (b) MC-182, (c) MC-454, (d) MC-433, (e) MC-431 (red arrows indicate apoptotic cells and yellow arrows indicate necrotic cell)

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Table 3: The percentage of apoptotic and necrotic cells (BT 474 and HeLa cell line)

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Determination of apoptosis and necrosis using flow cytometry (Jurkat cell line)

MC-433 and MC-454, which exhibited the best results (based on IC50 value) on Jurkat cell line among the tested sydnone compounds, were used to study apoptosis and necrosis assays against Jurkat cell line at their IC50 concentrations for 72 h of treatment. As Jurkat cells are suspension lymphoblast, it was analyzed through flow cytometry. The principle of Annexin V-FITC/PI flow cytometry assay is Annexin V can bind to phosphatidylserine (PS) and PI can bind to DNA through the damaged cell membrane. Early apoptosis is distinguished by the subsidence of membrane asymmetry, with translocation of PS from the inner to the outer membrane before the loss of membrane integrity. Hence, this assay can discriminate the live cells (Annexin-V−/PI−), early apoptotic (Annexin-V+/PI−), late apoptotic (Annexin-V+/PI+), or necrotic (Annexin-V−/PI+). In [Figure 3], MC-433 induced 27.2% early apoptotic cells and 15.85% late apoptotic cells. No considerable changes were seen in the necrotic population. Compound MC-454 induced 52.93% early apoptotic cell. No significant changes were observed in late apoptotic and necrotic populations. In [Figure 4], the overlay of the results was (the % of cells undergone apoptosis in the untreated and test compounds, namely MC-433 and MC-454 treated Jurkat cells) plotted on a bar graph.
Figure 3: Determination of apoptosis and necrosis using flow cytometry in Jurkat cell line. Cells were stained with V-FITC/PI and assayed by flow cytometry. A dot plot is shown below. The right quadrant of each diagram (Annexin-V+/PI−), (Annexin-V+/PI+), represents apoptotic cells. (a) Untreated or control, (b) MC-433, (c) MC-431

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Figure 4: Overlay of the results (the % of cells of undergone apoptosis in the untreated and test compounds, namely MC-433 and MC-454 treated Jurkat cells) plotted in bar graph

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Optimization of sydnone derivatives by Lipinski Rule

Lipinski's rule of five” is a thumb rule to evaluate the drug-likeness of a chemical compound with a specific pharmacological property that would enforce an orally active drug. The conditions of this rule are as follows (i) hydrogen-bond donors should be <5, (ii) hydrogen-bond acceptors should be less than10, (iii) a molecular mass no more than 500 Dalton, and (iv) octanol-water partition coefficient (log P) not >5. All compounds of the present study met the requirements of “Lipinski's rule of five”. The other significant property is the total polar surface area (TPSA), TPSA of a compound should be <140Å. “Lipinski rule of five” properties of sydnone compounds are listed in [Table 4].
Table 4: Lipinski rule of five

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Molecular docking analysis

The molecular docking study was carried out by sydnone derivatives with tumor-associated proteins like EGF-TK, TNF-α, and Caspase3, which is highly expressed during uncontrolled cell proliferation. The minimum binding energy indicated that the target proteins EGF-TK, TNF-α, and Caspase3 bound with high affinity to Sydnone compounds. The docking results showed more interaction and more inhibitory activity with the targeted protein. The molecular docking study showed good interaction of sydnone derivatives with targeted cancer-inducing proteins which was compared with the commercially available anticancer drug compound anastrozole. Anastrozole is a nonsteroidal inhibitor of estrogen synthesis that resembles paclitaxel in chemical structure.

Epidermal growth factor domain receptor tyrosine kinase

The predicted active site amino acids in EGF-TK are 694 LEU, 694 LEU, 694 LEU, 694 LEU, 694 LEU, 695 GLY, 696 SER, 696 SER, 696 SER, 697 GLY, 697 GLY, 698 ALA, 699 PHE, and 699 PHE.

The molecular docking analysis of sydnone compounds with EGF-TK showed high binding energy compounds MC-454 (−6.99), MC-431 (−6.99) > MC-433 (−5.59) > MC-182 (−5.28). The detailed results of molecular confirmations, hydrophobic interactions, and hydrogen bond interactions are tabulated in [Table 5], [Table 6], [Table 7], respectively, the molecular interaction and 3D confirmation of 3-(4-propylbiphenyl-4'-yl) (MC-454) sydnone (binding energy-6.99) and 3-(4-methylbiphenyl-4'-yl) sydnone (MC 431) (binding energy-6.99) with EGF-TK are shown in [Figure 5] and [Figure 6].
Table 5: Binding energy and hydrogen bond interaction of sydnone derivatives and anastrozole docked with epidermal growth factor receptor tyrosine kinase, tumor necrosis factor caspase3, and tumor necrosis factor-alpha

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Table 6: Hydrophobic interactions of high binding energy sydnone compound with epidermal growth factor receptor tyrosine kinase, tumor necrosis factor-alpha, and caspase 3

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Table 7: Hydrogen interactions of high binding energy sydnone compound with epidermal growth factor receptor tyrosine kinase, tumor necrosis factor-alpha, and caspase 3

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Figure 5: The molecular interaction and three-dimensional confirmation of 3-(4-propylbiphenyl-4'-yl) (MC-454) sydnone with epidermal growth factor domain receptor tyrosine kinase (binding energy -6.99) (a) Surface interaction (b) Molecular interaction (c) Protein-Ligand interaction profiler view

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Figure 6: The molecular interaction and three-dimensional confirmation of 3-(4-methylbiphenyl-4'-yl) (MC-431) sydnone with epidermal growth factor domain receptor tyrosine kinase (binding energy-6.99), (a) Surface interaction (b) Molecular interaction (c) Protein-Ligand interaction profiler view

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Tumor necrosis factor-alpha

The predicted active site amino acids in TNF-α are 15HIS, 17VAL, 32ARG, 33ALA, 34ASN, 35ALA, 148GLY, and 151TYR. The molecular docking analysis of sydnone compounds with TNF-α was shown high binding energy compounds MC-431 (−5.47) > MC-454 (−5.38) > MC-433 (−4.98) > MC-182 (−2.66). The molecular interaction and 3D confirmation of 3-(4-propylbiphenyl-4'-yl) (MC-454) (binding energy-5.38) and 3-(4-methylbiphenyl-4'-yl) sydnone (MC-431) sydnone (binding energy-5.47) sydnone with TNF-α are shown in [Figure 7] and [Figure 8].
Figure 7: The molecular interaction and three-dimensional confirmation of 3-(4-propylbiphenyl-4'-yl) (MC-454) sydnone with tumor necrosis factor-alpha ((binding energy -5.38)), (a) Surface interaction (b) Molecular interaction (c) Protein-Ligand interaction profiler view

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Figure 8: The molecular interaction and three-dimensional confirmation of 3-(4-methylbiphenyl-4'-yl) sydnone (MC-431) sydnone with tumor necrosis factor-alpha (binding energy-5.47), (a) Surface interaction (b) Molecular interaction (c) Protein-Ligand interaction profiler view

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Caspase3

The predicted active site amino acids in caspase3 are 29SER, 29SER, 30GLY, 31ILE, 32SER, 32SER, 33LEU, 33LEU, 33LEU, 33LEU, 33LEU, 33LEU, 34ASP, 35ASN, 35ASN, 36SER, 36SER and 36SER. The molecular docking analysis of sydnone compounds with Caspase3 showed high binding energy compounds MC-431 (-8.02) > MC-454 (-6.38) > MC-433 (-4.80) > MC-182 (-2.14). The detailed molecular confirmations, hydrophobic interactions, and hydrogen bond interactions are tabulated in [Table 5], [Table 6], [Table 7], respectively. The molecular interaction and 3D confirmation of 3-(4-propylbiphenyl-4'-yl) (MC-454) (binding energy-6.38) and 3-(4-methylbiphenyl-4'-yl) sydnone (MC-431) sydnone (binding energy-8.02) sydnone with Caspase3 is shown in [Figure 9] and [Figure 10].
Figure 9: The molecular interaction and three-dimensional confirmation of 3-(4-propylbiphenyl-4'-yl) (MC-454) sydnone with Caspase3 (binding energy-6.38), (a) Surface interaction (b) Molecular interaction (c) Protein-Ligand interaction profiler view

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Figure 10: The molecular interaction and three-dimensional confirmation of 3-(4-methylbiphenyl-4'-yl) sydnone (MC-431) sydnone with Caspase3 (binding energy-8.02), (a) Surface interaction (b) Molecular interaction (c) Protein-Ligand interaction profiler view

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Absorption, distribution, metabolism, excretion, and toxicology analysis

ADMET properties are evaluated routinely at an early stage of drug development to reduce the failure rate in drug discovery processes because most of the clinical trial failures have been reported due to ADMET issues.[19],[20] The potential ADMET studies of sydnone compounds were carried out by pkCSM online server with the proteins EGF-TK, TNF-α, and Caspase 3. It was analyzed that the sydnone compounds were promising pharmacological active compounds. The detailed ADMET study results of sydnone compounds are listed in [Table 8].
Table 8: Absorption, distribution, metabolism, excretion, and toxicology profile of high energy docked sydnone compounds

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The insilico absorption prediction of sydnone compounds in human epithelial colorectal adenocarcinoma cells Cacao– 2 permeability is >0.90% which will be considered to be properly absorbed. Predicted absorption values are MC-454 (1.296), MC-431 (1.257), MC-433 (1.418), and MC-182 (1.206) which is proper for absorption. The detailed predicted absorption results like Intestinal absorption (human) and Skin Permeability are shown in [Table 8].

The circulation of the compound through the body was screened as VDss (human), fraction unbound (human), BBB permeability, CNS permeability parameters. When the VDss (volume distribution in steady state) is <0.71 L/kg, VDss will be considered as low. When VDss is higher than 2.81 L/kg (log VDss >0.45), the VDss will be likely to be relatively high.[36] Brain–blood permeability is measured in vivo in animal models as logBB, based on the predicted values, the compounds showed poor distribution to the brain as shown in [Table 8]. Metabolic processes and liability can cause various problems like poor bioavailability, toxic effects which are caused by reactive metabolites, and (drug–drug interactions) drug interactions.[37]

The inhibitors of the cytochrome p450's (essential enzyme for drug metabolism in the kidney) can alter the structural characteristics of drugs. From [Table 8], it is seen that sydnone compounds do not affect cytochrome P450. Organic cation transporter 2 (OCT2) is a transporter that plays an important role in the disposition and clearance of drugs and endogenous compounds in the kidney. OCT2 substrate also has the potential to cause side interactions when given together with OCT2 inhibitors. From [Table 8], it is seen that all the compounds studied did not affect the OCT2 substrate. Ames toxicity test is used to assess the mutagenic potential of compounds using bacteria. Positive test results indicate that the compound is mutagenic and therefore, can act as a carcinogen. From [Table 8], it is seen that MC-431, MC-433 showed positive results and the other two sydnone compounds (MC-454 and MC-182) showed negative results and they are not mutagenic. The predicted ADMET values of all sydnone compounds were compared with commercial breast cancer Anastrozole drug molecule. The details of each parameter are shown in [Table 8].


 > Conclusion Top


Most heterocyclic compounds present in most commercial pharmaceuticals, besides their inherent versatility and unique structural traits, have made these molecules an essential element of medical chemistry. Aside from the existing drugs, there are several other compounds that are being studied against several cancer diseases.[38]

In this study, we have evaluated four sydnone compounds for antiproliferative activity against three cancer cell lines. All the compounds were able to induce apoptosis in BT 474 breast cancer, HeLa human cervical carcinoma, and Jurkat cell lines.

In the molecular docking study, sydnone compounds showed a decent interaction affinity with receptor proteins, but among all four sydnone molecules, two compounds MC-454(−6.99) and MC-431(−6.99) found to have a better docking score with EGF-TK, that was comparable with the standard drug (anastrozole). Drug-like property was predicted by ADME toxicity study and by Lipinski rule of five and ADME toxicity studies. Therefore, the improved potential and selectivity of these sydnone derivatives need further preclinical evaluation.

Acknowledgement

The authors thank Principal Dr. V R Manjunath, Dr. H G Nagendra, Prof & Head, Dept. of Biotechnology, Sir M Visvesvarya institute of Technology and Sri Krishnadevaraya Educational Trust (Sri KET), Bengaluru for their support and encouragement.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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