Int J Pharm Pharm Sci, Vol 7, Issue 12, 113-117Original Article


DOCKING AND CYTOTOXICITY STUDIES OF 2-VINYLCHROMONE DERIVATIVES ON HUMAN BREAST CANCER CELL LINES

SWATI KAUSHIK#, MEGHA RIKHI#, SEEMA BHATNAGAR*

Novel Molecule Synthesis Laboratory, Amity Institute of Biotechnology, Amity University, Noida 201303, Uttar Pradesh, India
Email: sbhatnagar1@amity.edu

Received: 16 Aug 2015 Revised and Accepted: 27 Oct 2015

ABSTRACT

Objective: Estrogen receptor (ER) is over-expressed in 70% of breast cancers. The ER has two isoforms, ERα and ERβ. The ER ligand binding domain (LBD) has been the target for hormone-responsive breast cancer. Due to tissue-specific effects currently available drugs for hormone positive breast cancer presents serious limitation. The dynamic and plastic nature of ER LBD plays a crucial role in ligand design that discriminates between the ER subtypes. Agents that selectively target ER isoform are a formidable challenge to researchers. The chromone scaffold is a privileged scaffold for exploration of anticancer agents. The objective of the present study was to evaluate the anticancer activity of a small library of 2-vinylchromones in human breast cancer cell lines MCF-7 and MDA-MB-231.

Methods: The compounds were synthesized by the reported procedures. Docking studies of the substituted 2-vinylchromone was performed using GLIDE tool in Maestro 8.0. The compounds were evaluated for anticancer activity against MCF-7 (ERα positive), MDA-MB-231 (ERβ positive) and MRC-5 (ERα, β negative) cell lines using MTT assay.

Results: The in silico studies indicated that substituted 2-vinylchromones, 1(a-c) and 2(a-b) exhibited comparable docking score at LBD of ERα and ERβ. However, the binding affinity of the compounds for the allosteric binding site in ERβ was negligible. The dose-dependent studies using MTT assay depicted that compounds 1(a-c) and 2(a-b) exhibited anticancer activity in ERα positive cell line MCF-7 as compared to ERβ positive cell line MDA MB 231. The most potent anticancer activity was observed for compound 2b against MCF-7 cells with IC50 value of 15.625 μg/ml.

Conclusion: The present investigation indicated that 2-vinylchromone derivatives exhibited ER isoform selectivity and the presence of bulky group in 2-vinylchromones resulted in significantly higher cytotoxicity in ERα positive cell lines as compared to the ERβ positive cell line.

Keywords:Estrogen receptor, 2-vinylchromone, Docking, Anticancer, MCF-7, MDA-MB-231.

© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

INTRODUCTION

Chromones are an important class of oxygen-containing heterocyclic compounds. Synthetic and natural chromone derivatives possess important biological activities such as antitumor [1], antihepatotoxic [2], antioxidant [3], anti-inflammatory [4], antiallergic [5], estrogenic [6] and antibacterial [7] activities. The anticancer activity of chromones has also been related to its structural similarity with flavonoids.

Several compounds such as quercetin, genistein, daidzein, apigenin, biochanin A, kaempferol and naringenin that have chromone scaffold are known to bind to ER isoforms. Genistein, apigenin and kaempferol have a higher binding affinity 20 to 30-fold more for ERβ than ERα as indicated by a solid-phase binding assay [8-9].

The above literature precedence prompted us to investigate the docking and cytotoxicity of 2-vinylchromones on human breast cancer cell lines. Previous work in our own group had led to the synthesis of novel chromone derivatives such as 3,4-dihydro-2H-1-benzopyran-3,4 dione 1(a-c) using NaBH4(unpublished results)and in another study E-3-aminochromen-4-one 2(a-b) were synthesized(unpublished results). Herein, we report the in silico studies of chromone derivatives along with an assessment of cytotoxic activity of the compounds on MCF-7, MDA-MB-231 and MRC-5 cells.

MATERIALS AND METHODS

Determination of anticancer activity

MTT assay

5x103 cells/well were seeded in 96-multiwell flat bottom microtitre plate and cultured overnight in a humidified atmosphere of 5% CO2 at 37 °C. The test compounds were serially diluted to the wells. Different concentrations ranging from 125µg/ml to 0.98µg/ml were analyzed. The cells were incubated for 24 h with test compounds with 5% CO2 at 37 °C. After 24 h, 10 µl of MTT (5 mg/ml in 1X PBS) was added to each well. The cells were incubated for 3 h and the centrifuged at 1500 rpm for 10 min. The media were discarded and 150 µl of DMSO was added to each well. Optical density was measured using a microplate reader at 570 nm with reference optical density at 630 nm. IC50 values were calculated as the concentrations that show 50% inhibition of proliferation on the tested cell line.

Docking methodology

The compounds were modelled using the BUILD application of Maestro 8.0. The geometry was optimized by molecular mechanics using IMPACT in a dynamic environment using the standard TIP4P water model. The energy minimization was done using Optimized Potentials for Liquid Simulations 2005 (OPLS 2005) force field. Energy minimization was done using Polak-Ribier conjugate gradient and Truncated Newton conjugate gradient algorithms. The convergence threshold used was rms gradient of 0.01.

Conformational models of the ligands were generated. Docking of the ligands was carried out using an extra precision (XP) method called GLIDE (Grid-based Ligand Docking with Energetics) for flexible ligand docking. The ligands were prepared for docking using LIGPREP from Schrodinger’s molecular modeling software [10]. The PDB entries chosen for the above studies were 2QTU, 2FSZ and 3ERT. The ligand interaction map was used to study the interactions of novel molecules with the residues of ER.

Statistical analysis

The experimental data obtained for each set of the experiment were expressed as the mean of change±SEM and analyzed by one-way and two-way ANOVA followed by a bonferroni post-test. Level of significance was set to P≤ 0.05. All the statistical calculations were performed using the evaluation version of Graph Pad Prism 5.1 statistical software.

RESULTS

The synthesized compounds were evaluated for their binding affinity towards ERα and ERβ by docking studies. The PDB entry chosen for the docking studies were 2QTU [11], 2FSZ [12] and 3ERT [13]. The PDB entry 3ERT is a complex of ERα with hydroxy-tamoxifen (HT). The selection of 2QTU entry was based on structural similarity of the synthesized ligands with benzopyranones. Native ERβ pdb entry is not available therefore 2FSZ was selected which is a complex of HT with ERβ. The compounds were modelled using the BUILD application of Maestro 9.6. The energy minimization was performed using optimized potentials for liquid simulations 2005 (OPLS 2005) force fields. In case of ERβ isoform, the docking studies were performed at the cognate site (2FSZ-101) as well as the allosteric binding site (2FSZ-103) of HT. The docking score of compounds 1(a-c) and 2(a-b) are indicated in table 1.

The docking studies indicated that 1(a-c) and 2(a-b) exhibited comparable docking score at LBD of ERα and ERβ. However, the binding affinity of the compounds for the allosteric binding site in ERβ [12] was negligible. Amongst the analogues synthesized, best docking score for ERα was exhibited by compound 3-(butylamino)-2-[(E)-2-(2-methoxyphenyl) ethenyl]-4H-chromen-4-one (2b) containing aminoalkyl group and 2-[2-(2-methoxy phenyl)ethyl]-3,4-dihydro-2H-1-benzopyran-3,4 dione (1b) having a keto group at C-3 position. A comparison of the ligand interaction map represented in fig. 1 indicated that majority of the poses obtained for 2b share the following common residues: Glu 353, Arg 394, Trp 383, Ala 350, Met 343, Leu 391, Ile 424, Leu 525, Leu 346, Leu 387 as compared with HT. Compound 2b also depicted other significant interactions with the following residues: Asp 351, Leu 349, Leu 354, Leu 536, Met 528, Thr 347, His 524, Leu 428, Met 388, Leu 384, Phe 404, Ala 350.

Table 1: Docking score and glide energy of the compounds 1(a-c) and 2(a-b) against ERα and ERβ

S. No.

Compound

Structure

3ERT

2QTU

2FSZ (101)

2FSZ (103)
     

Docking

Score

Glide energy of themodel (Kcal/mol)

Docking

Score

Glide energy of themodel (Kcal/mol)

Docking Score

Glide energy of themodel (Kcal/mol)

Docking Score

Glide energy of the

model (Kcal/mol)

1

 1a

-5.19

-36.75

-7.8

-40.29

-6.57

-34.87

-3.42

-23.76

2

1b

-7.1

-27.4

-7.78

-38.64

-6.7

-36.56

-4.25

-29.98

3

1c

-6.14

-26.81

-6.03

-30.17

-5.23

-27.11

-3.42

-20.63

4

2a

-6.4

-34.35

-3.09

-21.86

-6.83

-43.35

-4.61

-30.49

5

2b

-8.27

-55.59

-3.87

-24.97

-5.84

-39.08

-3.39

-26.98

3ERT is a complex of ERα with HT. 2QTU pdb entry is a complex of benzopyranones with ERβ. 2FSZ is a complex of HT with ERβ where docking studies performed at the cognate site of HT was labeled as 2FSZ-101 and the allosteric binding site was labeled as 2FSZ-103.

The glide energy of the compound 1b and 2b with ERα was found to be -27.40Kcal/mol and -55.59Kcal/mol with -7.01 and -8.27 docking score. Fig. 1 (A and B) illustrates the interaction of compound 1b and 2b with amino acid residues of ERα (PDB ID 3ERT).

The dose-dependent studies using MTT assay depicted that compounds 1(a-c) and 2(a-b) exhibited anticancer activity in ERα positive cell line MCF-7 as compared to ERβ positive cell line MDA-MB-231. The compound 1b, 2a and 2b exhibited anticancer activity in ERα positive cell line. Amongst them compound 2b was most potent. Remarkable differences in the IC50 value were seen for 1b, 2a and 2b in MCF-7 and MDA-MB-231. The IC50 value for 1b, 2a, 2b were 62.5μg/ml, 31.25μg/ml and 15.625μg/ml, respectively in MCF-7 cell line. All the compounds exhibited an IC50 value of more than 125μg/ml in MDA-MB-231 cell line as indicated in table 2.

Table 2: Comparative IC50 value of compounds in MCF-7 and MDA-MB-231 cell lines

Compound

IC50-MCF-7 (μg/ml)

IC50-MDA-MB-231 (μg/ml)

1a

125

>125

1b

62.5

>125

1c

>125

>125

2a

31.25

>125

2b

15.62

>125

[A]


[B]

Fig. 1: [A] Interactions of compound 1b; [B] Interactions of compound 2b

The docking results of compound 1b, 2a and 2b on ERβ (PDB ID 2FSZ) on the other hand did not exhibit interactions with the significant residues in the ERβ LBD. The results are presented in fig. 2.



[A]


[B]

[C]

Fig. 2: [A] Interactions of compound 1b; [B] Interactions of compound 2a; [C] Interactions of compound 2b

The three-dimensional structure of ERα docked with compound 1b and 2b are represented in fig. 3 which also displays the superimposition of compound 1b and 2b with HT in the ligand binding pocket of ERα.


Fig.3: [A] Three-dimensional structure of ERα docked with compound 1b; [B] Three-dimensional structure of ERα docked with compound 2b; where HT is represented in red and compound 1b and 2b in green

In an attempt to corroborate the results of docking studies for compounds 1(a-c) and 2(a-b) MTT assay were performed in ERα and ERβ positive cell lines. The cell lines chosen for the studies were MCF-7 (ERα positive), MDA-MB-231 (ERβ positive) and MRC-5 (ERα,β negative). The results of these studies are depicted in fig. 4.

Fig.4: Comparative graph for 1(a-c) and 2(a-b) compounds with % change in optical density in respective cell lines plotted against different concentrations of compounds. The data are mean±SEM from 2 samples for each group and analyzed by two-way ANOVA followed by Bonferroni Post-test where * P<0.01 significant from control

DISCUSSION

The LBD of ERα and ERβ has 60% homology [14]. The dynamic and plastic nature of ER LBD plays a crucial role in ligand design that discriminates between the ER subtypes.

The interior of the ligand binding pocket of ERα and ERβ have been reported to have 22-24 residues which are identical and involved in interactions with ligands [15]. The two pockets are however different in size and flexibility and this aspect is crucial for the development of selective subtype agents [16].

Apart from this the discovery of allosteric binding pocket in the coactivator groove of ERβ further provides avenues for identification of agents known as coactivator binding inhibitors [12, 17]. In the present exploratory work we have attempted to identify ER isoform-selective compounds from a small library of 2-vinylchromones. For this purpose docking studies have been carried at both ERα and ERβ LBD and allosteric binding pocket of ERβ. The findings of docking studies have been correlated with MTT assay on ERα, ERβ and ER α, β negative cell lines.

The in silico studies revealed that the synthesized compounds preferentially binds to the ligand binding domain of ERα and ERβ with a comparable docking score except for 2b which exhibits preferential docking to ERα. It was also found that these compounds exhibited poor binding to the allosteric binding site of ERβ. Highest docking score amongst the series was also observed for compound 2b. Prominent interacting residues including Glu 353, Arg 394and Asp351 as compared to HT clearly indicate high-affinity binding which is further corroborated with a high docking score of 2b. A comparison of docking residues of ERβ, on the other hand, did not reveal an interaction with these residues.

Further, the evaluation ofthe cytotoxic activity indicated that these novel compounds have exhibited significant cytotoxicity in ERα positive cell line instead of ERβ positive cell line. Studies on ligands such as genistein have clearly established that although it exhibits similar binding mode when complexed to ERα and ERβ [18] it exhibits 40-fold selectivity for ERβ [19]. Similarly, electron density maps of HT complexed to ERβ indicate one of the HT molecules is located in the cognate ligand binding pocket with a confirmation indistinguishable from ERα/HT structure [20]. However; a comparison of IC50 values of HT in ERα and ERβ indicates ERα selectivity [21]. These observations suggest the involvement of different residues of ERα and ERβ LBD that may contribute towards isoform selectivity.

In our studies, it was observed that in all cases the chromones with methoxy substitution 1b, 2a and 2b exhibited the highest cytotoxicity. The presence of a bulky aminoalkyl group at C-3 of the chromone ring as compared to the keto group as in the case of 2a and 2b respectively led to significantly higher cytotoxicity. Further; structure-activity relationship studies will help to correlate contribution of long chain aminoalkyl substitution to cytotoxicity.

CONCLUSION

2-vinylchromone derivatives exhibited higher cytotoxicity in ERα positive cell lines as compared to the ERβ positive cell line. Our results also indicated that these derivatives exhibited ER isoform selectivity and presence of bulky group as in the case of 2a and 2b resulted in a significant increase in cytotoxic activity.

ACKNOWLEDGEMENT

The authors gratefully acknowledge Amity University for infrastructure and facilities. Author, MR thanks financial assistance through Amity Science, Technology and Innovation Foundation (ASTIF) fellowship provided by Amity University. Author, SK thanks Indian Council of Medical Research (ICMR) for financial support through Senior Research Fellowship (SRF).

CONFLICT OF INTERESTS

Declared None

REFERENCES

  1. Marten’s S, Mithofer A. Flavones and flavone synthases. Phytochemistry 2005;66:2399-407.
  2. Fernandes E, Carvalho M, Carvalho F, Silva AMS, Santos CMM, Pinto DCGA, et al. Hepatoprotective activity of polyhydroxylated 2-styrylchromones against tert-butyl-hydroperoxide induced toxicity in freshly isolated rat hepatocytes. Arch Toxicol 2003;77:500-3.
  3. Kuroda M, Uchida S, Watanabe K, Mimaki K. Chromones from the tubers of eranthis cilicica and their antioxidant activity. Phytochemistry 2009;70:288-93.
  4. Gabor M. Anti-inflammatory and anti-allergic properties of flavonoids. Prog Clin Biol Res 1986;213:471-80.
  5. Doria G, Romeo C, Forgione A, Sberze P, Tibolla N, Corno ML, et al. Antiallergic agents. III. Substituted trans-2-ethenyl-4-oxo-4H-1-benzopyran-6-carboxylic acids. Eur J Med Chem 1979;14:347-51.
  6. Maggioline M, Bonofiglio D, Marisco S, Panno ML, Cenni B, Picard D, et al. Estrogen receptor alpha mediates the proliferative but not the cytotoxic dose-dependent effects of two major phytoestrogens on human breast cancer cells. Mol Pharmacol 2001;60:595-602.
  7. Grindlay D, Reynolds T. The aloe vera phenomenon: a review of the properties and modern uses of the leaf parenchyma gel. J Ethnopharmacol 1986;16:117-51.
  8. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, et al. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology 1997;138:863-70.
  9. Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, Van der Saag PT, et al. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 1998;139:4252-63.
  10. Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, et al. Glide: a new approach for rapid, Accurate docking and scoring. 1. Method and assessment of docking accuracy. J Med Chem 2004;47:1739-49.
  11. Richardson TI, Dodge JA, Wang Y, Durbin JD, Krishnan V, Norman BH. Benzopyrans as selective estrogen receptor beta agonists (SERBAs). Part 5: Combined A-and C-ring structure-activity relationship studies. Bioorg Med Chem Lett 2007;17:5563-6.
  12. Wang Y, Chirgadze NY, Briggs SL, Khan S, Jensen EV, Burris TP. A second binding site for hydroxytamoxifen within the coactivator-binding groove of estrogen receptor beta. Proc Natl Acad Sci 2006;103:9908-11.
  13. Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, et al. The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 1998;95:927-37.
  14. Petit FG, Valotaire Y, Pakdel F. The analysis of chimeric human/rainbow trout estrogen receptors reveals amino acid residues outside of P-and D-boxes important for the transactivation function. Nucleic Acids Res 2000;28:2634-42.
  15. Pike AC, Brzozowski AM, Hubbard RE, Bonn T, Thorsell AG, Engstrom O, et al. Structure of the ligand-binding domain of estrogen receptor beta in the presence of a partial agonist and a full antagonist. EMBO J 1999;18:4608–18.
  16. Katzenellenbogen JA, Muthyala R, Katzenellenbogen BS. The nature of the ligand-binding pocket of estrogen receptor alpha and beta: the search for subtype-selective ligands and implications for the prediction of estrogenic activity. Pure Appl Chem 2003;75:2397-403.
  17. Sun A, Moore TW, Gunther JR, Kim MS, Rhoden E, Du Y, et al. Discovering small-molecule estrogen receptor α/coactivator binding inhibitors: high-throughput screening, ligand development, and models for enhanced potency. ChemMedChem 2011;6:654-66.
  18. Manas ES, Xu ZB, Unwalla RJ, Somers WS. Understanding the selectivity of genistein for human estrogen receptor-beta using X-ray crystallography and computational methods. Structure 2004;12:2197-207.
  19. Poluzzi E, Piccinni C, Raschi E, Rampa A, Recanatini M, Ponti FD. Phytoestrogens in postmenopause: the state of the art from a chemical, pharmacological and regulatory perspective. Curr Med Chem 2014;21:417-36.
  20. Kojetin DJ, Burris TP, Jensen EV, Khan SA. Implications of the binding of tamoxifen to the coactivator recognition site of the estrogen receptor. Endocr-Relat Cancer 2008;15:851–70.
  21. Weatherman RV, Clegg NJ, Scanlan TS. Differential SERM activation of the estrogen receptors (ERα and ERβ) at AP-1 sites. Chem Biol 2001;8:427-36.