Thienopyrimidine-Chalcones Hybrid Molecules Inhibits Fas-activated

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Thienopyrimidine-Chalcones Hybrid Molecules Inhibits Fas-activated Serine/Threonine Kinase: An Approach to Ameliorates Antiproliferation in Human Breast Cancer Cells Nashrah Sharif Khan, Parvez Khan, Mohammad Fawad ANSARI, SAURABHA SRIVASTAVA, Gulam Mustafa Hasan, Mohammad Husain, and Md Imtaiyaz Hassan Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00566 • Publication Date (Web): 24 Jul 2018 Downloaded from http://pubs.acs.org on July 25, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Molecular Pharmaceutics

1 Thienopyrimidine-Chalcones Hybrid Molecules Inhibits Fas-activated Serine/Threonine 2 3 Kinase: An Approach to Ameliorates Antiproliferation in Human Breast Cancer Cells 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Nashrah Sharif Khan, Parvez Khan, Mohammad Fawad Ansari, Saurabha Srivastava, Gulam Mustafa Hasan, Mohammad Husain and Md. Imtaiyaz Hassan

FASTK (10) O Cl

Binding affinity Estimation: ITC

HN N N

KD = 2.26 µM (ITC)

S

Apoptosis

IC50 = 0.17 µM Kinase assay

High Binding-affinity

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FASTK Inhibition: Apoptosis in MCF-7 Cells

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Thienopyrimidine-Chalcones Hybrid Molecules Inhibits Fasactivated Serine/Threonine Kinase: An Approach to Ameliorates Antiproliferation in Human Breast Cancer Cells Nashrah Sharif Khan1, Parvez Khan2, Mohammad Fawad Ansari3, Saurabha Srivastava2, Gulam Mustafa Hasan4, Mohammad Husain1,* and Md. Imtaiyaz Hassan2,* 1

Department of Biotechnology, Jamia Millia Islamia, Jamia Nagar, New Delhi, 110025 India. Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi-110025, India. 3 Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi, 110025 India. 4 Department of Biochemistry, College of Medicine, Prince Sattam Bin Abdulaziz University, Al-Kharj, Saudi Arabia. 2

Running head: FASTK Inhibitors

*Correspondence Professor Mohammad Husain Department of Biotechnology Jamia Millia Islamia, New Delhi-110025, India Tel.: 011-26981717, 3426 (Extn) E-mail: [email protected] Dr. Md. Imtaiyaz Hassan Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi-110025, India Tel.: +91-9990323217 E-mail: [email protected]

1 ACS Paragon Plus Environment

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ABSTRACT Apoptotic evasion by cancerous cells being one of the striking hallmarks of cancer has turned into a new arena of exploration for drug discovery. A number of pathways reported to govern the apoptotic evasion. Fas-activated Serine/Threonine Kinase (FASTK) is a member of Ser/Thr kinase family, has been implicated in apoptotic evasion, and hence development of cancers. Keeping this in view, a series of novel thienopyrimidine based chalcones have been synthesized and evaluated to modulate the FASTK mediated apoptotic evasion. Initial screening was done by enzyme inhibition assay and binding studies which showed that out of 15 synthesized compounds, three thienopyrimidine based chalcone derivatives possesses considerably high binding affinity and enzyme inhibitory potential (nM range) for FASTK. Cell proliferation assessment of selected compounds was performed on HEK-293 and MCF-7 cells. For MCF-7 cells, compound 2, 10 and 12 show an IC50 value of 20.22±1.50 µM, 6.52±0.82 µM and 8.20±0.61µM, respectively. Annexin-V and PI-staining suggested that these molecules induce apoptosis in MCF-7 cells, arrest cell cycle in G0/G1 phase and subsequently inhibit cell migration presumably by inhibiting FASTK and reactive oxygen species production. In conclusion, we have successfully designed, synthesized and characterized thienopyrimidine based chalcones which inhibits FASTK and induce apoptosis, and these compounds may be exploited as potential anticancer agents. KEYWORDS: Fas-activated Serine/Threonine Kinase; apoptosis; chalcones; kinase inhibitors and cell-cycle arrest



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INTRODUCTION Breast cancer is a second foremost reason of mortality (after lung cancer) in women with an estimated 1.67 million new cancer cases (25% of all the cancer) 1. Despite current efforts to control this disease its risk is estimated to upsurge in coming years

2-5

. Risk factors responsible

for the increased incidents of the disease include lesser physical activities, higher consumption of alcohol, different hormone therapies, early menarche, later menopause, late age of first pregnancy, family history and shorter or no periods of breast feeding

5, 6

. Commonly used

treatments for breast cancer include surgery and/or radiotherapy and adjuvant chemo- or hormone therapies 7. In spite of recent advancements in the conventional treatment of breast cancer, patient’s survival rate remains very low 8. These conventional treatments are hindered by unwanted side effects, including treatment resistance, disease recurrence, and metastasis. Thus, there is an urgent need of innovative and operative therapies for breast cancer 9.

Fas-activated serine/threonine phosphoprotein/kinase (FASTK) is an important protein, inhibiting Fas and UV-induced apoptosis and found associated with nucleus and mitochondria 10, 11

. The amino terminus of FASTK codes for a mitochondrial-targeting motif that helps to pass

through the outer mitochondrial membrane, whereas the carboxyl terminus interacts with BCLXL, which mainly responsible for outer mitochondrial membrane interactions

10,

11

.

Overexpression of recombinant FASTK inhibits Fas- and UV-induced apoptosis. In Jurkat T cells, ligation of Fas triggered caspase-8 which results in the cleavage of BH3-only protein BID 12

. Truncated BID (tBID) collaborate with BCL-2 and BCL-XL as it is transported from

cytoplasm to mitochondrial membrane 13.


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It was noticed that if neutralizing potential of BCL-2 and BCL-XL enhanced, then tBID acted on the BAX and BAK proapoptotic proteins, which than facilitate the transport of apoptotic effectors molecules from mitochondrial stocks

14

. The discharge of smac/DIABLO

induces apoptosis by IAP proteins (E3 ligases family proteins) inhibition that prevent the activation of apoptotic caspases

15

. FAST is found to be dephosphorylated in the Fas-induced

apoptotic Jurkat cells 16. After release from the mitochondria FASTK binds to the cytoplasmic Tcell intracellular antigen-1 (TIA-1), latter act as a repressor of protein translation

11, 16

. TIA-1

inhibition supports the overexpression of cIAP and XIAP which ultimately results in the inhibition of apoptosis 11, that was regulated by the NF-κB pathway 17. FASTK gene is identified as a negative gene whose expression is associated with shorter survival rate of lymphoblastic leukemia cases

18

. Recently, FASTK family members are emerged as key regulators of post-

transcriptional gene expression in mitochondria

19

. These proteins are architecturally correlated

RNA-binding partners with diverse functions in the regulation of RNA biology, gene expression regulation and function within the mitochondria 19, 20.

Homozygous nonsense mutations in FASTKD2 have been associated with mitochondrial encephalomyopathy, which are further linked with insufficiency of cytochrome-c oxidase activity

21, 22

. Tissue expression atlases (https://www.ebi.ac.uk) and the human protein

atlas (http://www.proteinatlas.org/cell) suggesting that over-expression of FASTK in breast cancer tissues or derived cell lines. Cancerous cells are smart as they evade the apoptosis which helps them to survive and multiply continuously. So, apoptotic evasion becomes an important hallmark of cancer that has been used for the identification of novel anticancer therapeutic molecules

8, 23

. Furthermore, over-expression of FASTK inhibits the cell apoptosis 11, and leads



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to several diseases including cancer, and thus it becomes an important drug target for the development of anticancer molecules.

Chalcones are naturally occurring plant products widely distributed in spices, tea, beer, fruits and vegetables 24, 25. Chalcone derivatives (molecules comprising 1,3-diphenyl- 2-3propen1-one groups) are precursors in the biosynthesis of flavonoids/ isoflavonoids, consisting of an open-chain flavonoid moiety associated with two aromatic rings that are further linked by a three-carbon α, β-unsaturated carbonyl system

26, 27

. These are precursor of several biologically

active compounds and major components of natural products that has been evaluated as potential inhibitors against different signalling pathways or enzymes

28-32

. Despite its numerous

applications, limited reports are available on the use of chalcones in cancer therapy. Similarly, pyrimidine nucleus representing a class of heterocyclic nitrogen containing compound with a broad spectrum of biological utilities including anticancer properties

33-35

. Hence, we have

incorporated pyrimidinyl group to the chalcone core, and thus novel chalcone hybrid molecules bearing anticancerous properties were synthesized, which possesses the dual moiety, one is pyrimidinyl (anticancerous moiety) and other is chalcone core (widely present in natural compounds and used as anchoring molecule, non-toxic in nature).

Since, FASTK is involved in apoptosis and cancer; therefore we will try to inhibit its activity using novel chalcones-pyrimidine hybrid analogues As no crystal structure is available for human FASTK, the structure of FASTK was modelled and compared with the reported crystal structures of the same family and their known inhibitors were taken into the consideration for the synthesis of newer chalcone derivatives. Further, major tools of structure based drug design and discovery was exploited to find the possible hits to synthesize thienopyrimidine-


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chalcones hybrids, which may have the potential to inhibit the enzyme activity of FASTK. Binding of synthesized compounds to FASTK was evaluated by fluorescence emission spectra measurements and isothermal titration calorimetry (ITC). Finally, anticancerous activities of these compounds were estimated through cell proliferation, cell cycle analysis, apoptosis (propidium iodide and annexin-V staining) and cell migration based assays.

MATERIAL AND METHODS Synthesis protocols The chemicals used for synthesis were procured from Sigma Aldrich, USA and Merck Darmstadt, Germany. Thin layer chromatographic plates (Silica gel 60 F254) were used for chromatographic analysis. IR spectra were recorded on Bruker FT-IR spectrophotometer, Massachusetts, United States. The mass spectra of synthesized compounds were taken with the help of ESI-MS, AB-Sciex 2000, Applied Biosystem California, United States. The

13

C NMR

and 1H NMR were acquired on Bruker Spectrospin DPX 75 MHz and Bruker Spectrospin DPX 300 MHz instruments. CDCl3 and DMSO-d6 were used as a solvent while trimethylsilane (TMS) was used as internal control. The following codes were assigned to define the splitting pattern of peaks; s (singlet); d (doublet); m (multiplet). The values of chemical shift are in ppm.

General method for the synthesis of 5,6,7,8-tetrahydrobenzo [4,5]theino[2,3d]pyrimidine

based

chalcones.

A

mixture

of

1-(4-((5,6,7,8-

tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)phenyl) ethan-1-one(1 eq.), aromatic benzaldehydes (1.5 eq.), and 2 mL, 40% aqueous solution of sodium hydroxide in 10 mL of ethanol was stirred up at room temperature for 24 h. At each step, the progress of reaction was 6 ACS Paragon Plus Environment


monitored with the help of TLC. Once the reaction was completed, the solvent was evaporated in vacuo. Further, the reaction concentrate was extracted with the help of dichloromethane and dried using anhydrous sodium sulphate. The final reaction product was recrystallized using methanol.

1-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)phenyl) ethan-1-one (1). Yield: 87%; m.p. 238°C; Anal. Calc. C18H17N3OS: C 66.85, H 5.30, N 12.99, S 9.91%. found: C 66.59, H 5.48, N 13.16, S 10.07%; 1H NMR (CDCl3) δ (ppm): 8.46 (s, 1H, Ar-H), 8.36 (s, 1H, NH), 8.05 (d, 2H, J=7.8 Hz, Ar-H), 7.88 (d, 2H, J=8.4 Hz, Ar-H), 3.25 (s, 2H, CH2), 2.88 (s, 2H, CH2), 2.01 (s, 4H, CH2);

13

C NMR (CDCl3) δ (ppm): 201.32, 162.56, 159.99, 152.35,

146.75, 141.42, 138.40, 133.96, 131.86, 127.52, 122.95, 31.38, 30.45, 30.30, 26.98, 26.83; ESIMS: m/z = 323.1.

3-phenyl-1-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino) phenyl)prop-2-en-1-one (2). Yield: 73%; m.p. 204°C; Anal. Calc. C25H21N3OS: C 72.97, H 5.14, N 10.21, S 7.79%. found: C 72.71, H 5.37, N 10.39, S 7.55%; FT-IR νmax (cm-1): 1648 (C=O), 1553 (C=C); 1H NMR (CDCl3) δ (ppm): 8.58 (s, 1H, Ar-H), 8.09 (d, 2H, J=8.4 Hz, ArH), 7.87 (d, 2H, J=8.7 Hz, Ar-H), 7.85 (d, 1Hα, J=15.3 Hz), 7.66 (m, 2H, Ar-H), 7.58 (d, 1Hβ, J=15.6), 7.43 (m, 3H, Ar-H), 7.39 (s, 1H, NH), 3.10 (d, 2H, J=5.7 Hz, CH2), 2.87 (s, 2H, CH2), 2.00-1.95 (m, 4H, CH2);

13

C NMR (CDCl3) δ (ppm): 188.58, 166.68, 154.12, 152.15, 144.23,

143.05, 135.67, 135.00, 132.85, 130.42, 129.95, 128.94, 128.39, 124.46, 121.74, 119.50, 117.15, 26.47, 25.57, 22.51, 22.33; ESI-MS: m/z = 412.4.

3-(4-methylphenyl)-1-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4yl)amino)phenyl)prop-2-en-1-one (3). Yield: 75%; m.p. 196°C;Anal. Calc. C26H23N3OS: C 7 ACS Paragon Plus Environment

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73.38, H 5.45, N 9.87, S 7.54%. found: C 73.51, H 5.63, N 9.62, S 7.75%;FT-IR νmax (cm-1): 1649 (C=O), 1551 (C=C);1H NMR (CDCl3) δ (ppm): 8.57 (s, 1H, Ar-H), 8.07 (d, 2H, J=8.7 Hz, Ar-H), 7.86 (d, 2H, J=8.7 Hz, Ar-H), 7.83 (d, 1Hα, J=16.5 Hz), 7.55 (d, 2H, J=7.5 Hz, Ar-H), 7.53 (d, 1Hβ, J=15.6), 7.37 (s, 1H, NH), 7.24 (d, 2H, J=7.8 Hz, Ar-H), 3.09 (d, 2H, J=5.7 Hz, CH2), 2.88 (d, 2H, J=5.4 Hz, CH2), 2.39 (s, 3H, CH3), 2.02-1.94 (m, 4H, CH2);

13

C NMR

(CDCl3) δ (ppm): 187.90, 166.18, 153.61, 151.36, 143.45, 142.78, 140.28, 134.63, 132.13, 131.56, 129.14, 129.07, 127.79, 124.58, 120.19, 119.19, 116.90, 25.62, 24.98, 21.84, 21.77, 20.88; ESI-MS: m/z = 425.89.

3-(4-ethylphenyl)-1-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4yl)amino)phenyl)prop-2-en-1-one (4). Yield: 78%; m.p. 198°C; Anal. Calc. C27H25N3OS: C 73.77, H 5.73, N 9.56, S 7.29%. found: C 73.91, H 5.94, N 9.67, S 7.51%; FT-IR νmax(cm-1): 1647 (C=O), 1549 (C=C);1H NMR (CDCl3) δ (ppm): 8.57 (s, 1H, Ar-H), 8.09 (d, 2H, J=8.7 Hz, Ar-H), 7.87-7.79 (m, 3H, 2Ar-H, 1Hα), 7.59 (d, 2H, J=8.1 Hz, Ar-H), 7.54 (d, 1Hβ, J=15.6 Hz), 7.38 (s, 1H, NH), 7.27-7.24 (m, 3H, 2Ar-H), 3.12 (t, 2H, J=5.7 Hz, CH2), 2.90 (t, 2H, J=6.0 Hz, CH2), 2.73 (m, 2H, CH2), 2.03 (m, 4H, CH2), 1.29 (t, 3H, J=7.5, CH3);

13

C NMR (CDCl3) δ

(ppm): 188.82, 166.81, 154.22, 152.24, 147.24, 144.40, 142.95, 135.67, 133.17, 132.56, 129.97, 128.50, 124.44, 120.93, 119.60, 117.15, 28.85, 26.56, 25.59, 22.55, 22.37, 15.30; ESI-MS: m/z = 440.4.

3-(4-methoxyphenyl)-1-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4yl)amino)phenyl)prop-2-en-1-one (5). Yield: 77%; m.p. 222°C; Anal. Calc. C26H23N3O2S: C 70.72, H 5.25, N 9.52, S 7.26%. found: C 70.83, H 5.63, N 9.77, S 7.55%; FT-IR νmax (cm-1): 1647 (C=O), 1548 (C=C); 1H NMR (CDCl3) δ (ppm): 8.57 (s, 1H, Ar-H), 8.07 (d, 2H, J=8.4 Hz,



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Ar-H), 7.86 (d, 2H, J=8.74 Hz, Ar-H), 7.82 (d, 1Hα, J=15.9 Hz), 7.62 (d, 2H, J=8.4 Hz, Ar-H), 7.46 (d, 1Hβ, J=15.6), 7.37 (s, 1H, NH), 6.95 (d, 2H, J=8.7 Hz, Ar-H), 3.86 (s, 3H, OCH3), 3.11 (d, 2H, J=5.1 Hz, CH2), 2.89 (t, 2H, J=5.7 Hz, CH2), 2.02-1.95 (m, 4H, CH2);

13

C NMR

(DMSO-d6) δ (ppm): 187.72, 166.87, 161.69, 154.48, 152.32, 144.41, 143.58, 134.39, 132.46, 131.09, 129.87, 127.95, 126.98, 120.51, 120.01, 118.19, 114.85, 55.83, 25.69, 25.59, 22.62, 22.42; ESI-MS: m/z = 442.4.

3-(4-ethoxyphenyl)-1-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4yl)amino)phenyl)prop-2-en-1-one (6). Yield: 74%; m.p. 230°C; Anal. Calc. C27H25N3O2S: C 71.18, H 5.53, N 9.22, S 7.04%. found: C 71.05, H 5.72, N 9.55, S 7.35%; FT-IR νmax (cm-1): 1650 (C=O), 1553 (C=C); 1H NMR (CDCl3) δ (ppm): 8.57 (s, 1H, Ar-H), 8.08 (d, 2H, J=8.1 Hz, Ar-H), 7.86-7.77 (dd, 3H, 2Ar-H J=8.1 Hz, 1Hα, J=15.9 Hz), 7.61 (d, 2H, J=8.4 Hz, Ar-H), 7.46 (d, 1Hβ, J=15.6 Hz), 7.38 (s, 1H, NH), 6.94 (d, 2H, J=8.1 Hz, Ar-H), 4.12-4.05 (m, 2H, CH2), 3.10 (d, 2H, J=5.7 Hz, CH2), 2.89 (t, 2H, J=5.7 Hz, CH2), 2.03 (m, 4H, CH2), 1.46 (t, 3H, J=6.9 Hz, CH3);

13

C NMR (DMSO-d6) δ (ppm): 187.69, 166.86, 160.99, 154.46, 152.30, 144.38,

143.60, 134.37, 132.46, 131.08, 129.85, 127.79, 126.95, 120.48, 119.89, 118.16, 115.22, 63.78, 25.69, 25.59, 22.61, 22.42, 15.01; ESI-MS: m/z = 456.4.

3-(4-propoxyphenyl)-1-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4yl)amino)phenyl)prop-2-en-1-one (7). Yield: 75%; m.p. 188°C; Anal. Calc. C28H27N3O2S: C 71.61, H 5.80, N 8.95, S 6.83%. found: C 71.94, H 6.01, N 9.09, S 6.54%; FT-IR νmax (cm-1): 1650 (C=O), 1552 (C=C); 1H NMR (CDCl3) δ (ppm): 8.56 (s, 1H, Ar-H), 8.06 (d, 2H, J=8.1 Hz, Ar-H), 7.84 (d, 2H, J=8.7 Hz, Ar-H), 7.81 (d, 1Hα, J=16.8 Hz), 7.59 (d, 2H, J=7.8 Hz, Ar-H), 7.44 (d, 1Hβ, J=15.6 Hz), 7.35 (s, 1H, NH), 6.93 (d, 2H, J=8.1 Hz, Ar-H), 3.98 (t, 2H, J=6.0 Hz,


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CH2), 3.08 (s, 2H, CH2), 2.85 (s, 2H, CH2), 1.99-1.95 (m, 4H, CH2), 1.86-1.79 (m, 2H, CH2), 1.07 (t, 3H, J=6.9, CH3);

13

C NMR (CDCl3) δ (ppm): 193.28, 171.46, 165.94, 158.94, 156.78,

148.78, 147.81, 140.08, 137.76, 134.87, 134.46, 132.14, 129.65, 124.42, 123.98, 122.07, 119.64, 74.35, 31.06, 30.30, 27.18, 27.08, 15.20; ESI-MS: m/z = (470.03).

3-(4-isopropylphenyl)-1-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4yl)amino)phenyl)prop-2-en-1-one (8). Yield: 79%; m.p. 208°C; Anal. Calc. C28H27N3OS: C 74.14, H 6.00, N 9.26, S 7.07%. found: C 74.32, H 6.19, N 9.39, S 7.33%; FT-IR νmax (cm-1): 1651 (C=O), 1550 (C=C); 1H NMR (CDCl3) δ (ppm): 8.57 (s, 1H, Ar-H), 8.08 (d, 2H, J=8.4 Hz, Ar-H), 7.87 (d, 2H, J=8.4 Hz, Ar-H), 7.84 (d, 1Hα, J=15.9 Hz), 7.60 (d, 2H, J=8.1 Hz, Ar-H), 7.54 (d, 1Hβ, J=15.9 Hz), 7.39 (s, 1H, NH), 7.30 (d, 2H, J=7.8 Hz, Ar-H), 3.11 (s, 2H, CH2), 3.00-2.90 (m, 1H, CH), 2.88 (s, 2H, CH2), 2.03-1.95 (m, 4H, CH2), 1.29 (d, 6H, J=6.9 Hz, CH3); 13

C NMR (CDCl3) δ (ppm): 187.83, 166.89, 154.46, 152.31, 151.70, 144.56, 143.68, 134.44,

133.00, 132.26, 129.96, 129.37, 127.33, 126.99, 121.57, 120.49, 118.24, 33.88, 25.68, 25.59, 24.08, 22.61, 22.41; ESI-MS: m/z = (454.01).

3-(4-(dimethylamino)phenyl)-1-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4yl)amino)phenyl)prop-2-en-1-one (9). Yield: 77%; m.p. 170°C; Anal. Calc. C27H26N4OS: C 71.34, H 5.76, N 12.32, S 7.05%. found: C 71.51, H 5.84, N 12.61, S 7.09%; FT-IR νmax (cm-1): 1649 (C=O), 1551 (C=C); 1H NMR (CDCl3) δ (ppm): 8.57 (s, 1H, Ar-H), 8.09 (d, 2H, J=8.7 Hz, Ar-H), 7.86 (d, 2H, J=6.3 Hz, Ar-H), 7.83 (d, 1Hα, J=15.9 Hz), 7.57 (d, 2H, J=9.0 Hz, Ar-H), 7.39-7.34 (m, 3H, NH and Ar-H), 6.72 (d, 2H, J=8.7 Hz, Ar-H), 3.13 (s, 2H, CH2), 3.07 (s, 6H, CH3), 2.90 (s, 2H, CH2), 1.99 (m, 4H, CH2);

13

C NMR (DMSO-d6) δ (ppm): 188.01, 167.35,



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155.31, 152.35, 151.51, 144.99, 144.05, 134.35, 133.09, 131.01, 129.62, 129.31, 127.01, 126.06, 120.61, 119.76, 112.24, 39.91. 26.67, 25.73, 22.63, 22.46; ESI-MS: m/z = (455.5).

3-(4-chlorophenyl)-1-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4yl)amino)phenyl)prop-2-en-1-one (10). Yield: 77%; m.p. 225°C; Anal. Calc. C25H20ClN3OS: C 67.33, H 4.52, N 9.42, S 7.19%. found: C 67.39, H 4.71, N 9.55, S 7.01%; FT-IR νmax (cm-1): 1651 (C=O), 1551 (C=C); 1H NMR (CDCl3) δ (ppm): 8.57 (s, 1H, Ar-H), 8.08 (d, 2H, J=8.7 Hz, Ar-H), 8.00-7.97 (m, 1H, Ar-H), 7.88-7.83 (m, 2H, Ar-H), 7.79 (d, 1Hα, J=15.6 Hz), 7.59 (d, 1H, J=8.7 Hz, Ar-H), 7.55 (d, 1Hβ, J=15.6 Hz), 7.41-7.31 (m, 3H, Ar-H), 3.11 (m, 2H, CH2), 2.88 (s, 2H, CH2), 2.04-1.96 (m, 4H, CH2);

13

C NMR (DMSO-d6) δ (ppm): 187.49, 166.83, 154.56,

152.34, 144.76, 144.04, 134.32, 133.02, 131.05, 129.61, 127.01, 122.66, 120.57, 118.14, 116.62, 112.22, 25.71, 25.60, 22.62, 22.43; ESI-MS: m/z = (446.04).

3-(2,5-dimethoxyphenyl)-1-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4yl)amino)phenyl)prop-2-en-1-one (11). Yield: 76%; m.p. 160°C; Anal. Calc. C27H25N3O3S: C 68.77, H 5.34, N 8.91, S 6.80%. found: C 69.01, H 5.42, N 9.03, S 6.74%; FT-IR νmax (cm-1): 1650 (C=O), 1554 (C=C); 1H NMR (CDCl3) δ (ppm): 8.57 (s, 1H, Ar-H), 8.10-8.05 (m, 3H, ArH), 7.86 (d, 2H, J=8.4 Hz, Ar-H), 7.64 (d, 1Hα, J=15.9 Hz), 7.38 (s, 1H, NH), 7.17 (s, 1H, Ar-H), 6.96-6.86 (m, 2H, Ar-H), 3.88 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.12 (t, 2H, J=5.4 Hz, CH2), 2.89 (t, 2H, J=6.0 Hz, CH2), 2.02-1.94 (m, 4H, CH2);

13

C NMR (CDCl3) δ (ppm): 189.11,

166.69, 154.16, 153.51, 153.32, 152.16, 142.87, 139.56, 135.56, 133.14, 129.96, 124.61, 124.49, 122.83, 119.48, 117.12, 117.05, 113.88, 112.44, 56.11, 55.84, 26.48, 25.56, 22.51, 22.33; ESIMS: m/z = 472.4.


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3-(3,4-dimethoxyphenyl)-1-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4yl)amino)phenyl)prop-2-en-1-one (12). Yield: 75%; m.p. 214°C; Anal. Calc. C27H25N3O3S: C 68.77, H 5.34, N 8.91, S 6.80%. found: C 68.85, H 5.47, N 9.02, S 6.99%; FT-IR νmax (cm-1): 1647 (C=O), 1551 (C=C); 1H NMR (CDCl3) δ (ppm): 8.57 (s, 1H, Ar-H), 8.08 (d, 2H, J=8.7 Hz, Ar-H), 7.87 (d, 2H, J=8.7 Hz, Ar-H), 7.80 (d, 1Hα, J=15.3 Hz), 7.44 (d, 1Hβ, J=15.9 Hz), 7.23 (m, 2H, NH+Ar-H), 6.92 (d, 1H, J=8.4 Hz, Ar-H), 3.96 (s, 3H, OCH2), 3.93 (s, 3H, OCH3), 3.10 (s, 2H, CH2), 2.87 (s, 2H, CH2), 2.01-1.96 (m, 4H, CH2);

13

C NMR (CDCl3) δ (ppm): 188.67,

166.71, 154.15, 152.15, 151.37, 149.26, 144.39, 142.86, 135.61, 133.15, 129.86, 128.00, 124.47, 123.02, 119.73, 119.51, 117.13, 111.16, 110.18, 55.99, 26.48, 25.57, 22.51, 22.33; ESI-MS: m/z = 472.4.

3-(3,4,5-trimethoxyphenyl)-1-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4yl)amino)phenyl)prop-2-en-1-one (13). Yield: 81%; m.p. 179°C; Anal. Calc. C28H27N3O4S: C 67.70, H 5.43, N 8.38, S 6.39%. found: C 67.52, H 5.63, N 8.42, S 6.48%; FT-IR νmax (cm-1): 1650 (C=O), 1550 (C=C); 1H NMR (CDCl3) δ (ppm): 8.57 (s, 1H, Ar-H), 8.09 (d, 2H, J=8.4 Hz, Ar-H), 7.89 (d, 2H, J=8.4 Hz, Ar-H), 7.76 (d, 1Hα, J=15.6 Hz), 7.45-7.40 (m, 2H, 1Hβ and NH), 3.93 (s, 6H, OCH3), 3.91 (s, 3H, OCH3), 3.12 (s, 2H, CH2), 2.88 (s, 2H, CH2), 2.01-1.97 (m, 4H, CH2);

13

C NMR (CDCl3) δ (ppm): 188.78, 154.21, 153.51, 152.20, 144.47, 143.01, 135.75,

133.07, 130.51, 130.01, 124.41, 121.31, 119.62, 117.17, 105.73, 60.99, 56.52, 26.52, 25.59, 22.55, 22.36; ESI-MS: m/z = 502.4.

3-(furan-2-yl)-1-[4-(5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4ylamino)phenyl]prop-2-en-1-one (14). Yield: 73%; m.p. 163°C; Anal. Calc. C23H19N3O2S: C 68.81, H 4.77, N 10.47, S 7.99%. found: C 69.09, H 4.55, N 10.59, S 8.17%; FT-IR νmax (cm-1):



1651 (C=O), 1549 (C=C); 1H NMR (CDCl3) δ (ppm): 8.58 (s, 1H, Ar-H), 8.11 (d, 2H, J=8.7 Hz, Ar-H), 7.85 (d, 2H, J=8.4 Hz, Ar-H), 7.64 (d, 1Hα, J=14.7 Hz), 7.54-7.49 (m, 2H, Ar-H), 7.40 (s, 1H, NH), 6.72 (s, 1H, Ar-H), 6.52 (s, 1H, Ar-H), 3.12 (s, 2H, CH2), 2.88 (s, 2H, CH2), 2.01-1.97 (m, 4H, CH2);

13

C NMR (CDCl3) δ (ppm): 187.24, 166.88, 154.41, 152.28, 151.74, 146.43,

144.58, 134.45, 132.04, 130.20, 129.78, 126.97, 120.48, 119.21, 118.24, 117.06, 113.51, 25.66, 25.58, 22.60, 22.40; ESI-MS: m/z = 402.4.

3-(3-Methylthiophen-2-yl)-1-[4-(5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-d]pyrimidin-4ylamino)phenyl]prop-2-en-1-one (15). Yield: 78%; m.p. 157°C; Anal. Calc. C24H21N3OS2: C 66.79, H 4.90, N 9.74, S 14.86%. found: C 66.88, H 4.71, N 9.58, S 15.03%; FT-IR νmax (cm-1): 1649 (C=O), 1551 (C=C); 1H NMR (CDCl3) δ (ppm): 8.57 (s, 1H, Ar-H), 8.09-8.03 (m, 3H, ArH), 7.87 (d, 2H, J=8.4 Hz, Ar-H), 7.39 (m, 3H, NH and 1Hβ), 6.92 (m, 2H, Ar-H), 3.11 (s, 2H, CH2), 2.88 (s, 2H, CH2), 2.40 (s, 3H, CH3), 2.03-1.97 (m, 4H, CH2); 13C NMR (CDCl3) δ (ppm): 187.93, 166.70, 154.14, 152.17, 142.94, 142.57, 135.61, 135.04, 134.67, 132.96, 131.44, 129.80, 127.15, 124.47, 119.51, 117.13, 26.49, 25.58, 22.52, 22.34, 14.31; ESI-MS: m/z = 432.4.

3-Benzo[1,3]dioxol-5-yl-1-[4-(5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-d]pyrimidin4ylamino)-phenyl]-propenone (16). Yield: 78%; m.p. 232°C; Anal. Calc. C26H21N3O3S: C 68.55, H 4.65, N 9.22, S 7.04%. found: C 68.67, H 4.71, N 9.01, S 7.29%; FT-IR νmax (cm-1): 1650 (C=O), 1558 (C=C); 1H NMR (CDCl3) δ (ppm): 8.57 (s, 1H, Ar-H), 8.07 (d, 2H, J=7.8 Hz, Ar-H), 7.86 (d, 2H, J=7.5 Hz, Ar-H), 7.77 (d, 1Hα, J=15.6 Hz), 7.41 (m, 2H, NH+Ar-H), 7.177.11 (m, 2H, Ar-H), 6.86 (d, 1H, J=7.8 Hz, Ar-H), 5.92 (s, 2H, CH2), 3.10 (d, 2H, J=6.0 Hz, CH2), 2.89 (t, 2H, J=6.0 Hz, CH2), 2.02-1.94 (m, 4H, -CH2); 13C NMR (CDCl3) δ (ppm): 188.53, 166.73, 152.23, 149.82, 148.40, 144.11, 142.91, 135.68, 133.17, 129.90, 129.51, 125.07, 124.44,


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119.85, 119.59, 119.49, 108.66, 106.66, 101.61, 26.54, 25.59, 22.54, 22.36; ESI-MS: m/z = 456.42.

BIOLOGICAL EVALUATIONS The bacterial culture medium, Luria broth and Luria agar were obtained from Himedia (Mumbai, India). For gene cloning, NdeI and XhoI, restriction enzymes, Taq DNA polymerase and phusion polymerase, DNA-ligase were purchased from Invitrogen, California, United States. Ni-NTA resin column and gel filtration column (Superdex-75) were procured from GE healthcare (GE Healthcare Life Sciences, Uppsala, Sweden). Reagent and solvents for chemical synthesis were obtained from Merck. Human breast cancer cell line (MCF-7) and human embryonic kidney (HEK-293) cell lines were obtained from National Centre for Cell Sciences, Pune, India. Fetal bovine serum, antibiotic cocktail, propidium iodide and Dulbecco’s modified eagle’s media were obtained from Gibco-life technologies Gaithersburg, MD, USA. MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide), PMSF and trypsin-EDTA solution were obtained from Sigma (St. Louis, MO). FITC-Annexin-V staining kit was purchased from BD-Pharmingen, BD Biosciences (USA).

Cloning, expression and purification of FASTK protein. The full-length gene of human

FASTK

was

procured

from

(http://plasmid.med.harvard.edu/PLASMID).

the For

Harvard protein

University expression,

Plasmid the

Repository

FASTK

gene

corresponding to the kinase domain (residue 355 to 444) were cloned in pET28b expression vector and transformed into E. coli BL21 (DE3) cells. Individual colonies were selected and allowed to grow for overnight in Luria broth with 50 µg/mL kanamycin, at 37°C, 180 rpm. On the next day, secondary cultures were established by using primary culture as inoculum (induced 14 ACS Paragon Plus Environment


by 0.5 mM isopropyl-β-D-thiogalactopyranoside). Consequently temperature was fixed at 16°C and finally, the cells were harvested after 14–16 h by centrifugation (5000 rpm, 20 min).

The final cell pellet was homogenized in 50 mM Tris–buffer, pH 8.0 having 10 mM 1,4dithiothreitol and 15 mM sodium azide. Resultant cell suspension was lysed by sonication, (ultrasonicator, Labsonic-P, Sartorious Stedium Biotech) in cell lysis buffer containing 1% deoxycholate, 5% glycerol, 1% Triton X-100, 0.5 mg/ml lysozyme, 250 mM NaCl and 1 mM PMSF. Followed sonication, the supernatant was separated by centrifugation (12000 rpm for 30 min). The solubilized protein was instantaneously purified from supernatant by using Ni-NTA resin column; elutions were made with 50 mM Tris, 150 mM NaCl, 5% glycerol, 250 mM imidazole. Fractions with FASTK protein were collected and its purity was checked with the help of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Finally, the purified protein was further confirmed through Western blot by using protein specific primary antibodies and respective peroxidase conjugates 36.

Kinase assay. Enzyme activity profile of purified FASTK was studied by using Malachite Green (Biomol Green reagent, Enzo Life Sciences) based microtitre-plate assay. Enzymatic reactions (in a final volume of 50µl) were established on a 96-well plate. A typical reaction consisting of FASTK (200 ng) and substrate (ATP) were established and incubated for 10-15 min at 25 °C with increasing concentrations (5-200 µM) of synthesized chalcones derivatives, at pH 8.0. Finally, the reactions were terminated by the addition of 100µl of Biomol Green reagent and the plate was further incubated for 30 min at 25 °C. The absorbance of each well was measured at 620 nm in a multiplate ELISA reader (BioRad). In order to quantify the free phosphate levels, the phosphate standard curve was prepared by using inorganic phosphate


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standard solutions as supplied in the Biomol Green reagent kit. All data points represent triplicate measurements from at least three independent experiments.

Molecular docking. The crystal structure of FASTK is not determined so far thus we have modelled its kinase domain by homology modelling

37

, which was further rectify by using

steepest descent method from Gromacs 4.5.5. The two-dimensional and three-dimensional structures of all the chalcones derivatives were drawn by using Chembio Draw ultra. Further estimations and file preparations are completed by using our previously published protocol 38, 39. After preparing the FASTK coordinate files and respective chalcones derivative, it was subjected to docking by means of AutoDock 4 package

40

. The interaction between FASTK and the

respective chalcone derivative was interpreted using the Lamarckian genetic algorithm (LGA). The total binding energy was estimated using electrostatic interactions, van der Waals and hydrogen bonding. Finally, the best docked complexes of FASTK-compound were further reformed, validated and analyzed using “Receptor–Ligand Interactions” modules of Discover Studio 4.0 41 and visualized by using PyMOL.

Fluorescence measurements. Binding of synthesized chalcones derivatives with recombinant FASTK kinase domain was performed by measuring fluorescence intensity of protein by following our previously published protocol

38, 42

. The experiment was done in

triplicates and their average was used for analysis. The decreased intensity of fluorescence on increasing the concentration of synthesized chalcones derivatives helped in calculating binding constant (Ka) and binding sites number (n) in protein by following the Stern-Volmer equation43 :

log (Fo-F)/F = log Ka + nlog[L] 16 ACS Paragon Plus Environment

(1)


where, Fo = Fluorescence intensity (native protein), F = Fluorescence intensity in presence of ligand, Ka = Binding constant, n = number of binding sites, L = ligand concentration. The values for binding constant (Ka, intercept) and number of binding sites (n, slope) were calculated.

Isothermal titration calorimetry. ITC studies were carried out on a VP-ITC microcalorimeter (MicroCal, Inc. GE, MicroCal, USA) by following our previously published protocols

38

. For sample preparation, protein was comprehensively dialyzed in 20 mM Tris

buffer, pH 8.0 and the ligands were dissolved in last dialyzing buffer. After dialysis, the concentration of protein was measured with the help of absorption spectroscopy (Bradford’s method) and fixed accordingly. The final titration data was analysed using MicroCal Origin 7.0 software provided with the instrument. Thermodynamic parameters of binding named enthalpy change (∆H), stoichiometry of binding (n), and association constant (Ka) were estimated by fitting the binding isotherm into ‘one-set of sites’ binding model.

Cell culture. MCF-7 and HEK293 cell lines were cultured in a humidified incubator with 5% CO2 at 37°C and were cultured in DMEM complemented with 10% heat-inactivated fetal bovine serum (Gibco) and 1% streptomycin, penicillin solution. Cells were regularly maintained and sub-cultured not more than 30 passages.

Cell proliferation assay. To assess the effect of synthesized chalcones on cell viability and proliferation, standard MTT method was performed. Cells were plated (6000-8000/well) in 96well plates and incubated overnight. The cells were incubated with increasing concentrations (0.1–80 µM) of synthesized chalcones in 200 µl final volume for 48 h at 37º C in a humidified chamber. At the end of time point to each well of plate, 20µl of MTT solution (from a 5mg/ml


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stock solution in PBS) was added and further incubated for 4-5 h at 37ºC in a humidified chamber. After 4-5 h the supernatant were removed and formazan crystal (colored) formed from MTT was solubilized in 100µl DMSO. Absorbance (A) was taken at 570nm in a multiplate ELSIA reader (BioRad). Cell viability percentage was calculated to interpret the IC50 (50% inhibitory concentration) values of particular inhibitor. For cell proliferation and anticancer activities paclitaxel was estimated as positive control.

Cell apoptotic assay. The apoptotic potential of synthesized chalcones derivatives were accessed by Annexin-V staining. Briefly, MCF-7 cells were dosed with IC50 concentration with the synthesised compounds for a period of 24 h at 37ºC, while the control cells were given treatment only with the media. After incubating for 24 hrs, approximately 2.5x106 cells were trypsinized and collected by centrifuging at 1800 rpm for 4 min. Consecutively, cells were washed two times with 5 ml PBS and stained with FITC-Annexin-V staining using FITCAnnexin-V kit according to manufacturer’s guidelines (BD-Biosciences, USA). 10,000 events for every sample were analyzed using flow cytometry on BD LSR II Flow Cytometry Analyzer.

RNA isolation and qPCR assay. MCF-7 cells were dosed with IC50 concentration of each compound for 24 h at 37ºC, and the control cells were given treatment with media. Total RNA was isolated by using RNA-isolation kit as per the given protocol (Roche Molecular Diagnostics, Mannheim Germany). The concentration of RNA was measured spectrophotometrically and the purity was confirmed by taking the ratio of absorbance (A260/A280). cDNA was synthesized using 1 µg of total RNA using oligo-dT primers and cDNA synthesis kit (Thermo Scientific). Synthesis was confirmed using β-actin primers for PCR reaction. Quantitative real time-PCR (qPCR) was done for FASTK with each sample in triplicate, on a Roche LC-96 machine (Roche



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Diagnostics, Mannheim, Germany). The primers, FASTK-Forward-gaactgactccccacgtgat, FASTK-Reverse-aaagggcaggaccaacttct, β-actin-Forward-ctctgctcctcctgttcgac, β-actin-reversegcccaatacgaccaaatc, were designed using primer3 tool by taking sequence of FASTK gene from NCBI database. All amplification curves were analyzed using LC96-software.

Cell cycle analysis by Propidium iodide staining. Cell cycle was analyzed by Propidium iodide (PI) staining. For this, MCF-7 cell lines were dosed with IC50 concentrations of selected chalcones derivatives for about 24 h at 37 °C and the control cells were given treatment only with the media. After 24 hrs. cells were collected by trypsinization and 2×106 cells were washed twice using 5 ml PBS, at 37 °C using centrifugation at about 1800 rpm, for 4-5 min. Fixation of homogenous cell suspension was carried out using 70% ethanol (chilled) by gentle mixing and was incubated overnight at 4 °C. The fixed cells were washed twice with 5ml of PBS by centrifuging at 1800 rpm for 5 min. Finally, cells were resuspended using 50 µl of PBS, 200 µg/ml RNase A was also added to them and then incubated at 37 °C for 45 min. PI (2 µg/ml) of about 50µl was mixed with citrate buffer (450 µl) and then kept for incubation with cells for 5 min. Nearly, 10,000 events were collected for each sample by BD LSR II Flow Cytometry Analyzer, and data was analyzed with the help of FACS DIVA software.

Reactive oxygen species determination by DCFDA staining. Reactive oxygen species (ROS) content inside the cells was quantified using the DCFDA staining. This assay helps to measures innumerable ROS such as H2O2 and hydroxyl radicals

44

. Briefly, in 24-well

culture plates the 70-80% confluent MCF-7 cells were incubated with IC50 dose of each compound and positive control H2O2, respectively. After 5-6 h treatment of compounds to MCF7 cells, cells were washed with 500µl Kreb’s Ringer buffer (20 mM HEPES, 2 mM MgSO4, 10 19 ACS Paragon Plus Environment

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mM dextrose, 127 mM NaCl, 1 mM CaCl2 and 5.5 mM KCl), pre warmed at 37ºC and 10 µM DCFDA (Invitrogen Grand Island, NY) has been added and incubated for 30 min in dark at 37ºC in a humidified incubator. Subsequently the cells were trypsinized and assayed for ROS levels with the help of flow cytometry. Approximately 10,000 events for each sample of cells were evaluated by BD LSR II Flow Cytometry Analyzer, and data analysis was performed with help of FACS DIVA software. Similarly, for intact cell imaging, cells were washed with PBS and subsequently imaged for ROS levels estimation using the DCFDA fluorescent dye. Fluorescence images were taken on Nikon-Eclipse TS100 microscope.

Wound healing assay. Migration of MCF-7 cells was assessed by an in vitro wound healing assay. Cells were seeded in a 6-well cell culture plate. When they should reach ~60% confluence the scratch was introduced by scraping the monolayer with a 200 µl sterile micropipette tip. To eliminate the detached cells floating after scratching, cells were washed twice with incomplete medium/PBS. Cells were replenished with fresh complete medium containing selected compounds (IC50 dose) and were incubated for 24 hours. Cells were then washed twice with PBS and fixed with 4% paraformaldehyde for 30 min in a wet chamber. Each well of plate were photographed by phase contrast inverted microscope and mobility of the cells was measured by their ability to close the wound.

Statistical analysis. All the data were expressed as mean ± standard error from a minimum three independent experiments. The statistical analysis of each data was performed using the two-tailed Student t-test for unpaired samples. Differences were considered significant at P 3.84 x 103 M-1) and also inhibit the proliferation of MCF-7 23 ACS Paragon Plus Environment

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cells (IC50: 6.52-20.22 µΜ). The results of molecular docking also correlates with the results of binding and enzymatic assays, and showed that the compounds 2, 10 and 12 have the most promising binding energies.

Isothermal titration calorimetry measurements. Enzyme inhibition, docking and fluorescence binding studies suggest that compound 2, 10 and 12 interact with FASTK and reduces its enzyme activity. Thus, actual binding affinity and stoichiometry of these compounds with the recombinant FASTK was measured by the isothermal titration calorimetry (ITC). A typical ITC isotherm was obtained after titration of FASTK with compound 2, 10 and 12 (Figure 4 A-C). The upper panel of each measurement with negative heat pulses indicates exothermic binding. The extent of heat generated as a result of each injection further provides the molar ratio of studied compound to that of FASTK. The results shown in Figure 4 A-C were obtained from one-site fitting model. Thermodynamic parameters associated with binding of compound 2, 10 and 12 (∆H, enthalpy change and ∆S, entropy change) is shown in Table 1. Results of ITC infers that though three studied compounds appreciably binds to FASTK but as compared to compound 2, compound 10 and 12 shows relatively high binding affinity (as shown by their high binding constant).

Structure activity relationship. Chalcones-pyrimidine hybrid analogues were designed, synthesized and screened against FASTK. Among all the compounds, only three compounds (2, 10 & 12) of the series were found to inhibit the enzyme activity of FASTK. These compounds showed IC50 values in the range of 0.15 – 0.32 µM (Table S1). Compound 2 having unsubstituted phenyl ring exhibited IC50 of 0.32 µM, whereas the compound 12 having 3,4dimethoxy substitution at phenyl ring displayed most promising activity against FASTK (IC50 =



0.15 µM) followed by compound 10 having chlorine at p-position. (IC50 = 0.17 µM). The better activity of compounds 10 and 12 was further confirmed by their strong binding with the enzyme as shown by ITC and fluorescence data. Molecular docking data also suggested that compound 10 and 12 form more number of interactions with FASTK residues including hydrogen bonding with Thr37, Ser83, Arg41 and Ala81 in the active site of the enzyme compared to compound 2, which displays less number of interactions with FASTK. Methoxy group at m-position in compound 12 showed interaction with Arg41 and the other methoxy group was well tolerated as not in the case of compounds 11 and 13. Therefore, in terms of structure activity relationship (SAR), it can be concluded that activity of this class of compound was substituent as well as position dependent.

Cell proliferation assay. Three selected chalcones derivatives were evaluated on MCF-7 and HEK293 cell lines by MTT assay. The cells were incubated with 0-200 µM concentration range, and treatments were given for 24 and 48 h. Cell viability results revealed that compound 2, 10 and 12 elicited better toxicity in a concentration dependent manner on MCF-7 cells (Figure 4 D) and in the tested concentration range these compounds did not show any considerable cytotoxicity towards HEK293 cell lines (see lower panel of Figure 4 D). The IC50 values for compound 2, 10 and 12 were found to be 20.22±1.50 µM, 6.52±0.82µM and 8.20±0.61µM for MCF-7 cell lines (Figure 4 D). Further, the cytotoxic attributes of these selected chalcone derivatives against normal cells (HEK-293) were also studied at their corresponding IC50 value, and it was observed that more than 80% cells are viable even after 72 h of


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incubation. As these compounds are non-toxic to normal cells and specifically shown toxicity towards cancerous cells, compound 2, 10 and 12 were taken for further studies such as apoptosis, cell cycle analysis and reactive oxygen species (ROS) production.

Apoptosis assay. Apoptosis is an indispensible process that regulates and controls abnormal growth of cells, but due to impaired signaling cancerous cells escape apoptosis. FASTK over expression also down regulates the process of apoptosis. That’s why a probability was studied that whether inhibition of FASTK leading the induction of apoptosis. MCF-7 cells were starved in reduced serum medium and treated with IC50 dose of each thienopyrimidine based chalcones derivative for 24 hand annexin-V staining was used to evaluate the apoptosis. After 24 h cells were washed twice with PBS and subsequently incubated with FITC labelled annexin-V. Stained cells were analyzed by flow cytometry. It was found that treatment of compound 2, 10 and 12 were considerably induces apoptosis in the MCF-7 cells (Figure 5 A). Flow cytometry analysis reveals that treatment of compound 2, 10 and 12 induces apoptosis in 11.0 %, 19.9 % and 17.5 % of MCF-7 cells, respectively as compared to the control cells (Figure 5 B). These observations clearly indicate that these three compounds induce apoptosis in MCF-7 cell. Further, the mRNA expression level of FASTK after the treatment of compound 2, 10 and 12 was studied by qPCR. Following the treatment, mRNA expression was assessed for FASTK and changes in the mRNA expression were plotted after the samples were normalized to the β-actin control (Figure 5 C). The expressions of FASTK were found to be decreased in MCF-7 cells after the treatment of selected compounds as compared to control one (Figure 5C). These results suggested that down regulation of FASTK might be responsible for apoptosis.



Chalcone derivatives induce cell cycle arrest in G0/G1-phase. Variations in cell cycle progression of cancer cells is considered as a potential target for the treatment of human malignancies as unregulated and defective cell cycle is also a major hallmarks of human cancer 8. In order to see the cell cycle variations in MCF-7 cells after the treatment of compound 2, 10 and 12 flow cytometry coupled PI-staining has been performed. After the treatment of MCF-7 cells with compound 2, 10 and 12 for stipulated time periods, cells were harvested and processed for flow cytometry. Incubation of cells with the IC50 dose of compounds resulted in atypical cell cycle profile (Figure 5 D), which might be the result of their strong toxic effects as evident from the increased cell death. The histogram representation of cell cycle distribution showed that the fraction of G1-arrested cells increases with all the three compounds (Figure 5 D). Interestingly consistent with the binding and apoptosis studies, here in cell cycle studies we observed that compound 10 and 12 induces the G0/G1 cell cycle arrest more prominently than compound 2 (Figure 5 E). Taken together, these results suggesting the inhibitory effect of these compounds might be responsible for the induction of G0/G1-phase arrest.

Synthesized chalcones enhance level of reactive oxygen species. The MCF-7 cells were treated with IC50 dose of compound 2, 10 and 12 for 5-6 h incubation (Figure 6). ROS levels were measured by flow cytometry by using 2-Dichlorofluorescein diacetate (DCFDA) staining. Treatment of compound 2, 10 and 12 increases the production of ROS. Histogram presentation of ROS quantification suggests that incubation of MCF-7 cells with compound 2, 10 and 12 shifts the position of histogram towards right, which indicates an increase in the levels of ROS (Figure 6 A). Besides flow cytometry, levels of ROS were also measured by fluorescence spectroscopy, after the treatment of cells with compound 2, 10 and 12, DCFDA staining was performed and images of cells has been taken on a fluorescence microscope (Figure 6 B). Here 27 ACS Paragon Plus Environment

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we can see in the Figure 6B that the intensity of green fluorescence increases in case of treated cells, which means that ROS levels has been increased. Thus, results of ROS measurements clearly indicates that compound 2, 10 and 12 considerably increases the levels of ROS that might be also a reason for cellular death.

Synthesized chalcones inhibit migration of MCF-7 cells. Cell migration of MCF-7 was examined after the IC50 dose treatment of compound 2, 10 and 12. The results of wound healing assay are shown Figure 6 C, for cell migration studies, the treatment were given up to 48 h and width was measured pre and post treatment. Cell migration results show a clear inhibition in the migration rate after compound 2, 10 and 12 treatment in MCF-7 cells (Figure 6 C). Our results showed that the untreated cells migrated to fill up space of scratch more rapidly as compared to the treated cells (Figure 6 C).

DISCUSSION Breast cancer is the major cause of mortality in female. In an attempt to identify a drug target from the intricate networks of cancer, protein kinases become the first choice of researchers. Different kinases such as CAMKIV, MARK4, FASTK and several other kinases have been identified as the potential drug targets for anticancer therapies

38, 45-49

. Our group previously

identified some natural or synthetic molecules as potential inhibitors against CAMKIV and MARK4

38, 45, 46, 49

. FASTK is an important drug target as its overexpression helps the cancer

cells to evade the apoptosis, which is a prominent hallmark of all types of cancer 8. Hence, FASTK have been taken as potential drug target to develop novel pharmacophore molecules against breast cancer.



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In the field of drug design, generally researchers go for chemical synthesis. However, toxicity of synthesized compounds is a major limiting factor. In the other hand, use of natural products has gained attention because of fewer side-effects

38, 50

. Keeping cytotoxicity problem

into consideration, chalcone moiety has been selected as basic synthesis skeleton, as it is a naturally occurring moiety that does not confirms toxicity. Since, FASTK provides proliferative advantage to cancer cells by helping them to overcome the apoptosis, chalcones based hybrid molecules were evaluated for their antitumor/apoptotic activities by targeting FASTK.

Following synthesis; enzyme activity, binding and docking studies were carried out. Docking studies indicates a relatively strong binding affinity of chalcones with the FASTK. The docked complex of chalcones-FASTK was stabilized by enormous number of covalent interactions (Figure 3). To further corroborate, the docking results were validated by fluorescence and ITC measurements performed on purified recombinant human FASTK showing that chalcones binds with a high affinity (µM range) to the FASTK (Figure 4). Binding of chalcones derivative inhibit the enzyme activity of FASTK and it was noticed that three compounds appreciably inhibits the activity of FASTK, with IC50 value in nM range. On the basis of binding and enzyme activity it was clearly observed that binding of these synthesized molecules leads to the inhibition of FASTK, and thus these three molecules are further evaluated for functional biological assays.

To determine the effect of chalcones on the proliferation of MCF-7 and HEK-293 cell lines MTT assay was performed. Selected chalcones derivatives were found to be nontoxic to HEK293 cells and showed cytotoxic effect to MCF-7 cells only (Figure 4). Results of cytotoxicity suggested that the synthesized compounds don’t show any cytotoxicity towards HEK-293 cells


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in the tested concentration range. This non-toxic behaviour of these compounds validate our criteria for the selection of natural compound moiety (chalcone moiety) which helps to reduce the cytotoxicity of synthesized compounds. Based on the preliminary outcomes of enzyme activity, binding and cell proliferation experiments we presumed that selected thienopyrimidine based chalcone derivatives studied here may be used as a promising anti-cancer molecule because these are non-toxic human cells in sub-micromolar concentration.

As it was a

fundamental phenomenon that cancer cells showed uncontrolled proliferation, migration, invasiveness and very smartly they evade the apoptotic signals, which allow the cells to proliferate without differentiation 8. Apoptosis is a natural phenomenon which removes dead or injured cells, and thus maintains homeostasis. FASTK is an important supervisor of apoptosis and other mitochondrial functions 20, 51, 52. To check the effect of FASTK inhibition on apoptotic death of the cells annexin-V and PI-staining was performed (Figure 5). The results of apoptosis suggest that inhibition of FASTK induces the apoptosis in MCF-7 cells. It can be inference from these observations that inhibition of FASTK does not allow the cancerous cell to evade the process of programme cell death and this property of synthesized hybrid molecules (compound 2, 10 and 12) may be very helpful to overcome the uncontrolled division of cancerous cell. The interpretation of results that inhibition of FASTK leads to the induction of apoptosis are found to be consistent with the earlier reports performed on astrocytoma cells and breast cancer cells 51, 53.

In the present study, we found that selected chalcones hybrids effectively obstruct the cell cycle progression of highly aggressive MCF-7 cells. Unusual progression of cell cycle is also a characteristic of cancer cells 8. To maintain the proper division of cells, different cell cycle check-points are evolved that ensures the efficient and optimized progression of cell cycle54. But these check-points are compromised in case of cancer cell division 30 ACS Paragon Plus Environment

8, 55

. The G0/G1 check-point


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becomes a striking chemotherapeutic target for anticancer therapies. Retraction of cancer cells from the G0/G1 check-point of cell cycle prevents the cancer cells to repair DNA and consecutively inhibits their S-phase entry. Interestingly, it was observed that treatment of synthesized thienopyrimidine chalcones derivatives arrested the MCF-7 cells in G0/G1 phase of cell cycle and induces the apoptosis (Figure 5). These results of G0/G1 arrest by the treatment of anticancer compounds were found to be consistent with previous findings in other cancer cells 56, 57

.

Other important aspect of the study turns towards the estimation and rationale of ROS after the treatment of synthesized hybrid molecules. Generally, cancer cells as compared to the normal cells are under increased oxidative stress accompanied with oncogenic transformation, alterations in metabolic activity, and increased production of ROS

58, 59

. High levels of ROS in

cancer cells may result from increased metabolic activity, oncogene activity, peroxisome activity, mitochondrial dysfunction, increased cellular receptor signalling or increased activity of oxidases 60, 61. Though ROS plays a major role in cancer progression, but if it exceeds to certain levels than it causes harm to cancerous cells too, because these cells setup their own ROS environment. The mitochondrial respiratory cycle is the foremost source of ROS, the ROS has the capacity to induce cell apoptosis 62. ROS production and accumulation resulted in oxidative stress and play a decisive role in determining cancer cell behaviour. Heightened levels of ROS triggered a sequel of pro-apoptotic pathways, like ER stress and mitochondrial disfunctioning and, which finally leads to disintegration of cellular functions and apoptosis 62. It was found that these chalcone derivatives increased ROS levels in MCF-7 cells as measured by DCF fluorescence (Figure 6 A & B). High levels of ROS put up the cancerous cells on heavy oxidative stress, which ultimately leads to cell death. Cell migration and metastasis is the other 31 ACS Paragon Plus Environment

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characteristics of cancerous cells that adds to the invasion of cancerous cells to other body parts 8

. Cell migration assay showed that chalcones inhibit the migratory properties of MCF-7 cells

(Figure 6C). Reductions in cell migration behaviour of any type of cancer cell also an important aspect for the development of novel inhibitors against different anticancer drug targets. These results of inhibitions of cell migration by chalcone derivatives are also found to be consistent with the studies published by other groups on the development of anticancer drug molecules 56.

CONCLUSION Conclusively, in this study, treatment of chalcone derivatives leads to the inhibition of FASTK, which inhibit the cell proliferation, induce apoptosis, enhance ROS generation, arrest the cell cycle in G0/G1 and reduced the cell migration of cancerous cell, clearly supports the notion that FASTK will be the effective anticancer drug target. On the basis of results obtained from this study, mechanism of action of synthesized thienopyrimidine based chalcones on the activity of FASTK has been proposed (Figure 6D). It has been proposed that these molecules inhibit the activity of FASTK that ultimately leads to the induction of apoptosis directly or indirectly through the production of ROS (as shown by dashed green colour lines in Figure 6D). All these observations speculate that chalcone based hybrid molecules will become a potential ligand which can be further employed for the development of novel pharmacophore against FASTK.

SUPPORTING INFORMATION: Additional Supplementary Data: Table S1, 1H and 13C NMR spectra of compounds 2, 10 and 12 and Figure S1.

ACKNOWLEDGEMENTS: NSK and SS are thankful to the University Grant Commission (UGC) India, for the award of UGC Non-NET fellowship and DSKPDF fellowship, 32 ACS Paragon Plus Environment


respectively. PK thanks to the Department of Biotechnology (DBT) for financial support. We sincerely acknowledge Harvard University-plasmid providing facility for providing the FATSK gene. Authors thank to the Department of Science and Technology, Government of India for the FIST support (FIST program No. SR/FST/LSI-541/2012).

AUTHOR CONTRIBUTIONS: Conceived and designed the experiments: NSK, PK and MIH. NSK and PK performed the experiments and contributed equally. MFA synthesized the compounds. SS helps in cloning and protein purification. Analyzed the data: SHK, PK, GMH, MH and MIH. Wrote the paper: NSK, PK and MIH.

CONFLICTS OF INTEREST: Authors have declared that there is no any conflict of interest.

FUNDING: This work is supported by the Science & Engineering Research Board, Department of Science and Technology, Government of India, grant to MIH (Project no: EMR/2015/002372).

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50. Fulda, S. Modulation of apoptosis by natural products for cancer therapy. Planta medica 2010, 76, (11), 1075-1079. 51. Zhi, F.; Zhou, G.; Shao, N.; Xia, X.; Shi, Y.; Wang, Q.; Zhang, Y.; Wang, R.; Xue, L.; Wang, S.; Wu, S.; Peng, Y.; Yang, Y. miR-106a-5p inhibits the proliferation and migration of astrocytoma cells and promotes apoptosis by targeting FASTK. PLoS One 2013, 8, (8), e72390. 52. Boehm, E.; Zornoza, M.; Jourdain, A. A.; Delmiro Magdalena, A.; Garcia-Consuegra, I.; Torres Merino, R.; Orduna, A.; Martin, M. A.; Martinou, J. C.; De la Fuente, M. A.; Simarro, M. Role of FAST Kinase Domains 3 (FASTKD3) in Post-transcriptional Regulation of Mitochondrial Gene Expression. J Biol Chem 2016, 291, (50), 25877-25887. 53. Zheng, Y. Z.; Xue, M. Z.; Shen, H. J.; Li, X. G.; Ma, D.; Gong, Y.; Liu, Y. R.; Qiao, F.; Xie, H. Y.; Lian, B.; Sun, W. L.; Zhao, H. Y.; Yao, L.; Zuo, W. J.; Li, D. Q.; Wang, P.; Hu, X.; Shao, Z. M. PHF5A Epigenetically Inhibits Apoptosis to Promote Breast Cancer Progression. Cancer Res 2018, 78, (12), 3190-3206. 54. Elledge, S. J. Cell cycle checkpoints: preventing an identity crisis. Science 1996, 274, (5293), 1664-72. 55. Kastan, M. B.; Bartek, J. Cell-cycle checkpoints and cancer. Nature 2004, 432, (7015), 31623. 56. Hafeez, B. B.; Ganju, A.; Sikander, M.; Kashyap, V. K.; Hafeez, Z. B.; Chauhan, N.; Malik, S.; Massey, A. E.; Tripathi, M. K.; Halaweish, F. T.; Zafar, N.; Singh, M. M.; Yallapu, M. M.; Jaggi, M.; Chauhan, S. C. Ormeloxifene suppresses prostate tumor growth and metastatic phenotypes via inhibition of oncogenic beta-catenin signaling and EMT progression. Mol Cancer Ther 2017. 57. Neumann, J.; Boerries, M.; Kohler, R.; Giaisi, M.; Krammer, P. H.; Busch, H.; Li-Weber, M. The natural anticancer compound rocaglamide selectively inhibits the G1-S-phase transition in cancer cells through the ATM/ATR-mediated Chk1/2 cell cycle checkpoints. Int J Cancer 2014, 134, (8), 1991-2002. 58. Pelicano, H.; Carney, D.; Huang, P. ROS stress in cancer cells and therapeutic implications. Drug Resistance Updates 2004, 7, (2), 97-110. 59. Moreno‐Sánchez, R.; Rodríguez‐Enríquez, S.; Marín‐Hernández, A.; Saavedra, E. Energy metabolism in tumor cells. The FEBS journal 2007, 274, (6), 1393-1418. 60. Storz, P. Reactive oxygen species in tumor progression. Front Biosci 2005, 10, (1-3), 18811896. 61. Szatrowski, T. P.; Nathan, C. F. Production of large amounts of hydrogen peroxide by human tumor cells. Cancer research 1991, 51, (3), 794-798.



62. Circu, M. L.; Aw, T. Y. Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic Biol Med 2010, 48, (6), 749-62.

Figure legends: Figure 1: Expression, purification and enzyme activity of FASTK. (A) SDS-PAGE of purified FASTK. Lane 1-3 represents the increasing concentration of FASTK, lane 4 is protein marker. (B) Western blot of purified FASTK. Approximately 10µg of purified FASTK has been separated on 15% SDS-PAGE and transferred by blotting. Blot was developed by anti-His primary antibodies. Lane 1-3 shows the FASTK probed with anti-His antibodies. (C) Standard curve phosphate hydrolysis curve. (D) ATPase inhibition (% hydrolysis of phosphate) with increasing concentrations of compound 2, 10 and 12 are shown as a function of concentration calculated by comparing with standard phosphate hydrolysis curve. Figure 2: Binding studies of compound 2, 10 and 12 with FATSK using fluorescence spectroscopy. Fluorescence emission spectra of FASTK (10 µM) with the increasing concentration of; (A) compound 2 (B) compound 10 (C) compound 12. Excitation wavelength was fixed at 280nm and emission was recorded in the range 300-400 nm. Modified Stern-Volmer plot showing fluorescence quenching of FASTK by (D) compound 2 (E) compound 10 (F) compound 12, used to calculate binding affinity (Ka) and number of binding sites (n). Figure 3: Molecular docking studies of selected compounds: (A-B) Pocket view of FASTK binding with compound 2 shows the hydrogen bond donor-acceptor residues and hydrophobic surface, respectively. (C) 2D schematic diagram of docking model of compound 2 with FASTK. Residues involved in hydrogen bonding, charge or polar interactions, van der Waals interactions are represented by respective colour (see inset). (D-E) Pocket view of FASTK binding with compound 10 shows the hydrogen bond donor-acceptor residues and hydrophobic surface, respectively. (F) 2D 39 ACS Paragon Plus Environment

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schematic diagram of docking model of compound 10 with FASTK. Residues involved in hydrogen bonding, charge or polar interactions, van der Waals interactions are represented by respective colour (see inset). (G-H) Pocket view of FASTK binding with compound 12 shows the hydrogen bond donor-acceptor residues and hydrophobic surface, respectively. (I) 2D schematic diagram of docking model of compound 12 with FASTK. Residues involved in hydrogen bonding, charge or polar interactions, van der Waals interactions are represented by respective colour (see inset). Figure 4: ITC measurement showing the titration of selected thienopyrimidine based chalcones derivatives with FASTK and cell proliferation studies. (Top panel) Raw data plot of heat produced against time for the titration of 800-1200µM (A) compound 2, (B) compound 10, (C) compound 12, into 14-20 µM FASTK. (Bottom panel) Corresponding binding isotherm obtained after integration of peak area and normalization to yield a plot of molar enthalpy change against each compoundFASTK ratio. The one-site fit curve is displayed as a thin red colour line. (D) Effect of selected thienopyrimidine based chalcones on the viability of MCF-7 cells; Cells were treated with increasing concentrations of thino-pyrimidine based chalcones (050 µM) for 48 h. Cell viabilities were presented as a percentage of the number of viable cells to that of the control. Each data point shown is the mean ± SD from n=3.(For anticancer activities paclitaxel has been taken as positive control). Figure 5: FASTK inhibition by thino-pyrimidine based chalcones induces apoptosis and G0/G1 cell cycle arrest in MCF-7 cells. MCF-7 cells were treated with IC50 concentrations of each compound for 24 h and processed for apoptosis analysis using Annexin V-PI apoptosis kit and cell cycle analysis by PI-staining and were quantified by flow cytometry. (A) Histogram showing the anti-FITC-Annexin-V stained cells after the treatment of each compound as indicated in each histogram. (B) Bar graphs represents the percentage of apoptotic MCF-7 cells stained with Annexin-V for duplicate measurements ± SD. **p < 0.001, as compared to control (untreated cells). (C) qPCR analysis of FASTK in MCF-7 cells (D) Histogram showing the cell cycle distribution of MCF-7 cells after different experimental treatments as mentioned on 40 ACS Paragon Plus Environment


each histogram. (E) Graphical presentation of cell cycle distribution in each phase, respective treatment shown on horizontal axis. Statistical analysis was done using two-tailed Student t-test for unpaired samples. (For anticancer activities paclitaxel has been taken as positive control). Figure 6: Effect of synthesized chalcones on the ROS production and cell migration. (A) Histogram showing the fluorescence emission intensity of DCF as measured by flow cytometry, MCF-7 cells were treated with IC50 concentrations of each compound for 5-6 h and processed for ROS measurements using DCFDA staining and were quantified by flow cytometry. (B) Representative images of MCF-7 cells stained with DCFDA captured on fluorescence microscope for the assessment of ROS after the treatment of respective compound and intensity of green colour represents the levels of ROS. (C) Effect of respective chalcone derivative on cell migration of MCF-7 cells as determined by wound healing assay. Representative images (20X original magnification) showing inhibition of MCF-7 cells migration by wound healing assay. (D) Proposed mechanism by which thieno-pyrimidine based chalcones may induces apoptosis by inhibiting the activity of FASTK and prodcution of ROS.


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O

HN

Cl a)

N S

N S

N

N (1)

O

R

HN b) N S

N (2-16)

Scheme 1: Synthesis of 5,6,7,8-tetrahydrobenzo [4,5]theino[2,3-d] pyrimidine based chalcones (2-16). Reagents and conditions: a) Ethanol, reflux, 12-14 h, b) Ethanol, NaOH, r.t. 14-16 h.

TABLES Table 1: Thermodynamic parameters and functional activity concentrations obtained from ITC, kinase and MTT assays. Compound number

§

Ka, (M-1)

∆H, cal/mol

KD, (µM)

3.84× 104 ± 1.79 × 103

-3.51 × 107± 3.87× 103

4.42× 105 ± 7.3× 102

3.63 ×105 ± 4.2× 103

§

¥

KD

IC50, (µM), FASTK

26.04

1.3 µM

0.32±0.02

20.22±1.50

-7.54 × 108± 4.65× 103

2.26

86 nM

0.17±0.01

6.52±0.82

-5.65 × 107± 4.69 × 103

2.75

34 nM

0.15±0.01

8.20±0.61

¶

#

IC50, (µM),

2

10

12 §=From ITC, ¶= from fluorescence, ¥= from enzyme inhibition, #=from MTT



Page 44 of 50

Table2: Molecular docking parameters of FASTK with selected thienopyrimidine derivatives.

Compound number

Binding Energy (kcal/mol)

No. of hydrogen bonds

Hydrogen bond forming residues

Distances (Å)

Other interacting residues

-7.4

1

Ala81

3.5

Leu14, Pro15, Ile36, Ala40, Ser82, Ser83, Arg41, Thr37, Ala81, Gly85, Leu79, Val10

-7.7

2

Thr37 Ser83

3.0 3.63

Lys43, Arg41, Thr37, Ile36, Ala81, Ala40, Leu79, Gly85, Val10, Val87

-7.6

2

Arg41 Ala81

3.3 2.2

Pro15, Leu14, Ile36, Ser83, Thr37, Ser82, Ala40, Ala81, Gly85, Leu79, Val10,Val87

2

10

12


C

0.26 Molecular Pharmaceutics

Page 45 of 50

0.24 0.22

1

2

3

0.20

4

OD 620nm

A

0.18 0.16 0.14

55kDa

0.12

45kDa

0.10

29kDa

0.0

0.5

D

1.0

1.5

2.0

375

500

phosphate (nmol)

1500

5kDa

B

1

2

3

phosphate released (pmol)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Purified 21 FASTK 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

2 10 12 1000

500

0 ACS Paragon Plus Environment 0

Figure 1

50

125

250

Concentration (nM)

Molecular Pharmaceutics 300

A

Native 1.0 M 2.0 M 3.0 M 4.0 M 5.0 M 6.0 M 7.0 M 8.0 M 9.0 M

D

0.0

-0.2

logFo-F/F

Fluorescence intensity

-0.4

-0.6

-0.8

-1.0 -6.2

320

340

360

380

-6.0

-5.8

-5.6

400

-5.4

-5.2

-5.0

-4.8

log[Ligand]

Wavelength, nm 1.0

B

E

0.8 0.6 0.4

logFo-F/F


Native 1.0 M 2.0 M 3.0 M 4.0 M 5.0 M 6.0 M 7.0 M 8.0 M 9.0 M 10.0 M 11.0 M 12.0 M 13.0 M 14.0 M

0.2 0.0 -0.2 -0.4 -0.6 -0.8 -1.0 -6.2

320

340

360

380

-6.0

-5.8

400

-5.6

-5.4

-5.2

-5.0

-4.8

-4.6

-5.2

-5.0

-4.8

-4.6

log[Ligand]

Wavelength, nm 0.8

Native 1.0 M 2.0 M 3.0 M 4.0 M 5.0 M 6.0 M 7.0 M 8.0 M 9.0 M 10.0 M 11.0 M 12.0 M 13.0 M 14.0 M


C

F

0.6 0.4 0.2

logFo-F/F

1 250 2 3 4 200 5 6 150 7 8 100 9 10 11 50 12 13 0 14 15 16 17 18 200 19 20 21 150 22 23 24 100 25 26 27 50 28 29 30 31 0 32 33 34 35 36250 37 38 39200 40 41 42150 43 44 100 45 46 47 50 48 49 50 300 51 52 53 54 55 56

Page 46 of 50

0.2

0.0 -0.2 -0.4 -0.6 -0.8 -1.0 -1.2 -6.2

320

340

360

380

400

Wavelength, nm


Figure 2

-6.0

-5.8

-5.6

-5.4

log[Ligand]

A

B

D

E

G

H

Page 47 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55


F

I


Figure 3

C


A

B

Compound 10

Compound 2

C

D

100 MCF-7 cells

%age cell viability

80

60

40

120

Compound 12

2

1

-1

0

Compound 2 Compound 10 Compound 12

20

P HEK293 cells

100 80 60 40

log Concentration (PM)

Figure 4


2

-1

20

1

Compound 2 Compound 10 Compound 12

0

%age cell viability

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 50

Page 49 of 50


A

MCF-7 cells

5

2 C

C

om

po un d

po un d om

C

12

0

on tr ol

0%

***

10

om

100%

15

10

11.0%

***

C

89.0%

*** 20

po un d

Control

25

% increase (annexin-V + cells)

Compound 2

3 2

**

1

0

0

1

0/ G 0

0


Figure 5

12 un po

G

phase of cell cycle

phase of cell cycle

2

20

Compound 12

G

20

2

40

G

G2

40

1

S

G0/G1=66.9% S=14.2% G2=10.4%

60

0/ G

S

G0/G1=64.9% S=11.6% G2=12.9%

60

G

G2

80

Compound 10

1

80

S

Compound 12

0/ G

G1

S

Compound 10

om

G

G

phase of cell cycle

phase of cell cycle

G1

2

20

G

20

S

40

C

om

40

0/ G

S

G0/G1=66.9% S=11.4% G2=12.7%

60

Compound 2

2

G2

60

1

S

G0/G1=59.3% S=17.9% G2=18.1%

80

control

G

%age

G2

po om C

80

G1

S

Compound 2

Control

G1

d

d un

tr

E

10

2

ol

0

C on

D

**

**

d

17.5%

19.9%

FASTK-qPCR

4

un

82.5% 80.1%

5

po

Compound 12

C

Compound 10

Fold Change in Expression

C

%age

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

B


A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Compound 2 Compound 10

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Compound 12 Control (untreated) Unstained

B

C

control

Compound 2

Compound 10

D

Compound 12

Fas

Caspase-8 Bcl-2 family proteins

Thienopyrimidine based chalcones

ROS

FASTK

tBID

Caspase-3

TIA 1 1

2

3

PRD

FASTK

Apoptosis


Figure 6

Thienopyrimidine-Chalcones Hybrid Molecules Inhibits Fas-activated

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