Next Article in Journal
Chitosan Elicitation Impacts Flavonolignan Biosynthesis in Silybum marianum (L.) Gaertn Cell Suspension and Enhances Antioxidant and Anti-Inflammatory Activities of Cell Extracts
Previous Article in Journal
Metabolomics Insights of the Immunomodulatory Activities of Phlorizin and Phloretin on Human THP-1 Macrophages
Previous Article in Special Issue
Interrogating Plant-Microbe Interactions with Chemical Tools: Click Chemistry Reagents for Metabolic Labeling and Activity-Based Probes
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 26
Issue 4
10.3390/molecules26040783
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Novel Biphenyl Amines Inhibit Oestrogen Receptor (ER)-α in ER-Positive Mammary Carcinoma Cells

by
Basappa Basappa
1,2,
Baburajeev Chumadathil Pookunoth
2,
Mamatha Shinduvalli Kempasiddegowda
3,
Rangappa Knchugarakoppal Subbegowda
4,
Peter E. Lobie
3,5,6,* and
Vijay Pandey
3,5,*
1
Laboratory of Chemical Biology, Department of Studies in Organic Chemistry, University of Mysore, Manasagangotri, Mysore 570006, India
2
Department of Chemistry, Bangalore University, Bangalore 560001, India
3
Tsinghua Berkeley Shenzhen Institute, Tsinghua Shenzhen International Graduate School, Tsinghua University, Bei**g 518055, China
4
Institution of Excellence, University of Mysore, Manasagangotri, Mysore 570006, India
5
Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School, Tsinghua University, Bei**g 518055, China
6
Shenzhen Bay Laboratory, Shenzhen 518055, China
*
Authors to whom correspondence should be addressed.
Molecules 2021, 26(4), 783; https://doi.org/10.3390/molecules26040783
Submission received: 4 December 2020 / Revised: 20 January 2021 / Accepted: 23 January 2021 / Published: 3 February 2021
(This article belongs to the Special Issue 25th Anniversary of Molecules—Recent Advances in Chemical Biology)
Browse Figures
Review Reports Versions Notes

Abstract

:
Herein, the activity of adamantanyl-tethered-biphenyl amines (ATBAs) as oestrogen receptor alpha (ERα) modulating ligands is reported. Using an ERα competitor assay it was demonstrated that ATBA compound 3-(adamantan-1-yl)-4-methoxy-N-(4-(trifluoromethyl) phenyl) aniline (AMTA) exhibited an inhibitory concentration 50% (IC50) value of 62.84 nM and demonstrated better binding affinity compared to tamoxifen (IC50 = 79.48 nM). Treatment of ERα positive (ER+) mammary carcinoma (MC) cells (Michigan Cancer Foundation-7 (MCF7)) with AMTA significantly decreased cell viability at an IC50 value of 6.4 μM. AMTA treatment of MC cell-generated three-dimensional (3D) spheroids resulted in significantly decreased cell viability. AMTA demonstrated a superior inhibitory effect compared to tamoxifen-treated MC cell spheroids. Subsequently, by use of an oestrogen response element (ERE) luciferase reporter construct, it was demonstrated that AMTA treatment significantly deceased ERE transcriptional activity in MC cells. Concordantly, AMTA treatment of MC cells also significantly decreased protein levels of oestrogen-regulated CCND1 in a dose-dependent manner. In silico molecular docking analysis suggested that AMTA compounds interact with the ligand-binding domain of ERα compared to the co-crystal ligand, 5-(4-hydroxyphenoxy)-6-(3-hydroxyphenyl)-7- methylnaphthalen-2-ol. Therefore, an analogue of AMTA may provide a structural basis to develop a newer class of ERα partial agonists.

1. Introduction

17β-oestradiol (E2), a steroid hormone, plays important roles in the regulation of growth, differentiation, and function of a wide array of target tissues in both the male and female reproductive tracts, mammary glands, and skeletal and cardiovascular systems [1,2]. E2 exerts biological effects through at least two types of oestrogen receptors (ERs), namely ER-alpha (α) and ER-beta (β) [3,4]. These two receptors exhibit a high degree of homology in their ligand-binding and DNA-binding domains [5,6,7]. However, there are considerable differences in the N-terminus region of the two receptors [8]. The exact roles of ERβ are not clear, but it is known to counteract the activities of ERα [7].
Elevated levels of E2 are significantly associated with the neoplastic transformation and progression of female reproductive-related malignancies such as endometrial [9,10], ovarian [10], and breast cancer (BC) [11,12]. About two-thirds of human BC cases are ERα positive (+) [13]. Upon binding to E2, ERα dissociates from molecular chaperone complexes, dimerizes, migrates to the nucleus, and binds to specific DNA sequences (oestrogen response element (ERE)) that regulates the transcription of genes vital for BC cell survival [14] and metastasis [8,15]. In addition, E2 along with TGFβ1 enrich cancer stem cell populations in BC, leading to increased migration and resistance to therapy [16].
ERα is therefore an important therapeutic target in breast cancer with drugs limiting estrogenic activity to delay cancer progression [17,18,19]. Three approaches have been utilized clinically to inhibit ERα-related function. First, selective oestrogen modulators (SERMs) (e.g., tamoxifen, raloxifene) have been used to competitively bind to ER and displace E2, consequently inhibiting downstream signalling [17]. Second, selective ER degraders (SERDs) (e.g., fulvestrant) have been used to selectively promote ER degradation [18,19]. Finally, aromatase inhibitors have been used to inhibit aromatase enzyme activity and subsequently decrease the aromatization of androgens into oestrogens [20].
The majority of ERα-based anticancer agents such as tamoxifen, toremifene, raloxifene, and fulvestrant have been observed to be well tolerated in the clinic [21]. However, prolonged use of tamoxifen and toremifene in MC is associated with increased risk of endometrial cancer and eventual resistance to therapy leading to relapse [22,23]. Raloxifene use was also observed to be associated with increased risk of deep vein thrombosis in MC patients [24]. Therefore, to provide potential therapeutic alternatives, the discovery of novel ERα-targeting compounds is still required.
Synthetic oestrogens such as 4-(1-adamantyl) phenol (AdP) and 4,4-(1,3-adamantanediyl) diphenol (AdDP) bind to ERs and stimulate ER activity. Using structural references from synthetic oestrogens such as 4-(1-adamantyl) phenol (AdP) and 4,4-(1,3-adamantanediyl) diphenol (AdDP), a novel library of analogous AdDPs called adamantanyl-tethered-biphenyl amines (ATBAs) that potentially target ERα was designed [25]. We herein explored the in vitro and ex vivo inhibitory activity of newly designed ATBA compounds that bear phenolic amine as a linkage (unlike tamoxifen, GW368, and AdDP, small molecules which contain phenolic/ether linkages) as a new ligand that targets ERα in MC cells (Figure 1).

2. Results and Discussion

Diverse functionalized ATBA compounds were synthesized by treating adamantine bromo compound (1a) with substituted amines via an arylamination reaction (Scheme 1) [26]. The chemical synthesis and characterization of these compounds were reported recently [26].
First, to rank the novel biphenyl derivatives based on binding affinity to ERα, a commercially available fluorescence polarization assay kit (PolarScreen™ ER Alpha Competitor assay) was utilized. ATBA compounds were analysed for their capacity to displace fluorescently labelled E2 to form an ER-Fluormone ES2 complex. The smaller size of the free molecule can be discerned by increased mobility in the solution, which is detected by fluorescence polarization (FP). A shift in the FP value in the presence of the test compounds is used to determine the relative affinity of test compounds for ERα. The competitive binding affinity of the novel biphenyl derivatives is summarized in Table 1. Based on the FP value, the binding affinity of compound 4c was observed to be 62.84 nM, whereas for tamoxifen it was observed to be 79.48 nM. E2 exhibited a binding affinity of 6.27 nM. ATBA compound 4c exhibited higher binding affinity when compared to tamoxifen, but it was lower than that of E2. Therefore, the addition of –CF3, -OH, or -CH3 group tethered amines improved the binding affinity of the ATBA compounds to ERα.
Next, we determined the potential functional effects of novel ATBA compounds on the viability of MCF7 cells using an AlamarBlue® cell viability assay. The different ATBAs reduced the viability of MCF7 cells to differing degrees (Table 1). Paralleling the outcomes of the cell viability assay with ER-α binding, compounds 4a and 4b slightly reduced cell viability in MC cells with an IC50 greater than 50 µM. However, when the chlorine present in compound 4a or the methoxy group present in compound 4b was replaced with the -CF3 group in compound 4c, the loss of cell viability increased dramatically with an IC50 value of 6.29 µM. The presence of the hydroxyl group in compound 4d and methyl group in compound 4f also showed a better reduction of cell viability with IC50 at 8.81 µM and 12.3 µM, respectively. Similarly, the replacement of the benzene ring with a pyridine nucleus in compound 4g also showed activity with an IC50 of 12.09 µM. Other compounds 4h, 4i, 4k, and 4l reduced cell viability but with IC50 values close to 50 µM or higher. We also determined the functional efficacy of the 4c compound in additional ER+ MC cell lines using an AlamarBlue® cell viability assay. Compound 4c decreased cell viability of T47D cells with an IC50 value of 7.38 + 1.79 μM; BT474 cells with an IC50 value of 6.36 + 2.07 μM; and MDA-MB-361 cells with an IC50 value of 8.31 + 3.52μM.
Furthermore, we examined the effect of the novel biphenyl derivatives on preformed spheroids generated using MC cells in 3D Matrigel (Figure 2). Of the biphenyl derivatives tested, 4c-, 4d-, 4f-, and 4g-treated MC cell-generated spheroids exhibited significantly decreased cell viability compared to vehicle (DMSO) treated MC cell-generated spheroids. Among biphenyl derivatives, only the 4c derivative demonstrated a significant inhibitory effect compared to TAM-treated MC cell-generated spheroids.
Next, to evaluate the activity of the synthesized compounds as agonists or antagonists to ERα, a reporter assay was performed with ERE-luc [22,27]. The results of ERE activity (inhibition (upper chart) and activation (lower chart)) in response to the compounds are summarized in Figure 3. Of the biphenyl derivatives tested, 4c, 4d, 4f, and 4g exhibited the most potent antagonist activity against ERα in a dosage-dependent manner, represented by the most significantly decreased luciferase activity. Tamoxifen was used as positive control. The IC50 value of 4c was 0.83 ± 0.06 µM compared to TAM 2.07 ± 0.09 µM, as measured using ERE reporter assay. None of the tested biphenyl derivatives demonstrated a significant agonistic effect.
Increased expression of CCND1 is associated with approximately 50% of breast cancer cases and directly regulated by ER signalling [27,28]. To determine functional effects downstream of ERα, we examined the effect of ATBA compounds on hCCND1 protein expression in MC cells. Amongst the tested ATBA compounds, compound 4c most potently decreased the protein levels of CCND1 in MC cells in the nanomolar range when compared to other ATBA compounds (Figure 4).
Molecular docking analysis was performed by using the co-crystal structure of the hERα ligand-binding domain (LBD) with the naphthalene based small molecule, GW368 (5-(4-hydroxyphenoxy)-6-(3-hydroxyphenyl)-7-methylnaphthalen-2-ol; PDB ID: 3DT3) [31]. CDOCKER program of Accelrys DS version 2.5 was utilized. The receptor was energy-minimized and used for molecular docking studies as described previously [32,33]. The docking analysis observed that the most active compound 4c bound to the LBD of ERα with a CDOCKER interaction energy of 31kcal/mole and its binding mode was almost similar to GW368 with a perturbed trifluoromethyl (-CF3) group of compound 4c (Figure 5). The bulky adamantyl moiety of compound 4c may fill spaces in the LBD that are unoccupied when GW368-like molecules are bound. These relatively novel structural features of ATBAs could account for their high affinity for ERα. Thus, the addition of a -CF3 group to adamantyl-tethered-amino biphenylic, derivative 4c, shows improved efficacy and stability for further development as a potential therapeutic for ER + MC.
To determine the potential in vivo utility of the ATBA compounds, 15 properties associated with ADMET were determined by using the vNN-ADMET platform for the most active compounds (4c, 4d, 4g, 4h, and TAM as a comparison) [34]. The responses from the vNN-ADMET platform are tabulated in Table 2. The in silico analyses of ATBA compounds predicted that all of the active compounds will not be hepatotoxic, will not exhibit cytotoxicity, will not be metabolized rapidly by human liver membranes, will not inhibit drug metabolizing CYP450s, may pass through the blood-brain barrier (BBB), will not be a P-glycoptrotein inhibitor but may be a substrate, may exhibit hERG activity (as for TAM), will not impact matrix metalloproteinases, and will not exhibit chemical mutagenicity (Table 2). In addition, the predicted maximum recommended therapeutic dose of compound 4c was predicted to be approximately four times higher as compared to TAM.

3. Materials and Methods

The human Michigan Cancer Foundation-7 (MCF7) cell line was obtained from the American Type Culture Collection (ATCC, Rockville, Gaithersburg, MD, USA) and was cultured as per ATCC propagation instructions.
ER-alpha competitive binding assay: A competitive binding assay was performed using 17β-oestradiol and the PolarScreen™ ER Αlpha Competitor assay kit (Life TechnologiesTM, Carlsbad, CA, USA). The assay was performed as per the manufacturer’s instructions. The Fluorescence polarization value (mP) of each well on a fluorescence polarization plate was measured using fluorescence polarization Tecan infinite M1000PRO multimode microplate reader (Tecan, Switzerland). Data were modelled using GraphPad Prism® software from GraphPad Software, Inc. [27,30].
AlamarBlue®, 3D Matrigel, and luciferase assay: AlamarBlue® cell viability kits were obtained from ThemoFisher Scientific (Waltham, Massachusetts, USA), and the assay was performed as previously described [22,35]. 3D Matrigel assays were performed as previously described [30]. Luciferase assays were performed as previously described [22,35]. Briefly, transfections were carried out in triplicate using 1 μg of the appropriate luciferase reporter construct or empty vector along with 0.1 μg of Renilla luciferase construct as a control for transfection efficiency. Luciferase activities were measured using the Dual-Luciferase Assay System (Promega Corp, Madison, WI, USA).
Western blot analysis: Western blot analysis was performed as previously described [22,35], using a primary monoclonal antibody against CCND1 and β-ACTIN obtained from Cell Signalling, Danvers, Massachusetts, USA. Quantification of blot was performed by use of ImageJ software from NIH, USA (http://imagej.nih.gov/ij/) as described previously [27,29,30].
In silico studies: Molecular in silico interaction was carried out using 2DT3 protein ID and the ATBA compounds as ligands. The accelrys DS 2.5 was used for this study. After molecular redocking of GW368, we docked ATBAs to the ligand binding domain of ER using the CDOCKER programme as we reported in our previous publications [32]. Results were analysed using the accelrys visualization platform. The publicly available vNN Web Server was used for the ADMET predictions for the most active compounds and tabulated (https://vnnadmet.bhsai.org/).

4. Conclusions

It is herein disclosed that the ATBA compound 4c exhibits binding to ERα with promising inhibition of oestrogenic functions in hERα positive MC cells. In silico molecular docking studies revealed that compound 4c bound to the ligand-binding domain (LBD) of ERα strongly when compared to the co-crystal ligands. Further investigations of compound 4c are warranted to determine its pharmacological features and potential in vivo utility.

Author Contributions

B.B., B.C.P., and R.K.S. designed the experiments; B.B. and B.C.P. synthesized the compounds; B.B. performed the in silico analysis; V.P. performed the biological assays; B.B., M.S.K., V.P., and P.E.L. analysed the data; B.B., V.P., and P.E.L. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

The chemistry laboratory was supported by funding from the Council of Scientific and Industrial Research (No. 02(0291)17/EMR-II), Department of Biotechnology and the Vision Group on Science and Technology, Government of Karnataka. This work was supported by the Overseas Research Co-operation Project (HW2020008) (Tsinghua Shenzhen International Graduate School); the Shenzhen Development and Reform Commission Subject Construction Project ((2017)1434), the TBSI Faculty Start-up Funds, and the Shenzhen Bay Laboratory.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds ABTAs are available from Basappa.

References

  1. Hall, J.M.; Couse, J.F.; Korach, K.S. The multifaceted mechanisms of estradiol and estrogen receptor signaling. J. Biol. Chem. 2001, 276, 36869–36872. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Katzenelienbogen, J.A.; Katzenellenbogen, B.S. Nuclear hormone receptors: Ligand-activated regulators of transcription and diverse cell responses. Chem. Biol. 1996, 3, 529–536. [Google Scholar] [CrossRef] [Green Version]
  3. Srinivasan, S.; Nawaz, Z. 35—Molecular Biology of Estrogen Receptor Action” in Hormones, Brain and Behavior, 2nd ed.; Pfaff, D.W., Arnold, A.P., Etgen, A.M., Fahrbach, S.E., Rubin, R.T., Eds.; Academic Press: San Diego, CA, USA, 2009; pp. 1187–1220. [Google Scholar] [CrossRef]
  4. Grundy, J. Artificial Estrogens. Chem. Rev. 1957, 57, 281–415. [Google Scholar] [CrossRef]
  5. Fuentes, N.; Silveyra, P. Chapter Three—Estrogen receptor signaling mechanisms. In Advances in Protein Chemistry and Structural Biology; Donev, R., Ed.; Academic Press: Cambridge, MA, USA, 2019; Volume 116, pp. 135–170. [Google Scholar]
  6. Chang, M.-S. Tamoxifen resistance in breast cancer. Biomol. Ther. 2012, 20, 256–267. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  7. Alistair, R.; Mitch, D. Mechanisms of tamoxifen resistance. Endocr. Relat. Cancer 2004, 11, 643–658. [Google Scholar]
  8. Kuiper, G.G.; Enmark, E.; Pelto-Huikko, M.; Nilsson, S.; Gustafsson, J.A. Cloning of a novel receptor expressed in rat prostate and ovary. Proc. Natl. Acad. Sci. USA 1996, 93, 5925–5930. [Google Scholar] [CrossRef] [Green Version]
  9. Yang, C.; AlMomen, A.; Wee, Y.S.; Jarboe, E.A.; Peterson, C.M.; Janat-Amsbury, M.M. An estrogen-induced endometrial hyperplasia mouse model recapitulating human disease progression and genetic aberrations. Cancer Med. 2015, 4, 1039–1050. [Google Scholar] [CrossRef]
  10. Brown, S.B.; Hankinson, S.E. Endogenous estrogens and the risk of breast, endometrial, and ovarian cancers. Steroids 2015, 99, 8–10. [Google Scholar] [CrossRef]
  11. Yager, J.D. Mechanisms of estrogen carcinogenesis: The role of E2/E1–quinone metabolites suggests new approaches to preventive intervention—A review. Steroids 2015, 99, 56–60. [Google Scholar] [CrossRef] [Green Version]
  12. Chen, Y.; Wang, J.; Hong, D.-Y.; Chen, L.; Zhang, Y.-Y.; Xu, Y.-N.; Pan, D.; Fu, L.-Y.; Tao, L.; Luo, H.; et al. Baicalein has protective effects on the 17β-estradiol-induced transformation of breast epithelial cells. Oncotarget 2017, 8, 10470–10484. [Google Scholar] [CrossRef]
  13. O’Mahony, F.; Razandi, M.; Pedram, A.; Harvey, B.J.; Levin, E.R. Estrogen modulates metabolic pathway adaptation to available glucose in breast cancer cells. Mol. Endocrinol. 2012, 26, 2058–2070. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  14. Fiocchetti, M.; Nuzzo, M.T.; Totta, P.; Acconcia, F.; Ascenzi, P.; Marino, M. Neuroglobin, a pro-survival player in estrogen receptor α-positive cancer cells. Cell Death Dis. 2014, 5, e1449. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  15. Zeng, K.; He, B.; Yang, B.B.; Xu, T.; Chen, X.; Xu, M.; Liu, X.; Sun, H.; Pan, Y.; Wang, S. The pro-metastasis effect of circANKS1B in breast cancer. Mol. Cancer 2018, 17, 1–19. [Google Scholar] [CrossRef] [PubMed]
  16. Rodriguez, G.V.; Abrahamsson, A.; Jensen, L.D.E.; Dabrosin, C. Estradiol Promotes Breast Cancer Cell Migration via Recruitment and Activation of Neutrophils. Cancer Immunol. Res. 2017, 5, 234–247. [Google Scholar] [CrossRef] [Green Version]
  17. Katzenellenbogen, B.S.; A Katzenellenbogen, J. Estrogen receptor transcription and transactivation: Estrogen receptor alpha and estrogen receptor beta: Regulation by selective estrogen receptor modulators and importance in breast cancer. Breast Cancer Res. 2000, 2, 335–344. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  18. Lee, C.I.; Goodwin, A.; Freedman, O.; Clemons, M. Fulvestrant for hormone-sensitive metastatic breast cancer. Cochrane Database Syst. Rev. 2014, 1, 011093. [Google Scholar]
  19. Morris, C.; Wakeling, A. Fulvestrant (‘Faslodex’)—A new treatment option for patients progressing on prior endocrine therapy. Endocrine-Related Cancer 2002, 9, 267–276. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  20. Miller, W.R.; Larionov, A.A. Understanding the mechanisms of aromatase inhibitor resistance. Breast Cancer Res. 2012, 14, 1–11. [Google Scholar] [CrossRef] [Green Version]
  21. Sharma, D.; Kumar, S.; Narasimhan, B. Estrogen alpha receptor antagonists for the treatment of breast cancer: A review. Chem. Central J. 2018, 12, 1–32. [Google Scholar] [CrossRef] [Green Version]
  22. Pandey, V.; Zhang, M.; Chong, Q.-Y.; You, M.; Raquib, A.R.; Pandey, A.K.; Liu, D.-X.; Liu, L.; Ma, L.; Jha, S.; et al. Hypomethylation associated enhanced transcription of trefoil factor-3 mediates tamoxifen-stimulated oncogenicity of ER+ endometrial carcinoma cells. Oncotarget 2017, 8, 77268–77291. [Google Scholar] [CrossRef]
  23. Buzdar, A.U.; Hortobagyi, G.N. Tamoxifen and toremifene in breast cancer: Comparison of safety and efficacy. J. Clin. Oncol. 1998, 16, 348–353. [Google Scholar] [CrossRef] [PubMed]
  24. Deitcher, S.R.; Gomes, M.P. The risk of venous thromboembolic disease associated with adjuvant hormone therapy for breast carcinoma: A systematic review. Cancer 2004, 101, 439–449. [Google Scholar] [CrossRef] [PubMed]
  25. Wolfe, J.P.; Wagaw, A.S.; Buchwald, S.L. An Improved Catalyst System for Aromatic Carbon−Nitrogen Bond Formation:  The Possible Involvement of Bis(Phosphine) Palladium Complexes as Key Intermediates. J. Am. Chem. Soc. 1996, 118, 7215–7216. [Google Scholar] [CrossRef]
  26. Pookunoth, B.C.; Rao, S.E.; Deveshegowda, S.N.; Metri, P.K.; Fazl-Ur-Rahman, K.; Periyasamy, G.; Virupaiah, G.; Priya, B.S.; Pandey, V.; Lobie, P.E.; et al. Development of a New Arylamination Reaction Catalyzed by Polymer Bound 1,3-(Bisbenzimidazolyl) Benzene Co(II) Complex and Generation of Bioactive Adamanate Amines. Catalysts 2020, 10, 1315. [Google Scholar]
  27. Pandey, V.; Zhang, M.; You, M.; Zhang, W.; Chen, R.; Zhang, W.; Ma, L.; Wu, Z.; Zhu, T.; Xu, X.Q.; et al. Expression of two non-mutated genetic elements is sufficient to stimulate oncogenic transformation of human mammary epithelial cells. Cell Death Dis. 2018, 9, 1–18. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  28. Alao, J.P. The regulation of cyclin D1 degradation: Roles in cancer development and the potential for therapeutic invention. Mol. Cancer 2007, 6, 24. [Google Scholar] [CrossRef] [Green Version]
  29. Pandey, V.; Prabhu, J.S.; Payal, K.; Rajan, V.; Deepak, C.; Barde, S.; Jagannath, P.; Borges, A. Assessment of microsatellite instability in colorectal carcinoma at an Indian center. Int. J. Color. Dis. 2006, 22, 777–782. [Google Scholar] [CrossRef]
  30. Pandey, V.; Wang, B.; Mohan, C.D.; Raquib, A.R.; Rangappa, S.; Srinivasa, V.; Fuchs, J.E.; Girish, K.S.; Zhu, T.; Bender, A.; et al. Discovery of a small-molecule inhibitor of specific serine residue BAD phosphorylation. Proc. Natl. Acad. Sci. USA 2018, 115, E10505–E10514. [Google Scholar] [CrossRef] [Green Version]
  31. Fang, J.; Akwabi-Ameyaw, A.; Britton, J.E.; Katamreddy, S.R.; Navas, F.; Miller, A.B.; Williams, S.P.; Gray, D.W.; Orband-Miller, L.A.; Shearin, J.; et al. Synthesis of 3-alkyl naphthalenes as novel estrogen receptor ligands. Bioorg. Med. Chem. Lett. 2008, 18, 5075–5077. [Google Scholar] [CrossRef] [PubMed]
  32. Bharathkumar, H.; Mohan, C.D.; Ananda, H.; Fuchs, J.E.; Li, F.; Rangappa, S.; Surender, M.; Bulusu, K.C.; Girish, K.S.; Sethi, G.; et al. Microwave-assisted synthesis, characterization and cytotoxic studies of novel estrogen receptor α ligands towards human breast cancer cells. Bioorg. Med. Chem. Lett. 2015, 25, 1804–1807. [Google Scholar] [CrossRef] [PubMed]
  33. Bhuvanalakshmi, G.; Basappa; Rangappa, K.S.; Dharmarajan, A.; Sethi, G.; Kumar, A.P.; Warrier, S. Breast Cancer Stem-Like Cells Are Inhibited by Diosgenin, a Steroidal Saponin, by the Attenuation of the Wnt β-Catenin Signaling via the Wnt Antagonist Secreted Frizzled Related Protein-4. Front. Pharmacol. 2017, 8, 124. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Schyman, P.; Liu, R.; Desai, V.; Wallqvist, A. vNN Web Server for ADMET Predictions. Front. Pharmacol. 2017, 8, 889. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Pandey, V.; Wu, Z.; Zhang, M.; Li, R.; Zhang, J.V.; Zhu, T.; Lobie, P.E. Trefoil factor 3 promotes metastatic seeding and predicts poor survival outcome of patients with mammary carcinoma. Breast Cancer Res. 2014, 16, 429. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Molecules 26 00783 g001 550
Figure 1. Structures of the oestrogen receptor (ER) modulators used as ER ligands: (A) tamoxifen; (B) 5-(4-hydroxyphenoxy)-6-(3-hydroxyphenyl)-7- methylnaphthalen-2-ol; (C) 4,4-(1,3-adamantanediyl) diphenol; (D) 3-(adamantan-1-yl)-4-methoxy-N-(4-(trifluoromethyl) phenyl) aniline.
Figure 1. Structures of the oestrogen receptor (ER) modulators used as ER ligands: (A) tamoxifen; (B) 5-(4-hydroxyphenoxy)-6-(3-hydroxyphenyl)-7- methylnaphthalen-2-ol; (C) 4,4-(1,3-adamantanediyl) diphenol; (D) 3-(adamantan-1-yl)-4-methoxy-N-(4-(trifluoromethyl) phenyl) aniline.
Molecules 26 00783 g001
Molecules 26 00783 sch001 550
Scheme 1. Reported scheme to show the coupling of adamantane bromides with various aromatic amines to obtain adamantanyl-tethered-biphenyl amines (ATBAs). R.C: Reaction conditions; PS-Co (BBZN)Cl2 (12 mol %); CS2CO3 (3 Eq); BINAP (15 mol %); 1,4-dioxane (5 mL); N2 atmosphere 10 h; 100 °C.
Scheme 1. Reported scheme to show the coupling of adamantane bromides with various aromatic amines to obtain adamantanyl-tethered-biphenyl amines (ATBAs). R.C: Reaction conditions; PS-Co (BBZN)Cl2 (12 mol %); CS2CO3 (3 Eq); BINAP (15 mol %); 1,4-dioxane (5 mL); N2 atmosphere 10 h; 100 °C.
Molecules 26 00783 sch001
Molecules 26 00783 g002 550
Figure 2. Cell viability of MCF7 cell-generated spheroids after culture in 3D Matrigel for 10 days. Preformed MCF7 cell-generated spheroids were treated with compounds for 72 h before assessing cell viability. A concentration of 5 µM was used for the treatment. The cell viability of the MCF7 cell-generated spheroids was evaluated using an AlamarBlue® viability assay as described in the Materials and Methods section. Bars are the mean of triplicate experiments; bars, ±SD. ** P < 0.001, * P < 0.05.
Figure 2. Cell viability of MCF7 cell-generated spheroids after culture in 3D Matrigel for 10 days. Preformed MCF7 cell-generated spheroids were treated with compounds for 72 h before assessing cell viability. A concentration of 5 µM was used for the treatment. The cell viability of the MCF7 cell-generated spheroids was evaluated using an AlamarBlue® viability assay as described in the Materials and Methods section. Bars are the mean of triplicate experiments; bars, ±SD. ** P < 0.001, * P < 0.05.
Molecules 26 00783 g002
Molecules 26 00783 g003 550
Figure 3. Inhibition of 17β oestradiol (E2) stimulated oestrogen response element (ERE) transcrip Table 2. (lower panel). MCF7 cells were co-transfected with ERE-luc (firefly luciferase) and Renilla luciferase constructs as described in the Materials and Methods section. Cells were incubated with 17β oestradiol (E2) (10−8 M) (Upper panel) and ATBA compounds (10−10 to 10−4 M) and without oestradiol (lower panel). Results are shown as mean (+SD for triplicate transfection). TAM: tamoxifen.
Figure 3. Inhibition of 17β oestradiol (E2) stimulated oestrogen response element (ERE) transcrip Table 2. (lower panel). MCF7 cells were co-transfected with ERE-luc (firefly luciferase) and Renilla luciferase constructs as described in the Materials and Methods section. Cells were incubated with 17β oestradiol (E2) (10−8 M) (Upper panel) and ATBA compounds (10−10 to 10−4 M) and without oestradiol (lower panel). Results are shown as mean (+SD for triplicate transfection). TAM: tamoxifen.
Molecules 26 00783 g003
Molecules 26 00783 g004 550
Figure 4. Effect of selected ATBA compounds on the expression of CCND1 in MCF7 cells. Soluble whole-cell extracts were run on an SDS-PAGE and immunoblotted. β-ACTIN was used as input control for cell lysate (left side). The predicted sizes of detected protein bands in kDa are shown on the left side. The average of three independent densitometry analyses of CCND1 and β-ACTIN is shown above (right side) by use of Image J software from NIH, USA (http://imagej.nih.gov/ij/) as described previously [27,29,30].
Figure 4. Effect of selected ATBA compounds on the expression of CCND1 in MCF7 cells. Soluble whole-cell extracts were run on an SDS-PAGE and immunoblotted. β-ACTIN was used as input control for cell lysate (left side). The predicted sizes of detected protein bands in kDa are shown on the left side. The average of three independent densitometry analyses of CCND1 and β-ACTIN is shown above (right side) by use of Image J software from NIH, USA (http://imagej.nih.gov/ij/) as described previously [27,29,30].
Molecules 26 00783 g004
Molecules 26 00783 g005 550
Figure 5. Molecular docking analysis showing the most active compound 4c and its comparison with GW368 (upper panel); the interaction map of compound 4c at the ligand-binding domain (LBD) region of ER(lower panel) also shown using Accelrys DS visualization software.
Figure 5. Molecular docking analysis showing the most active compound 4c and its comparison with GW368 (upper panel); the interaction map of compound 4c at the ligand-binding domain (LBD) region of ER(lower panel) also shown using Accelrys DS visualization software.
Molecules 26 00783 g005
Table
Table 1. Competitive binding affinity of ATBA molecules to ERα and their functional effect on MCF7 cells.
Table 1. Competitive binding affinity of ATBA molecules to ERα and their functional effect on MCF7 cells.
EntryStructures of ATBACBA a IC50 + SD (nM)Cell Viability (MCF7 Cells)
IC50 ± SD (μM)
4a Molecules 26 00783 i001
3-(Adamantan-1-yl)-N-(4-chlorophenyl)-4-methoxyaniline		340.11 + 45.17>50
4b Molecules 26 00783 i002
3-(Adamantan-1-yl)-4-methoxy-N-(4-methoxyphenyl) aniline		202.19 + 21.32>50
4c Molecules 26 00783 i003
3-(Adamantan-1-yl)-4-methoxy-N-(4-(trifluoromethyl) phenyl) aniline		62.84 + 3.316.29 ± 2.41
4d Molecules 26 00783 i004
3-((3-(Adamantan-1-yl)-4-methoxyphenyl)amino) phenol		82.61 + 7.098.81 ± 1.85
4f Molecules 26 00783 i005
3-(Adamantan-1-yl)-4-methoxy-N-(p-tolyl) aniline		98.55 + 5.1812.3 ± 3.77
4g Molecules 26 00783 i006
N-(3-Adamantan-1-yl)-4-methoxyphenyl)pyridin-3-amine		129.45 + 12.8412.09 ± 3.07
4h Molecules 26 00783 i007
N-(3-Adamantan-1-yl)-4-methoxyphenyl)-5-methylpyridin-2-amine		189.28 + 10.6742.18 ± 9.31
4i Molecules 26 00783 i008
N-(3-Adamantan-1-yl)-4-methoxyphenyl)naphthalen-1-amine		264.28 + 24.5047.61 ± 7.48
TAM b79.48 + 4.516.92 ± 2.13
E2 c6.27 + 1.48NA
Note CBA a, competitive binding affinity; TAM b, tamoxifen; E2 c, 17β-oestradiol. Competitive binding affinity data generated using the PolarScreen™ ER Αlpha Competitor Assay; Green kit from Life TechnologiesTM. The fluorescence polarization value (mP) of each well was measured on a fluorescence polarization Tecan infinite M1000PRO multimode microplate reader. Values represent the average range or SD of three independent experiments. The effect of ATBA molecules on Michigan Cancer Foundation-7 (MCF7) cell viability was measured using AlamarBlue® cell viability assay. IC50 values were calculated using GraphPad Prism software (Version 5.0).
Table
Table 2. The vNN-ADMET predictions for the ATBA compounds.
Table 2. The vNN-ADMET predictions for the ATBA compounds.
QueryLiver
Toxicity		MetabolismMembrane
Transporters		Others
CYP Inhibitors for
DILICTHLM1A23A42D62C92C19BBBPgp SubPgp InhERGMMPAMESMRTD (mg/day)
4cN aNY bNNNNNYYNYNN856
4dNNYNNNNNYNYNNN1891
4fNNYNNNNNYYYNNY202
4gNNYNNNNNYNYNNN209
TAMYNNNYNNNYYYYNN197
Note: N a, No; Y b, Yes; DILI, drug-induced liver injury; CT, cytotoxicity; CYP, cytochrome P450; HLM, human liver microsomes; BBB; blood-brain barrier; Pgp, P-glycoprotein; Sub, substrate; In, inhibition; hERG, human ether-a-go-go-related gene; MMP, matrix metalloproteins; AMES; salmonella/microsome mutagenicity; MRTD, maximum recommended therapeutic dose. Online predictions and interpretations using a restricted/unrestricted applicability domain are represented.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Basappa, B.; Chumadathil Pookunoth, B.; Shinduvalli Kempasiddegowda, M.; Knchugarakoppal Subbegowda, R.; Lobie, P.E.; Pandey, V. Novel Biphenyl Amines Inhibit Oestrogen Receptor (ER)-α in ER-Positive Mammary Carcinoma Cells. Molecules 2021, 26, 783. https://doi.org/10.3390/molecules26040783

AMA Style

Basappa B, Chumadathil Pookunoth B, Shinduvalli Kempasiddegowda M, Knchugarakoppal Subbegowda R, Lobie PE, Pandey V. Novel Biphenyl Amines Inhibit Oestrogen Receptor (ER)-α in ER-Positive Mammary Carcinoma Cells. Molecules. 2021; 26(4):783. https://doi.org/10.3390/molecules26040783

Chicago/Turabian Style

Basappa, Basappa, Baburajeev Chumadathil Pookunoth, Mamatha Shinduvalli Kempasiddegowda, Rangappa Knchugarakoppal Subbegowda, Peter E. Lobie, and Vijay Pandey. 2021. "Novel Biphenyl Amines Inhibit Oestrogen Receptor (ER)-α in ER-Positive Mammary Carcinoma Cells" Molecules 26, no. 4: 783. https://doi.org/10.3390/molecules26040783

Article Metrics

Back to TopTop