title: In silico discovery and validation of potent small-molecule inhibitors targeting the activation function 2 site of human oestrogen receptor α.
abstract: BACKGROUND
  Current approaches to inhibit oestrogen receptor-alpha (ERα) are focused on targeting its hormone-binding pocket and have limitations. Thus, we propose that inhibitors that bind to a coactivator-binding pocket on ERα, called activation function 2 (AF2), might overcome some of these limitations.
  
  
  METHODS
  In silico virtual screening was used to identify small-molecule ERα AF2 inhibitors. These compounds were screened for inhibition of ERα transcriptional activity using stably transfected T47D-KBluc cell line. A direct physical interaction between the AF2 binders and the ERα protein was measured using biolayer interferometry (BLI) and an ERα coactivator displacement assay. Cell viability was assessed by MTS assay in ERα-positive MCF7 cells, tamoxifen-resistant (TamR) cell lines TamR3 and TamR6, and ERα-negative MDA-MB-453 and HeLa cell lines. In addition, ERα inhibition in TamR cells and the effect of compounds on mRNA and protein expression of oestrogen-dependent genes, pS2, cathepsin D and cell division cycle 2 (CDC2) were determined.
  
  
  RESULTS
  Fifteen inhibitors from two chemical classes, derivatives of pyrazolidine-3,5-dione and carbohydrazide, were identified. In a series of in vitro assays, VPC-16230 of the carbohydrazide chemical class emerged as a lead ERα AF2 inhibitor that significantly downregulated ERα transcriptional activity (half-maximal inhibitory concentration = 5.81 μM). By directly binding to the ERα protein, as confirmed by BLI, VPC-16230 effectively displaced coactivator peptides from the AF2 pocket, confirming its site-specific action. VPC-16230 selectively suppressed the growth of ERα-positive breast cancer cells. Furthermore, it significantly inhibited ERα mediated transcription in TamR cells. More importantly, it reduced mRNA and protein levels of pS2, cathepsin D and CDC2, validating its ER-directed activity.
  
  
  CONCLUSIONS
  We identified VPC-16230 as an ERα AF2-specific inhibitor that demonstrated promising antiproliferative effects in breast cancer cell lines, including TamR cells. VPC-16230 reduced the expression of ERα-inducible genes, including CDC2, which is involved in cell division. We anticipate that the application of ERα AF2 inhibitors will provide a novel approach that can act as a complementary therapeutic to treat ERα-positive, tamoxifen-resistant and metastatic breast cancers.
authors.forename: Kriti
authors.initials: K
authors.lastname: Singh
authors.forename: Ravi Shashi Nayana
authors.initials: RS
authors.lastname: Munuganti
authors.forename: Eric
authors.initials: E
authors.lastname: Leblanc
authors.forename: Yu Lun
authors.initials: YL
authors.lastname: Lin
authors.forename: Euphemia
authors.initials: E
authors.lastname: Leung
authors.forename: Nada
authors.initials: N
authors.lastname: Lallous
authors.forename: Miriam
authors.initials: M
authors.lastname: Butler
authors.forename: Artem
authors.initials: A
authors.lastname: Cherkasov
authors.forename: Paul S
authors.initials: PS
authors.lastname: Rennie
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry* / Q000494:pharmacology; D001665:Binding Sites; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003198:Computer Simulation; D055808:Drug Discovery / Q000379:methods; D019008:Drug Resistance, Neoplasm; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D004958:Estradiol / Q000378:metabolism; D020847:Estrogen Receptor Modulators / Q000737:chemistry* / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D004967:Estrogens / Q000378:metabolism; D005260:Female; D006801:Humans; D008024:Ligands; D061986:MCF-7 Cells; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D015203:Reproducibility of Results; D054852:Small Molecule Libraries; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D000970:Antineoplastic Agents; D002352:Carrier Proteins; D020847:Estrogen Receptor Modulators; D047628:Estrogen Receptor alpha; D004967:Estrogens; D008024:Ligands; D054852:Small Molecule Libraries; D004958:Estradiol
references.citation: J Biol Chem. 2000 May 26;275(21):16302-8
references.pmid: 10748020
references.citation: J Med Chem. 2011 Sep 22;54(18):6197-205
references.pmid: 21846139
references.citation: Endocrinology. 2003 Oct;144(10):4562-74
references.pmid: 12959972
references.citation: J Med Chem. 2004 Mar 25;47(7):1739-49
references.pmid: 15027865
references.citation: J Steroid Biochem Mol Biol. 2004 Apr;88(4-5):351-60
references.pmid: 15145444
references.citation: Cancer Res. 1995 Aug 1;55(15):3331-8
references.pmid: 7614468
references.citation: Clin Cancer Res. 2005 Jan 15;11(2 Pt 2):865s-70s
references.pmid: 15701879
references.citation: J Clin Oncol. 2005 Apr 10;23(11):2469-76
references.pmid: 15753463
references.citation: Clin Cancer Res. 2005 Apr 15;11(8):2822-31
references.pmid: 15837729
references.citation: Lancet. 2005 May 14-20;365(9472):1687-717
references.pmid: 15894097
references.citation: Cancer. 2005 Jul 15;104(2):236-9
references.pmid: 15937908
references.citation: Endocr Relat Cancer. 2005 Jul;12 Suppl 1:S113-7
references.pmid: 16113087
references.citation: Endocr Relat Cancer. 2005 Jul;12 Suppl 1:S99-S111
references.pmid: 16113104
references.citation: J Pathol. 2005 Oct;207(2):139-46
references.pmid: 16088978
references.citation: N Engl J Med. 2005 Oct 27;353(17):1784-92
references.pmid: 16251534
references.citation: N Engl J Med. 2006 Jan 19;354(3):270-82
references.pmid: 16421368
references.citation: CA Cancer J Clin. 2006 Mar-Apr;56(2):106-30
references.pmid: 16514137
references.citation: J R Soc Interface. 2006 Apr 22;3(7):215-33
references.pmid: 16849232
references.citation: Breast Dis. 2005-2006;24:93-105
references.pmid: 16917142
references.citation: Nat Genet. 2006 Nov;38(11):1289-97
references.pmid: 17013392
references.citation: Angew Chem Int Ed Engl. 2007;46(24):4471-3
references.pmid: 17487924
references.citation: J Mol Graph Model. 2007 Jul;26(1):198-212
references.pmid: 16860582
references.citation: Bioorg Med Chem Lett. 2007 Aug 1;17(15):4118-22
references.pmid: 17560105
references.citation: CA Cancer J Clin. 2007 Jul-Aug;57(4):190-205
references.pmid: 17626117
references.citation: Cancer Lett. 2007 Oct 18;256(1):1-24
references.pmid: 17475399
references.citation: Breast Cancer Res. 2007;9(5):112
references.pmid: 17980056
references.citation: ACS Chem Biol. 2008 May 16;3(5):282-6
references.pmid: 18484708
references.citation: J Med Chem. 2008 Oct 23;51(20):6512-30
references.pmid: 18785725
references.citation: Bioorg Med Chem. 2008 Dec 1;16(23):10075-84
references.pmid: 18976929
references.citation: ACS Chem Biol. 2009 Jun 19;4(6):435-40
references.pmid: 19441848
references.citation: Org Lett. 2009 Dec 3;11(23):5370-3
references.pmid: 19902964
references.citation: J Med Chem. 2011 Dec 22;54(24):8563-73
references.pmid: 22047606
references.citation: Breast Cancer Res. 2010;12(5):R75
references.pmid: 20863372
references.citation: Proc Natl Acad Sci U S A. 2012 Sep 11;109(37):14930-5
references.pmid: 22927406
references.citation: CA Cancer J Clin. 2013 Jan;63(1):11-30
references.pmid: 23335087
references.citation: J Med Chem. 2013 Feb 14;56(3):1136-48
references.pmid: 23301637
references.citation: Med Res Rev. 2013 Sep;33(5):1081-118
references.pmid: 23344935
references.citation: J Chem Inf Model. 2012 Jul 23;52(7):1757-68
references.pmid: 22587354
references.citation: Nat Genet. 2013 Dec;45(12):1446-51
references.pmid: 24185510
references.citation: Nat Genet. 2013 Dec;45(12):1439-45
references.pmid: 24185512
references.citation: Oncogene. 2010 Jan 14;29(2):201-13
references.pmid: 19838210
references.citation: Mod Pathol. 2010 Feb;23(2):205-12
references.pmid: 19898421
references.citation: Clin Cancer Res. 2010 Mar 1;16(5):1486-97
references.pmid: 20179226
references.citation: Breast Cancer Res Treat. 2010 Apr;120(3):603-12
references.pmid: 19495959
references.citation: Cancer Res. 2010 May 15;70(10):4092-101
references.pmid: 20460518
references.citation: Cancer Biol Ther. 2010 May 1;9(9):717-24
references.pmid: 20234184
references.citation: Clin Cancer Res. 2011 Mar 1;17(5):1147-59
references.pmid: 21220480
references.citation: ChemMedChem. 2011 Apr 4;6(4):654-66
references.pmid: 21365764
references.citation: Lancet. 2011 Aug 27;378(9793):771-84
references.pmid: 21802721
references.citation: Steroids. 2001 Dec;66(12):905-10
references.pmid: 11711119