Peptide antagonists of the human estrogen receptor

Science

Volumn 285, Issue 5428, 1999, Pages 744-746

  Norris, John D ^a   Paige, Lisa A ^b   Christensen, Dale J ^b   Chang, Ching Yi ^a   Huacani, Maria R ^a   Fan, Daju ^a   Hamilton, Paul T ^b   Fowlkes, Dana M ^b   McDonnell, Donald P ^a  

^a DUKE UNIVERSITY   (United States)

^b Novalon Pharmaceutical Corporation   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ANTIESTROGEN; ESTRADIOL; ESTROGEN RECEPTOR; FULVESTRANT; GLUCOCORTICOID RECEPTOR; GW 7604; HORMONE RECEPTOR AFFECTING AGENT; IDOXIFENE; MIFEPRISTONE; NAFOXIDINE; ONAPRISTONE; PEPTIDE; PROGESTERONE RECEPTOR; RALOXIFENE; TAMOXIFEN; UNCLASSIFIED DRUG;

ARTICLE; BREAST CANCER; CONFORMATIONAL TRANSITION; DRUG MECHANISM; DRUG RECEPTOR BINDING; GENE CONTROL; HUMAN; HUMAN CELL; LIGAND BINDING; PHAGE DISPLAY; PRIORITY JOURNAL; PROTEIN EXPRESSION; RECEPTOR BINDING; RECEPTOR BLOCKING;

AMINO ACID SEQUENCE; BINDING SITES; ESTRADIOL; ESTRADIOL ANTAGONISTS; ESTROGEN ANTAGONISTS; ESTROGEN RECEPTOR ALPHA; HUMANS; LIGANDS; MIFEPRISTONE; MOLECULAR SEQUENCE DATA; PEPTIDE LIBRARY; PEPTIDES; RECEPTORS, CYTOPLASMIC AND NUCLEAR; RECEPTORS, ESTROGEN; RECOMBINANT FUSION PROTEINS; TAMOXIFEN; TRANSCRIPTION FACTOR AP-1; TRANSCRIPTION, GENETIC;

EID: 0033618510     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5428.744     Document Type: Article

References (19)

1. J. A. Scott and W. L. McGuire, in Endocrine-Dependent Tumors, K. D. Voight and C. Knabbe, Eds. (Raven, New York, 1991), pp. 179-196.
2. C. K. Osborne, R. M. Elledge, S. A. W. Fuqua, Sci. Am. Sci. Med. 3, 32 (1996).
3. B. S. Katzenellenbogen, M. M. Montano, M. E. Ekena, E. M. McInerney, Breast Cancer Res. Treat. 44, 23 (1997).
4. M. Sato, M. K. Rippy, H. U. Bryant, FASEB J. 10, 905 (1996).
5. A. M. Brzozowski et al., Nature 389, 753 (1997); A. K. Shiau et al., Cell 95, 927 (1998).
6. A. M. Brzozowski et al., Nature 389, 753 (1997); A. K. Shiau et al., Cell 95, 927 (1998).
7. 1). Affinity selection was repeated three times, and individual phage were isolated from either the second or third round of amplification. Peptide sequences were then deduced by DNA sequencing.
8. D. M. Heery, E. Kalkhoven, S. Hoare, M. G. Parker, Nature 387, 733 (1997).
9. M. O. Imhof and D. P. McDonnell, Mol. Cell. Biol. 16, 2594 (1995).
10. TRF assays were performed at room temperature as follows: Costar (Cambridge, MA) high-binding 384-well plates were coated with streptavidin in 0.1 M sodium bicarbonate and blocked with BSA. Twenty microliters of biotinytated ERE (100 nM in TBST) was added to each well. After a 1-hour incubation, biotin (50 μM in TBST) was added to block any remaining binding sites. The plates were washed, and 20 μl of ERa (100 nM in TBST) was added to each well After a 1-hour incubation, the plates were washed, and 5 μl of 5 μM 4-OH tamoxifen was added to each well followed by 15 μl of solution containing the peptides conjugated to unlabeled streptavidin (prepared as described below) at a range of concentrations (from 1.67 μM in twofold dilutions). After a 30-min incubation with the 4-OH tamoxifen and conjugate, 5 μl of 400 nM europiumlabeled streptavidin (Wallac, Gaithersburg, MD)-biotinylated peptide conjugate (prepared as described below) was added and incubated for 1 hour. The plates were then washed, and the europium enhancement solution was added. Fluorescent readings were obtained with a POLARstar fluorimeter (BMG Lab Technologies, Durham, NC) with a <400-nm excitation filter and a 620-nm emission filter. The streptavidin-biotinylated peptide conjugates were prepared by adding 4 pmol of biotinylated peptide per picomole of streptavidin. After incubation on ice for 30 min, the remaining biotin-binding sites were blocked with biotin before addition to the ER-coated plate.
11. HepG2 cells were maintained in modified Eagle's medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Life Technologies). Transfections were performed as described [J. D. Norris et al., J. Biol. Chem. 270, 22777 (1995)]. pCMV-β-Gal and 5× GAL4-tata-Luc were described previously [B. L. Wagner, J. D. Norris, T. A. Knotts, N. L. Weigel, D. P. McDonnell, Mol. Cell. Biol. 18, 1369 (1998)]. Gal4 DBD-peptide fusions were created as follows: Peptide-coding sequences were excised from mBAX vector with Xho I-Xba I and subcloned into pM vector (Clontech, Palo Alto, CA) with a linker sequence to generate Sal I and Xba I sites for cloning. ERα-VP16 was generated by polymerase chain reaction (PCR) of human ERα-cDNA containing Eco RI sites flanking both 5′ and 3′ termini. The resultant PCR product was then subdoned into pVP15 (Clontech). All PCR products were sequenced to ensure the fidelity of the resultant construct. 17β-estradiol, 4-hydroxy-tamoxifen, and nafoxidine were purchased from Sigma.
12. HepG2 cells were maintained in modified Eagle's medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Life Technologies). Transfections were performed as described [J. D. Norris et al., J. Biol. Chem. 270, 22777 (1995)]. pCMV-β-Gal and 5× GAL4-tata-Luc were described previously [B. L. Wagner, J. D. Norris, T. A. Knotts, N. L. Weigel, D. P. McDonnell, Mol. Cell. Biol. 18, 1369 (1998)]. Gal4 DBD-peptide fusions were created as follows: Peptide-coding sequences were excised from mBAX vector with Xho I-Xba I and subcloned into pM vector (Clontech, Palo Alto, CA) with a linker sequence to generate Sal I and Xba I sites for cloning. ERα-VP16 was generated by polymerase chain reaction (PCR) of human ERα-cDNA containing Eco RI sites flanking both 5′ and 3′ termini. The resultant PCR product was then subdoned into pVP15 (Clontech). All PCR products were sequenced to ensure the fidelity of the resultant construct. 17β-estradiol, 4-hydroxy-tamoxifen, and nafoxidine were purchased from Sigma.
13. J. D. Norris and D. P. McDonnell, unpublished data.
14. J. D. Norris, D. Fan, B. L. Wagner, D. P. McDonnell, Mol. Endocrinol. 10, 1605 (1996).
15. C. Chang and D. P. McDonnell, unpublished data.
16. P. Webb, G. N. Lopez, R. M. Uht, P. J. Kushner, Mol. Endocrinol. 9, 443 (1995).
17. ERβ-VP16 was generated by PCR of ERβ cDNA, and the resultant product was cloned into pVP16. Dexamethasone and progesterone were purchased from Sigma.
18. ERα expression vector pRST7-hER is reported elsewhere [S. L. Dana, P. A. Hoener, D. L. Wheeler, C. L. Lawrence, D. P. McDonnell, Mol. Endocrinol. 8, 1193 (1994)].
19. Supported by grants to D.P.M. from the NIH (DK48807) and to M.R.H. from the Department of Defense (DAMD17-98-1-8072). ICI 182,780 was a gift from A. Wakeling (Zeneca Pharmaceuticals, Macclesfield, UK), raloxifene was a gift from E. Larson (Pfizer Pharmaceuticals, Groton, CT), idoxifene was a gift from M. Gowan (SmithKline Beecham Pharmaceuticals, King of Prussia, PA), GW 7604 was a gift from T. Willson (Glaxo Wellcome Research and Development Research Triangle Park, NC), PRB-VP16 and GR-VP16 were gifts from D. X. Wen and J. Miner (Ligand Pharmaceuticals, San Diego, CA), and RU 486 and ZK 98299 were gifts from Ugand Pharmaceuticals and Schering-AG Pharmaceuticals (Berlin, Germany), respectively.