(A) ERα transcriptional reporter gene assay in cells treated with BZA, FULV, and 4-OHT. (B) Relative cyclin D1 mRNA levels of in MCF-7 cells treated with E2 plus 4-OHT, FULV, or BZA vehicle and …
(A) Cell growth in MCF-7 cells with DOX-induced Y537S ERα expression. (B) Inhibition of ERα target genes in DOX-induced Y537S ERα expressed MCF-7 cells with increasing doses of BZA. (C) …
(A) Replicate immunoblot to that shown in Figure 1 probing for HA-ERα and actin in MCF-7 cells upon treatment. (B) Counts from immunoblot A normalized to actin. (C) Third replicate immunoblot to …
Numbers along the top indicate concentrations of BZA in nM.
(A) Cell growth inhibit with MCF7 breast cancer cells. (B) ZR75 breast cancer cells. (C) T47D breast cancer cells. (D) LTED breast cancer cells. (E) Non-induced MCF7 breast cancer cells with a …
(A) EC50 quartiles for cells treated with 4-OHT (red), BZA (blue), or FULV (green). (B) In vitro quantification of the effect of ligands on promoting (E2) or inhibiting (4-OHT, RAL, BZA) the binding …
(A) Overlay of BZA (cyan) with RAL (grey) X-ray crystal structures showing differences in H11-12 loop and H12 orientation. (B) Hydrogen bonds (dashed lines) formed by BZA and RAL in the binding …
Deuterium uptake for each peptide is calculated as the average of % D for the 6 time points (10 s, 30 s, 60 s, 300 s, 900 s and 3600 s), and the difference in average % D values between the Apo-ERα …
Deuterium uptake for each peptide is calculated as the average of % D for the 6 time points (10 s, 30 s, 60 s, 300 s, 900 s and 3600 s) and the difference in average % D values between the Apo-ERα …
Deuterium uptake for each peptide is calculated as the average of % D for the 6 time points (10 s, 30 s, 60 s, 300 s, 900 s and 3600 s) and the difference in average % D values between the Apo-ERα …
Deuterium uptake for each peptide is calculated as the average of % D for the 6 time points (10 s, 30 s, 60 s, 300 s, 900 s and 3600 s) and the difference in average % D values between the Apo-ERα …
(B) Overlaid representative calculated structure of Y537S-BZA (cyan) closest to the last 50-ns average from molecular dynamics (MD) simulation and the WT-BZA structure, vertical lines represent the …
Antiestrogen | Class | Approved clinical indications |
---|---|---|
![]() 4-Hydroxytamoxifen (4-OHT) | SERM | • Adjuvant treatment for ER + breast cancers (Early Breast Cancer Trialists' Collaborative Group., 1998). • Metastatic Breast Cancer (Lipton, 1982). • Ductal Carcinoma in Situ (Allred et al., 2012). • Reduction in Breast Cancer Incidence in High Risk Women (Visvanathan, 2009). |
![]() Raloxifene (RAL) | SERM | • Osteoporosis in postmenopausal women (Messalli and Scaffa, 2010). • Reduction in Breast Cancer Incidence in High Risk Women(Cauley et al., 2001). |
![]() Fulvestrant (FULV) | SERD | • First-line therapy for metastatic breast cancer (Howell et al., 2004). • Postmenopausal women with progressive breast cancer following other antiestrogen therapy (Osborne et al., 2002; Howell et al., 2002). |
![]() Bazedoxifene (BZA) | SERM/SERD | • In combination with conjugated equine estrogens (DUAVEE) to prevent postmenopausal osteoporosis (Tikoo and Gupta, 2015). |
Ligand/Mutant | Kd (nM)* | ||
---|---|---|---|
WT | Y537S | D538G | |
E2‡ | 0.22 ± 0.11 | 1.40 ± 0.54 | 1.77 ± 0.66 |
Ki (nM)† | |||
4-OHT‡ | 0.12 ± 0.003 | 2.64 ± 0.4 | 3.28 ± 0.7 |
RAL | 0.30 ± 0.05 | 3.59 ± 1.0 | 3.77 ± 1.0 |
BZA | 0.37 ± 0.01 | 3.50 ± 0.6 | 5.53 ± 0.7 |
Fulvestrant‡ | 0.13 ± 0.03 | 3.68 ± 0.8 | 5.06 ± 1.2 |
* Measured directly by Scatchard Analysis using [3H]E2.
† Calculated using the Cheng-Prusoff equation from the IC50 values determined in a competitive radiometric binding analysis using [3H]E2 as a tracer.
‡Indicates previously published data (Fanning et al., 2016; Zhao et al., 2017).
Reagent type (species) or resource | Designation | Source or reference | Identifier | RRID |
---|---|---|---|---|
Recombinant DNA reagent | Estrogen Receptor Alpha Ligand Binding Domain WT or Y537S | Fanning SW, Mayne CG, Dharmarajan V, Carlson KE, Martin TA, Novick SJ, Toy W, Green B, Panchamukhi S, Katzenellenbogen BS, Tajkhorshid E, Griffin PR, Shen Y, Chandarlapaty S, Katzenellenbogen JA, Greene GL. Estrogen receptor alpha somatic mutations Y537S and D538G confer breast cancer endocrine resistance by stabilizing the activating function-2 binding conformation. eLife. 2016;5:e12792. doi: 10.7554/eLife.12792. | ||
Strain, strain background (Protein Expression) | E.coli BL21(DE3) | https://www.neb.com/products/c2527-bl21de3-competent-e-coli#Product%20Information | ||
Cell line (Mammary Cells) | MCF7, MCF7 HA-Y537S, MCF7 HA-D538G, T47D, CAMA-1, ZR75-1, MDA-MB-361 | ATCC; this paper; this paper; ATCC; ATCC; ATCC; ATCC | ATCC HTB-22, ATCC HTB-23, ATCC HTB-21, ATCC CRL-1500, ATCC HTB-27 | CVCL_0031, CVCL_0553, CVCL_1115, CVCL_0588, CVCL_0620 |
Antibody | anti-ERα antibody F10 | Santa Cruz Biotechnology | sc-8002 | AB_627558 |
Antibody | anti-actin antibody | Santa Cruz Biotechnology | sc-69879 | AB_1119529 |
Antibody | anti-HA antibody | Cell Signaling | C29F4 | AB_1549585 |
Software | CCP4i | https://www.ccp4.ac.uk/ccp4i_main.php | SCR_007255 | |
Software | VMD | https://www.ks.uiuc.edu/Research/vmd/ | SCR_004905 | |
Software | Gaussion G09 | http://gaussian.com/ | SCR_014897 | |
Software | GaussView | http://gaussian.com/ | SCR_014897 | |
Software | HDX Workbench | http://hdxworkbench.com/ | ||
Software | Bionavigator | PamGene | ||
Commercial assay or kit | PamStation96 | PamGene | ||
Commercial assay or kit | MycoAlert Mycoplasma Detection Kit | Lonza | LT07-518 | |
Chemical compound, drug | protease inhibitor cocktail solution III | CalBiochem | 539134 | |
Chemical compound, drug | 4-hydroxytamoxifen | Tocris | 3412 | |
Chemical compound, drug | ICI 182–780 (fulvestrant) | Tocris | 1047/1 | |
Chemical compound, drug | Raloxifene | Sigma- Aldrich | R1402 | |
Chemical compound, drug | Estradiol | Sigma- Aldrich | E8875 |
ERα LBD-BZA | |
---|---|
Data Collection | |
Space Group | P1 |
a, b, c (Å) | 53.57, 59.17, 94.14 |
α, β, γ (°) | 86.76, 75.36, 63.03 |
Resolution Range | 50–2.49 |
Number of Reflections | |
(all/unique) | 63,978/29,080 |
I/σ (highest resolution) | 1.35 |
Rmerge | 8.0 |
Completeness (%) | 97.6 |
Redundancy | 2.2 |
Refinement | |
Rwork/Rfree | 21.1/29.3 |
No. Residues/Chain | |
ERα LBD D538G | 241 |
GRIP Peptide | 0 |
Water | 5 |
Ligand | 1 |
RMSD | |
Bond lengths (Å) | 0.010 |
Bond angles (°) | 1.575 |
Chiral volume | 0.1016 |
Ramachandran plot statistics | |
Preferred number (%) | 837 (97.44%) |
Additional allowed (%) | 18 (2.10%) |
Outliers (%) | 4 (0.47%) |