Compound (SMILES): Cn1c(=O)n(C2CCC(=O)NC2=O)c2cccc(C#CCN3CCN(c4ccc(C(=C5CC(C)(C)OC(C)(C)C5)c5ccc6[nH]nc(F)c6c5)cn4)CC3)c21
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): ESR1 (UniProt P03372)
POI sequence: MTMTLHTKASGMALLHQIQGNELEPLNRPQLKIPLERPLGEVYLDSSKPAVYNYPEGAAYEFNAAAAANAQVYGQTGLPYGPGSEAAAFGSNGLGGFPPLNSVSPSPLMLLHPPPQLSPFLQPHGQQVPYYLENEPSGYTVREAGPPAFYRPNSDNRRQGGRERLASTNDKGSMAMESAKETRYCAVCNDYASGYHYGVWSCEGCKAFFKRSIQGHNDYMCPATNQCTIDKNRRKSCQACRLRKCYEVGMMKGGIRKDRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: MCF-7 (Homo sapiens)
Cell line description: African=0.74%; Native American=0%; East Asian, North=4.2%; East Asian, South=0%; South Asian=0%; European, North=56.91%; European, South=38.15%.
Helen Marion Mallon (sister Catherine Frances), the patient from which this cell line is derived was a nun at the Immaculate Heart of Mary convent in Monroe, Michigan (CelloPub=CLPUB00336).
Gene deletion; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC
A*02 A*02 A*02
Stable (MSS).
Metastatic; Pleural effusion; 
Caucasian.
Space-flown cell line (cellonaut).
Cancer cell line
Assay: Western Blot
DC50: > 100 nM
Assay description: Degradation activity of the disclosed compounds against ERα in MCF7 cells
ERα-positive human breast cancer MCF-7 cells were cultured in 25 cm2 or 75 cm2 plastic tissue culture flasks using EMEM medium supplemented with 10% fetal bovine serum and 10 μg/mL bovine insulin at 37°C in 95% air and 5% CO?. The cells were subcultured two to three times per week.
Cells were seeded at a density of 1 × 10? cells/well in 6-well cell culture plates, using 1.8 mL/well, and cultured at 37°C in 95% air and 5% CO?. After 24 hours, test compounds were added: starting with 10 mM (dissolved in DMSO), the compounds were serially diluted 10-fold with DMSO to prepare gradient stock solutions. Complete culture medium containing 1% DMSO was prepared for further dilutions, temporarily referred to as "dilution buffer." Working solutions were prepared by diluting each stock solution with 99 volumes of dilution buffer: 1 μL of the 10 mM stock solution was added to 99 μL of dilution buffer, mixed thoroughly, and finally 10 μL of the working solution was added to the culture plate seeded with cells. The final DMSO concentration in the cell culture medium was 0.2%, and the final concentration of the test compound ranged from 1 nM to 100 nM. The cells were incubated at 37°C for 6 hours. The culture medium was aspirated, and the cells were washed twice with pre-chilled PBS. 1 mL of PBS was added to each well, and the cells were harvested into an EP tube using a cell scraper. The cells were centrifuged at 4000 rpm for 5 minutes at 4°C, and the PBS was aspirated. Total protein was extracted using Minute? Animal Cell/Tissue Total Protein Extraction Reagent. Protein concentration was determined by the BCA assay. 30 μg of protein was mixed with 5× SDS-PAGE loading buffer, boiled for 10 minutes, and then added to the wells of a precast gel comb. The sample was run at a constant voltage of 80 V for 30 minutes. After the sample entered the separating gel, the sample was run at 120 V. A Bio-Rad Transblot "sandwich" was assembled: sponge-filter paper-gel-membrane-filter paper-sponge. Transfer was performed at 4°C at 0.3 amp for 1 hour. Blocking was performed with 5% nonfat dry milk at room temperature for 1 hour. Estrogen Receptor alpha Antibody was then added with gentle shaking at 4°C overnight. Nonspecifically bound antibodies were removed from the membrane using TBST. A goat anti-mouse IgG-HRP secondary antibody was added and incubated at room temperature for 1 hour. ECL staining was performed. Protein degradation levels were calculated using grayscale.
Reference: PROTAC COMPOUND TARGETING ESTROGEN RECEPTOR, PREPARATION METHOD THEREFOR, AND USE THEREOF (patent)
Source database: TPDdb