Compound (SMILES): O=C1CCC(N2CC(Oc3cccc(C4CCN(CCCOc5ccc(C6c7ccc(O)cc7CCC6c6ccccc6)cc5)CC4)c3)=CC2=O)C(=O)N1
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: In-Cell Western/Western Blot
DC50: > 100 nM
Assay description: 2. Estrogen Receptor-alpha (ERa) degradation assay in MCF-7 cells using western blot method for Table 5.

The exemplary novel ERa degraders were assessed for their activity in degrading ERa in MCF-7 cells via western blot. The assay was carried out in the presence of 10 percent FBS or high percentage of human or mouse serum. Protocols of the western blot assay are described below.

MCF7 cells were grown in DME/F12 with 10 percent FBS and seeded at 24,000 cells per well in 100 microliters into 96-well clear tissue culture plates. The following day, the cells were treated with PROTACs in a 7-point concentration curve with 100 nM being the top concentration and serial dilutions to make the other concentrations (30 nM, 10 nM, 3 nM, 1 nM, and 0.3 nM). At all concentrations, 0.01 percent DMSO is the final concentration in the well. The following day, the plates are aspirated, washed with 50 microliters of cold PBS. The cells are lysed with 50 microliters per well 4 degrees Celsius Cell Lysis Buffer (20 mM Tris-HCL (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1 percent Triton, 2.5 mM sodium pyrophosphate, 1 mM B-glycerophosphate, 1 mM sodium vanadate, 1 ug/ml leupeptin). Lysates were clarified at 16,000 x g for 10 minutes, and 2 ug of protein was subjected to SDS-PAGE analysis and followed by immunoblotting according to standard protocols. The antibodies used were ERa and Tubulin. Detection reagents were Clarity Western ECL substrate.

Alternatively, MCF7 cells were grown in DMEM/F12 with 10 percent FBS and seeded at 24,000 cells per well in 500 microliters in 24-well clear tissue culture plates. The following day, the cells were treated with PROTACs in a 5-point concentration curve (100 nM, 33 nM, 11 nM, 3.7 nM, and 1.2 nM) in the presence of 0.01 percent DMSO. After 72 hours, the wells are aspirated and washed with 500 microliters of PBS. The cells are lysed with 100 microliters per well 4 degrees Celsius Cell Lysis Buffer (20 mM Tris-HCL (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1 percent Triton, 2.5 mM sodium pyrophosphate, 1 mM B-glycerophosphate, 1 mM sodium vanadate, 1 ug/ml leupeptin). Lysates were clarified at 16,000 x g for 10 minutes, and 2 ug of protein was subjected to SDS-PAGE analysis and followed by immunoblotting according to standard protocols. The antibodies used were ERa and Tubulin. Detection reagents were Clarity Western ECL substrate.

3. Estrogen receptor-alpha (ERa) degradation assay using In-Cell Western Assay for Table 5.

Degradation of ERa by claimed compounds were determined in MCF7 cells using an In-Cell Western assay. Briefly, MCF7 cells were plated in 96-well plates (2000 cells per well in 100 microliters media) and incubated at 37 degrees Celsius under an atmosphere of 5 percent CO2 in a humidified incubator overnight. One-hundred microliters of media containing test compound (at 2x concentration) was added to the appropriate wells to provide 11 serially decreasing concentrations (top final concentration, 1 microM then 3-fold less for the next 10 concentrations); a vehicle control (DMSO) was also added for each compound. For each experiment, all compounds were assayed in duplicate plates. Cells were then incubated for 3 or 5 days in the above-mentioned environment. The assay was terminated by removal of media, a single wash with ice-cold PBS and the addition of 50 microliters paraformaldehyde (4 percent in PBS). After 15 minutes in PFA at room temperature, the cells were permeabilized in Tris-phosphate-buffered saline with Tween (0.1 percent) supplemented with Triton X-100 (0.5 percent) for 15 minutes. Cells were then blocked in BSA (3 percent) for one hour. Primary antibodies for the detection of ERa (rabbit monoclonal, 1:1000) and tubulin (mouse monoclonal, 1:5000) in TBST with BSA (3 percent) were added. The cells were incubated overnight at 4 degrees Celsius. The cells were then washed thrice with TBST at room temperature and then incubated with anti-rabbit and anti-mouse fluorescently-labelled secondary antibodies in blocking buffer for one hour at room temperature. Following 3 washes with TBST, the buffer was removed and the plates were read on an Odyssey infrared imaging system at 700 nm and 800 nm. Using commercial software, the staining intensity for ERa and tubulin in each well was quantified and exported for analysis. For each data point, ERa intensity was normalized to tubulin intensity and for each compound all normalized intensity values were normalized to the vehicle control. DC50 and Dmax values were determined following a 4-parameter IC50 curve fit using ACAS dose response module.

4. BRD4 Western Protocol

VCaP cells were purchased from ATCC and cultured in Dulbecco's Modified Eagle's Medium, supplemented with 10 percent FBS and Penicillin/Streptomycin. DMSO control and compound treatments (0.003 microM, 0.01 microM, 0.03 microM and 0.1 microM) were performed in 12-well plates for 16h. Cells were harvested, and lysed in RIPA buffer (50 mM Tris pH8, 150 mM NaCl, 1 percent Tx-100, 0.1 percent SDS, 0.5 percent sodium deoxycholate) supplemented with protease and phosphatase inhibitors. Lysates were clarified at 16,000g for 10 minutes, and protein concentration was determined. Equal amount of protein (20 ug) was subjected to SDS-PAGE analysis and followed by immunoblotting according to standard protocols. The antibodies used were BRD4 and Actin. Detection reagents were Clarity Western ECL substrate.
Reference: Cereblon ligands and bifunctional compounds comprising the same (patent)
Source database: TPDdb