Compound (SMILES): Cn1c(=O)n(C2CCC(=O)NC2=O)c2ccc3c(c21)OCC31CCN(C2CC3(C2)CN(c2ccc([C@@H]4c5ccc(O)cc5CC[C@@H]4c4ccccc4)cc2)C3)CC1
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
Dmax:  25.5 %
Treatment concentration: 10 nM
Assay description: Effect of PROTAC compounds disclosed in the present invention on ERα degradation in MCF-7 cells MCF-7 (ATCC) cells were cultured in DMEM medium (11995-065, Gibco) containing 10% FBS at 37°C, 5% CO2 until they entered the logarithmic growth phase. MCF-7 cells were seeded at a density of 3×105 cells/well in 6-well plates and incubated at 37°C, 5% CO2 for 24 hours. After the cells attached, 1 μL of compound dimethyl sulfoxide solution (final concentration 0.15-1000 nM) was added and the cells were incubated in a 37°C, 5% CO2 incubator for another 4 hours. After 4 hours, the culture medium was discarded and each well was washed once with 1 mL of pre-chilled 1× DPBS. The cells were then lysed with 50 μL of RIPA lysis buffer and 1× protease inhibitor cocktail. After standing on ice for 20 minutes, the cells were centrifuged at 14,000 g at 4°C for 30 minutes. The supernatant was carefully collected and analyzed for ERα protein levels by immunoblotting.

Immunoblotting: The total protein concentration in the collected cell lysate supernatant was determined using the BCA protein concentration assay kit (enhanced). Based on the total protein concentration determined by BCA assay, the protein concentration was adjusted to 1 μg/μL using RIPA lysis buffer and 5× SDS-PAGE protein loading buffer. The protein was denatured in a 95°C water bath for 5 minutes. After denaturation, the condensed water on the centrifuge tube wall was collected by centrifugation and used as the loading sample. A 20 μL sample (20 μg total protein) was added to the sample well of a 12% precast gel (M00669, GenScript). Electrophoresis was performed at 100 V for 20 minutes, then switched to 150 V for 40 minutes. After electrophoresis, the proteins from the SDS-PAGE gel were transferred to a PVDF membrane and transferred at a constant voltage of 100 V for 60 minutes. After transfer, the PVDF membrane corresponding to the ERα protein and the internal control protein was cut out and incubated in 1× QμickBlock blocking buffer at room temperature on a shaker for 30 minutes. Following blocking, the ERα primary antibody (1:1000, 8644S, CST) was added and incubated on a shaker at 4°C overnight. After incubation, the PVDF membrane was washed three times with 1× TBST buffer (5 minutes each). Rabbit IgG (H+L) secondary antibody (1:7500, SA5-35571, Invitrogen) was added and incubated on a shaker for 1 hour at room temperature. The membrane was then washed again three times with 1× TBST buffer (5 minutes each). Finally, images were scanned using a dual-color infrared laser imaging system. Grayscale analysis of the protein profile was performed using Image Studio Lite analysis software. The grayscale correction value for each sample was calculated using the formula: grayscale correction value = grayscale value of the target protein / grayscale value of the corresponding internal reference. The grayscale correction values of the compound group were then compared with those of the control group to calculate the degradation rate of the compound.
Reference: CEREBELLAR PROTEIN E3 UBIQUITIN LIGASE INHIBITOR AND CHIMERIC COMPOUND TARGETING PROTEIN DEGRADATION (patent)
Source database: TPDdb