Compound (SMILES): Cn1c(=O)n(C2CCC(=O)NC2=O)c2cc3c(cc21)C1(CCN(C2CCN(c4ccc(C(=O)N[C@H]5CC[C@H](Oc6ccc(C#N)c(Cl)c6)CC5)nn4)CC2)CC1)CO3
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): AR (UniProt P10275)
POI sequence: MEVQLGLGRVYPRPPSKTYRGAFQNLFQSVREVIQNPGPRHPEAASAAPPGASLLLLQQQQQQQQQQQQQQQQQQQQQQQETSPRQQQQQQGEDGSPQAHRRGPTGYLVLDEEQQPSQPQSALECHPERGCVPEPGAAVAASKGLPQQLPAPPDEDDSAAPSTLSLLGPTFPGLSSCSADLKDILSEASTMQLLQQQQQEAVSEGSSSGRAREASGAPTSSKDNYLGGTSTISDNAKELCKAVSVSMGLGVEALEHLSPGEQLRGDCMYAPLLGVPPAVRPTPCAPLAECKGSLLDDSAGKSTEDTAEYSPFKGGYTKGLEGESLGCSGSAAAGSSGTLELPSTLSLYKSGALDEAAAYQSRDYYNFPLALAGPPPPPPPPHPHARIKLENPLDYGSAWAAAAAQCRYGDLASLHGAGAAGPGSGSPSAAASSSWHTLFTAEEGQLYGPCGGGGGGGGGGGGGGGGGGGGGGGEAGAVAPYGYTRPPQGLAGQESDFTAPDVWYPGGMVSRVPYPSPTCVKSEMGPWMDSYSGPYGDMRLETARDHVLPIDYYFPPQKTCLICGDEASGCHYGALTCGSCKVFFKRAAEGKQKYLCASRNDCTIDKFRRKNCPSCRLRKCYEAGMTLGARKLKKLGNLKLQEEGEASSTTSPTEETTQKLTVSHIEGYECQPIFLNVLEAIEPGVVCAGHDNNQPDSFAALLSSLNELGERQLVHVVKWAKALPGFRNLHVDDQMAVIQYSWMGLMVFAMGWRSFTNVNSRMLYFAPDLVFNEYRMHKSRMYSQCVRMRHLSQEFGWLQITPQEFLCMKALLLFSIIPVDGLKNQKFFDELRMNYIKELDRIIACKRKNPTSCSRRFYQLTKLLDSVQPIARELHQFTFDLLIKSHMVSVDFPEMMAEIISVQVPKILSGKVKPIYFHTQ
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: VCaP (Homo sapiens)
Cell line description: African=0%; Native American=0.05%; East Asian, North=1.4%; East Asian, South=0%; South Asian=0.98%; European, North=69.8%; European, South=27.77%.
Contains an integrated xenotropic MuLV-related virus (XMRV) Bxv-1.
Gene fusion; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC
Established from a xenograft produced by implantation of tumor cells in a SCID mouse.
Stable (MSS).
Metastatic; Bone, vertebra; 
Caucasian.
Cancer cell line
Assay: Western Blot
Dmax:  82.5 %
Treatment concentration: 5 nM
Assay description: The present invention discloses the degradation activity of AR protein in VCAP cells. AR-positive human prostate cancer cells VCaP were seeded in DMEM containing 10% FBS to a density of 3×105/well in a six-well microplate. After being cultured overnight in a carbon dioxide incubator (ESCO), the prepared compound of the present invention and the positive control sample ARV-766 solution at different concentrations were added at a volume of 1 μL/well to make the final concentrations of the compound and the positive control sample reach 5nM and 0.5nM, respectively. At the same time, the corresponding solvent control was set and cultured for 8 hours. After 8 hours of culture, the culture medium was removed, and the cells were washed with PBS. RIPA lysis buffer containing 1% Protease Inhibitor Cocktail was added. After lysis and centrifugation, a total protein extract was obtained, and the protein concentration in the extract was determined by BCA assay. Protein electrophoresis was performed using SDS-PAGE, and then the protein was transferred to a PVDF membrane at a constant voltage of 100V for 60 minutes. The PVDF membrane was placed in a 5% milk powder 1×TBST solution and blocked at room temperature for 1 hour. Prepare the primary antibody solution (1:2000 dilution) and incubate at 4°C overnight. Discard the primary antibody solution and wash the PVDF membrane with 1×TBST for 5 minutes each time, three times. Prepare the secondary antibody solution (1:7500 dilution) and incubate at room temperature for 1 hour. Discard the secondary antibody solution and wash the PVDF membrane with 1×TBST for 5 minutes each time, three times. Use a dual-color infrared laser imaging system to image the Western membrane. Use Image Studio software to perform grayscale analysis of the bands. Each sample also detects the GAPDH protein band as an internal reference. Calculate the AR protein degradation rate of the compound and the positive control sample based on the grayscale of the protein band.
Reference: CEREBELLAR PROTEIN E3 UBIQUITIN LIGASE INHIBITOR AND CHIMERIC COMPOUND TARGETING PROTEIN DEGRADATION (patent)
Source database: TPDdb