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: LNCaP clone FGC (Homo sapiens)
Cell line description: African=2.89%; Native American=0.31%; East Asian, North=0%; East Asian, South=2.4%; South Asian=8.61%; European, North=28.39%; European, South=57.41%.
Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC
Instable (MSI-high).
CVCL_0395 ! LNCaP
Metastatic; Left supraclavicular lymph node; 
Caucasian.
Cancer cell line
Assay: Western Blot
Dmax:  23.5 %
Treatment concentration: 100 nM
Assay description: The present invention discloses the degradation activity of compounds against AR protein in LNCaP cells. AR-positive human prostate cancer cells (LNCaP FGC) were seeded into six-well microplates in RPMI 1640 containing 10% FBS to a concentration of 1.5×10?/well. After the cells were cultured overnight in a carbon dioxide incubator (ESCO), various concentrations of the present compounds and a positive control, ARV-766, were added at a volume of 1 μL/well to achieve final concentrations of 100 nM and 10 nM, respectively. A corresponding vehicle control was also established. The cells were incubated for 8 hours. After 8 hours of culture, the culture medium was removed, the cells were washed with PBS, and RIPA lysis buffer containing 1% Protease Inhibitor Cocktail was added. After lysis and centrifugation, a total protein extract was obtained. The protein concentration in the extract was determined by the BCA assay. Protein electrophoresis was performed using SDS-PAGE, followed by 60 min of constant voltage electrophoresis at 100 V for protein transfer to a PVDF membrane. The PVDF membrane was blocked in a 5% milk powder/1×TBST solution for 1 h at room temperature. The primary antibody solution (Anti-Androgen receptor, Cell Signaling Technology catalog number: 5153S; 1:2000 dilution) was prepared and incubated overnight at 4°C. The primary antibody solution was discarded, and the PVDF membrane was washed three times with 1×TBST for 5 min each time. The secondary antibody solution was prepared and incubated at room temperature for 1 h. The secondary antibody solution was discarded, and the PVDF membrane was washed three times with 1×TBST for 5 min each time. The Western membrane was imaged using a dual-color infrared laser imaging system. The grayscale analysis of the bands was performed using Image Studio software. The GAPDH protein band was also detected for each sample as an internal reference. The AR protein degradation rate of the compound and the positive control was calculated 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