Compound (SMILES): Cn1c2c(c3ccc(N4CCN(CC5CCN(c6ccc(-c7ccc8c(c7)C(C)(C)C(=O)N8c7ccc(C#N)c(Cl)c7)nn6)CC5)CC4)cc31)C(=O)N(C1CCC(=O)NC1=O)C2=O
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
DC50: > 1000 nM
Assay description: Androgen Receptor Degradation Assay Using Western Blot Analysis
LNCaP. FGC cells grown in RPMI 1640 medium supplemented with 10%FBS, 10mM HEPES and 1mM sodium pyruvate were seeded at 2x105 cells per well in 24-well tissue culture plates. Cells were incubated at 37°C, 5%CO2 for 24 hours (hr) , then treated with 100 nanomolar concentrations (nM) any of the compounds 1 to 148 and ARV-110 for 24hr. After the treatment, the cells were harvested, washed by PBS, and lysed with RIPA lysis and extraction buffer supplemented with Halt Protease Inhibitor Cocktail to collect protein samples. Cells not treated with any of the above-mentioned compounds are used as a negative control.
The protein samples were separated by polyacrylamide gel electrophoresis, and then transferred to a piece of Immuno-Blot PVDF membrane . The presence of androgen receptor in the protein samples was detected by standard Western blotting procedure using an anti-AR antibody (1: 2000 dilution) and a goat anti-rabbit HRP-conjugated secondary antibody (1: 5000 dilution) . The internal loading control GAPDH was detected using a mouse monoclonal antibody (1: 5000) and a goat anti-mouse HRP-conjugated secondary antibody (1: 5000 dilution). Chemiluminescence signals were developed using Clarity Western ECL substrate and detected with digital imager iBright FL1500 .
Compounds 1-16 were selected and serial diluted (10x) with RPMI medium for treating LNCaP. FGC cells. A calibration curve was determined by the serial diluted samples for each compound, and the concentration needed for 50%AR degradation (AR DC50) for each compound was calculated. The results are shown in Table 2. "Cpd. ID" indicates the compound ID number in Table 1.
Reference: PROTEIN DEGRADATION COMPOUNDS AND METHODS OF USE (patent)
Source database: TPDdb