Compound (SMILES): N#Cc1ccc(N2CCc3c(ncnc3Nc3ccc(C(=O)NCC4CCN(c5ccc6c(c5)C(=O)N(C5CCC(=O)NC5=O)C6=O)CC4)c(F)n3)C2)cc1Cl
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 (Homo sapiens)
Cell line description: Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC
A*01
Instable (MSI).
Metastatic; Left supraclavicular lymph node; 
Caucasian.
Space-flown cell line (cellonaut).
Cancer cell line
Assay: ELISA
DC50: > 10000 nM
Assay description: Cell Degradation
Cell treatment
LNCaP Cells are collected by TrypLE and washed once and resuspended in assay medium (RPMI1640 +5%CSS) , 5×10 4/well/50μl, seed in 96-well plate , starve for 48h.
LNCaP cells are treated with compounds diluted in 0.2%DMSO and 0.1nM DHT, dilution is done according to the following protocol: (1) make 500×compounds solution in DMSO from 5mM by 5-fold dilution, total 7 doses were included; (2) Make assay medium containing 0.2nM DHT (2×) (3) make 2× compounds solution in assay medium by transferring 0.5μl 500×solution into 125μl assay medium containing 0.2nM DHT; (4) 50μl of 2×solution is added to cells and incubate for 6h, the final compound conc. is 10000, 2000, 400, 80, 16, 3.2 and 0.64nM, respectively. (5) For high control: Transfer 50μl medium to cells containing 0.4%DMSO and 0.2nM DHT. (6) For Low control: Transfer 10μl lysis buffer instead of cell lysate to plate wells when performing ELISA assay
ELISA assay
Cells in 96 well plate are discarded supernatant and wash once with ice-cold PBS. Then, 100μl 1×cell lysis buffer is added in each well. The cells are placed on a shaker for 20min at 4°C; 10ul lysate is transferred to 90μl sample diluent in ELISA plate, seal the plate incubate overnight at 4°C; sample is discarded from the plate, tap the plated on a paper towel, wash 3×200μl ELISA wash buffer, add 100μl/well 1 st capture antibody, incubate at 37°C, 1h; repeat wash with 3×200μl ELISA wash buffer, add 100μl/well 2 nd HRP-antibody, incubate at 37°C, 30min; repeat wash with 3×200μl ELISA wash buffer, add 100μl of TMB to each well and incubate the plate in the dark for 15min at room temperature or at 37°C, 10 min; add 100μl of STOP solution to each well; read absorbance at 450nm. The AR ELISA assay was performed by using PathScan Total Androgen Receptor Sandwich ELISA Kit.
Reference: DEGRADATION OF ANDROGEN RECEPTOR (AR) BY CONJUGATION OF AR ANTAGONISTS WITH E3 LIGASE LIGAND AND METHODS OF USE (patent)
Source database: TPDdb