Compound (SMILES): CC1(C)C(=O)N(c2ccc(C#N)c(Cl)c2)c2ccc(-c3ccc(N4CC(CN5CCN(c6ccc7c(c6)C(=O)N(C6CCC(=O)NC6=O)C7=O)CC5)C4)cc3)cc21
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: In-Cell Western
Dmax:  68 %
Assay description: Androgen Receptor In-Cell-Western Assay
This assay evaluates compound performance in VCap cells. Intracellular androgen receptor (AR) expression was assessed by in-cell-Western assay according to the following assay protocol.
Vcap cells were seeded at 50,000 cells/well in Vcap cell assay medium (DMEM with phenol red and fetal bovine serum (FBS)) in a poly-D-lysin-pretreated 96-well plate. The cells were cultured for at least two days.
1. Cells were first treated with the compound. Compounds were serially diluted in DMSO and cell culture medium to a 0.5% DMSO content in the cell culture plates using polypropylene plates according to the following protocol:
(1)(i) Prepare a 200× stock plate in DMSO; (ii) Dilute the 10 mM stock solution 1:4 in DMSO (10 μL stock solution + 40 μL DMSO) = 2000 μM and place it in row 2; (iii) Perform a 1:4 serial dilution from row 2 to row 9 (10 μL protac + 40 μL DMSO), reserving row 1 for the 2000 μM reference compound and row 10 for DMSO. (iv) A total of 8 concentrations (final concentrations on the 200× plate were 2000 μM, 400 μM, 80 μM, etc.). (2) (i) Prepare a 3× stock solution in culture medium; (ii) Transfer 3 μL of the 200× stock solution to 197 μL of culture medium (using a 12-channel pipette, from row 1 to row 10), i.e., a 3× stock solution plate. (iii) Mix the stock solution plate evenly. (3) (i) Replace the culture medium of the Vcap cells with fresh culture medium, 100 μL of culture medium. (ii) Transfer the mixed 3× stock solution to the cell culture plate (using a 12-channel pipette, from row 1 to row 10, transfer 50 μL of the stock solution). (iii) Culture the cells for 24 hours.
2. Detect the expression level of the androgen receptor in cells after compound treatment, and measure it according to the following method.
(1) (i) Add an equal volume of 8% paraformaldehyde to the cell culture plate to fix the cells. Discard the fixative in the cell plate and wash three times with PBS. (ii) Prepare Triton solution (dilute the stock solution 1:1000). Discard the solution in the cell plate and add 200 μL of Triton dilution to each well. (iii) Prepare 2× blocking solution (10× blocking stock solution diluted 1:4). Discard the solution in the cell plate and add 100 μL of 2× blocking solution to each well. (iv) Prepare primary antibody solution (1:1000 dilution). Discard the solution in the cell plate and add 100 μL of primary antibody dilution to each well and incubate overnight at 4°C. (v) Discard the primary antibody solution and wash the cell plate with 1× wash buffer. (vi) Prepare secondary antibody solution (Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP, Thermo Catalog No. 31460; 1:5000 dilution). Add 100 μL of secondary antibody dilution to each well and incubate. (vii) Discard the secondary antibody solution in the cell plate and wash the cell plate with 1× wash buffer. (viii) Prepare TMB colorimetric solution and add 100 μL of colorimetric solution to each well. (ix) Add 50 μL of stop solution to each well. (x) Read the absorbance at OD 450nm and 570nm using EnVision. (2) (i) Standardize the number of cells in each well. Discard the solution in the cell plate and wash the plate three times with wash buffer. (ii) Prepare Janus dilution solution (1:3 dilution). (iii) Add 50 μL of dilution solution to each well and incubate. (iv) Discard the solution in the plate and wash with deionized water. (v) Prepare 1M hydrochloric acid (concentrated hydrochloric acid diluted 1:24) and add 200 μL of hydrochloric acid dilution solution to each well to treat the cells. (vi) Read the absorbance at OD 595nm using a Flex Station. (vii) Based on the obtained readings, calculate the effect of the test compound on androgen receptor expression. The experimental results are shown in Table 3.
Reference: PROTEIN DEGRADATION AGENT COMPOUND PREPARATION METHOD AND APPLICATION (patent)
Source database: TPDdb