Compound (SMILES): CC1(C)C(=O)N(c2ccc(C#N)c(Cl)c2)c2ccc(-c3ccc(N4CC(CN5CCN(c6ccc7c(c6)CN(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: 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: In-Cell Western
DC50:  108.37 nM
Assay description: Androgen Receptor In-Cell-Western Assay
This assay evaluates compound performance in Lncap cells. Intracellular androgen receptor expression was measured by in-cell-Western assay according to the following assay procedure.
Lncap cells were seeded at 30,000 cells/well in a poly-D-lysin-pretreated 96-well plate in Lncap cell assay medium (DMEM with phenol red and fetal bovine serum (FBS)) at a volume of 100 μL/well. 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 1:4 in DMSO (10 μL stock + 40 μL DMSO) = 2000 μM and place 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 plate in culture medium. (ii) Transfer 3 μL of 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 Vcap cells with fresh 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 stock solution). (iii) Culture the cells for 24 hours.
2. Detect the expression level of 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 at 4°C overnight. (v) Discard the primary antibody solution and wash the cell plate with 1× wash buffer. (vi) Prepare secondary antibody solution (1:5000 dilution) and 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 color development solution and add 100 μL of color development 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) Normalize the analysis based on the number of cells in each well. Discard the solution in the cell plate and wash three times with wash buffer. (ii) Prepare Janus dilution (1:3 dilution). (iii) Add 50 μL of the dilution to each well and incubate. (iv) Discard the solution in the plate and wash with deionized water. (v) Prepare 1 M hydrochloric acid (concentrated hydrochloric acid diluted 1:24) and add 200 μL of the hydrochloric acid dilution to each well to treat the cells. (vi) Read the absorbance at OD 595 nm using a Flex Station. (vii) Based on the obtained reading, calculate the effect of the test compound on androgen receptor expression. The experimental results are shown in Table 1.
Reference: PROTEIN DEGRADATION AGENT COMPOUND PREPARATION METHOD AND APPLICATION (patent)
Source database: TPDdb