Compound (SMILES): COc1cc(O[C@H]2C(C)(C)[C@H](NC(=O)c3cnc(N4CCC(CN5CCc6c(ccc7c(C8CCC(=O)NC8=O)noc67)C5)CC4)nc3)C2(C)C)ccc1C#N
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: HTRF
DC50: < 5 nM
Assay description: Compounds' Effects on AR Degradation in Cells
1.1 Assay for AR Degradation in VCaP Cells
Detection was performed using the HTRF Human Androgen Receptor Detection Kit. 4× Supplemented Lysis Buffer was prepared using 100× Blocking Reagent Stock Solution and 4× Lysis Buffer Stock Solution. 1× Supplemented Lysis Buffer was prepared using 4× Supplemented Lysis Buffer and ddH2O. Premixed antibody solutions were prepared using Detection Buffer, Androgen Receptor Eu Cryptate Antibody, and Androgen Receptor d2 Antibody. Well-grown VCaP cells were washed with PBS, trypsinized, and terminated with phenol red-free DMEM supplemented with 1% CSS-FBS. The cells were harvested and transferred to a centrifuge tube. The cell density was adjusted to 5 × 105 cells/mL and seeded onto a 96-well plate (100 μL/well). The cells were cultured overnight in a cell culture incubator. Compounds were then added using a nanoliter pipette, with a five-fold serial dilution to a final concentration of 100 nM to 0.032 nM in duplicate. A control was also included. After 24 hours of incubation in the cell culture incubator, the medium was aspirated and cells were lysed with 40 μL/well of 1× Supplemented Lysis Buffer. After shaking at room temperature for 40 minutes, 16 μL of the cell lysate was transferred to a 384-well plate. 4 μL/well of the premixed antibody solutions were added and the plates were incubated at 25°C for 2 hours. Fluorescence was measured at 665 nm/620 nm using an Envision microplate reader. Four-parameter analysis was performed, and dose-response curves were fitted to calculate DC50 and Dmax. The results are shown in Table 1.
Reference: CYCLOBUTYL-CONTAINING COMPOUNDS (patent)
Source database: TPDdb