Compound (SMILES): N#Cc1ccc(O[C@H]2CC[C@H](NC(=O)c3ccc(N4CCC(CN5CCN(c6ccc(C(=O)NC7CCC(=O)NC7=O)cc6)CC5)CC4)nn3)CC2)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: 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: AR ELISA
DC50: < 1 nM
Assay description: 5. AR ELISA Assay Protocol
Compounds were evaluated in this assay in LNCaP and/or VCaP cells utilizing similar protocols. The protocols used with VCaP cells are described below. The androgen receptor ELISA assay was performed using PathScan AR Sandwich ELISA (Cell Signaling Catalog 12850) according to the following assay steps:

VCaP cells were seeded at 40,000 cells/well at a volume of 100 L well in VCaP assay medium [Phenol red free RPMI (Gibco Cat 11835-030); 5% Charcoal Stripped (Dextran treated) FBS (Omega Scientific, Cat FB-04); 1% penstrep (Life Technologies, Gibco Cat 10378-016)] in Corning 3904 plates. The cells were incubated for a minimum of 3 days. Cells were dosed with PROTACs diluted in 0.01% DMSO and the drug treatment was allowed for 5 hours.

AR ELISA (Cell Signaling) was performed as follows, lx Cell Signaling Cell lysis buffer was made (Catalogue 9803; comes with the kit). Media from the treated wells is aspirated, and 100 μl lx cell lysis buffer/well is added. The cells were placed on a shaker for 10 minutes at 4°C. Twenty microliters of lysate was transferred to 100 μl of Diluent in ELISA plate (0.15μg/ml - 0.075 μg/ml). The lysate-diluent mixture was shaken for 30 minutes at 37°C. Allow mouse AR antibody, anti-mouse antibody, TMB, and STOP solution to come to room temperature. The lx ELISA buffer included in kit was made and loaded in the reservoir. Media from the plates was discarded, the ELISA plate tapped hard on paper towel, and washed 4x 200 μl ELISA wash buffer using a plate washer.

One-hundred (100) μl/well of mouse AR detection Ab was added; the plates were covered and shaken at 37°C for 1 hour; media was discarded from the plates, the plates were tapped on a paper towel, washed 4x with 200 μl ELISA wash buffer with a plate washer.

One-hundred (100) μl/well of anti-mouse - HRP conjugated Ab (comes with the kit) was added; the plates were covered and shaken at 37°C for 30 minutes; the TMB reagent was allowed to come to room temperature; the media was discard from the plate, the plates were tapped on paper towel, washed 4x with 200 μl of ELISA wash buffer; the plates were tapped the plates on paper towel. One-hundred (100) μl of TMB was added and the plates shaken for 2 minutes - while watching for color development. One-hundred (100) μl Stop solution was added when light blue color developed. Plates were shaken and read at 450 nM.

Progression of prostate cancer in patients treated with anti-androgen therapy usually involves one of several mechanisms of enhanced Androgen Receptor (AR) signaling, including increased intratumoral androgen synthesis, increased AR expression and AR mutations. PROTACs (PROteolysis TArgeting Chimera), which use bi-functional molecules that simultaneously bind a target of choice and an E3 ligase, cause ubiquitination via induced proximity and degradation of the targeted, pathological protein. As opposed to traditional target inhibition, which is a competitive process, degradation is a progressive process. As such, it is less susceptible to increases in endogenous ligand, target expression, or mutations in the target. Thus, this technology appears to be ideal for addressing the mechanisms of AR resistance in patients with prostate cancer. Data was analyzed and plotted using GraphPad Prism software.
Reference: Cereblon ligands and bifunctional compounds comprising the same (patent)
Source database: TPDdb