Compound (SMILES): Cc1ccc([C@@H]2CCCN(c3cccc(OCCCCN4CCN(c5ccc6c(c5)CN(C5CCC(=O)NC5=O)C6=O)CC4)c3)C2)c2[nH]cc(C#N)c12
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: High-Content Analysis
DC50: > 3000 nM
Assay description: LNCaP Androgen Receptor Imaging Assay
This cell-based imaging assay uses immuno-fluorescence to measure endogenous nuclear AR staining in the prostate cancer cell line LNCaP (expressing AR-full length). The purpose of the assay is to identify compounds that regulate AR protein level according to the protocol laid out in the steps below. All work was performed in a sterile tissue culture hood up to the point of cell fixation. LNCaP cells were cultured at 6-8 ×10^6 per T175 flask and split every 3-4 days. Medium: RPMI (R7638, VWR Sigma) containing 10% FCS and 1% glutamine. Cells were plated in black wall transparent bottom plates at a density of 12,000 cells (CedexTM Cell counter, Beckmann & Coulter), 40μL per well, using a Multidrop (ThermoFisher). Cells were dosed with test compound diluted in DMSO using an Echo 555/655TM (Labcyte) and the final concentration of DMSO in the well was 0.3%. A neutral control (DMSO) and a positive AR degrader control at 3μM (a prior art AR PROTAC) were included. The Echo uses acoustic technology to perform direct microplate-to-microplate transfers of DMSO compound solutions to assay plates. The system can be programmed to transfer volumes as low as 2.5 nL in multiple increments between microplates and in so doing generates a serial dilution of compound in the assay plate which is then back-filled to normalise the DMSO concentration across the dilution range. Compounds were dispensed as a 12 point dose response with indicated concentrations. The plates were incubated at 37oC / 5% CO2 for 24h and then fixed and stained as below. 40μL of 8% paraformaldehyde solution (v/v) in phosphate buffered saline (PBS) was added on top of the media already in the well using a Multidrop to fix plates giving a final concentration of 4% PFA (v/v) for 20- 30 minutes (keep in fume hood to minimise PFA exposure). After 20-30 mins, the plates were washed with 3 × PBS using a BioTek washer in a fume hood. A shake out of remaining volume in the well was performed to ensure best removal of remaining PBS. At this plates could be stored in PBS after washing for at least 3 days at 4oC, if so, PBS was removed by the BioTek before continuing with the next step. The cells were incubated with a Mouse monoclonal Anti-AR antibody AR441 (DAKO M3562) (1:1000), diluted in modified blocking buffer (PBS+1% BSA+0.1% TritonX); 15 μL / well for 2h at RT. The plates were then washed with 3 × PBS using a BioTek washer. A shake out of remaining volume in the well was performed to ensure best removal of remaining PBS. Cells were then incubated with Goat anti-mouse secondary antibody 488 (Invitrogen A11001) (1:500) and nuclear stain, DRAQ5 (Abcam Ab108410) (1:2000) diluted in modified blocking buffer for 45 mins at room temperature 15 μL / well. Plates are then washed with 3 × PBS using a BioTek washer, without final aspiration, and sealed with black plate seals. Plates could be stored after staining for three days at 4°C before read if required. Plates are read using a Cellomics Cellinsight to measure the Androgen Receptor level in each well. The data is exported into Genedata to perform curve fitting analysis. All data are normalized as % of positive control and the BellFit method automatically determines the correct fit method based on the presence of hook effect, choosing the BellFit when there is a hook and HillFit when there is no hook. Down-regulation of AR is expressed as a DC50 value and determined by calculation of the concentration of compound that is required to give 50% of maximum degradation of AR as per the curve fit for that compound. The maximum degradation observed for each compound is expressed as Dmax. The AR degradation data for compounds is shown in the table below and may be a result from a single experiment or an average of two or more experiments.
LNCaP Androgen Receptor L702H Imaging Assay
While treatment with AR antagonists has been shown to be of patient benefit, most cancers eventually progress to castration resistant prostate cancer (CRPC). At this stage, anti-androgen therapy is largely ineffective due to AR amplification, overexpression or point mutation. One of the point mutations observed in patient populations is L702H in the AR Ligand Binding Domain. LNCAP cells have been engineered to introduce the mutation L702H into the AR gene using CRISPR/Cas9. The previously described imaging- based AR assay will monitor nuclear AR (L702H) levels in these cells to identify compounds that regulate AR (L702H). Down-regulation of AR (L702H) is expressed as a DC50 value and determined by calculation of the concentration of compound that is required to give 50% of maximum degradation of AR (L702H) as per the curve fit for that compound. The maximum degradation observed for each compound is expressed as Dmax. The AR and AR (L702H) degradation data for compounds is shown in the table below and may be a result from a single experiment or an average of two or more experiments.
Reference: BIFUNCTIONAL COMPOUNDS CAPABLE OF DEGRADING ANDROGEN RECEPTORS (patent)
Source database: TPDdb