Compound (SMILES): COc1cc(N2CCC(c3ccc(OCCCCN4CCN(c5ccc6c(c5)CN(C5CCC(=O)NC5=O)C6=O)CC4)cc3)CC2)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: 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: Fluorescence Imaging Assay
Dmax:  68 %
Assay description: LNCaP Androgen Receptor Imaging Assay
This cell-based imaging assay uses immuno-fluorescence to measure both cell number and 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 *106 per T175 flask and split every 3-4 days. Medium: RPMI 1640 containing 10% FCS and 1% glutamine. Cells were plated in assay ready plates at a density of 12,000 cells, 40LII , per well, using a Multidrop.
Cells were dosed with test compound diluted in DMSO using an Echo 555 and the final concentration of DMSO in the well was 0.3%. A neutral control (DMSO) and a positive AR degrader control at 3pM (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 onto the cell plates with a compound source plate prepared as above producing a 12 point dose response curve with 3-fold dilutions and one final 10-fold dilution, and a top concentration of 3 or 30LIM using the Integrated Echo workcell. The plates were incubated at 37°C / 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 x PBS using a BioTek washer.
The cells were incubated with a Mouse monoclonal Anti-AR antibody AR441 (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 x PBS using a BioTek washer. Cells were then incubated with Goat antimouse secondary antibody 488 (Invitrogen Al 1001) (1 :500) and nuclear stain, DRAQ5 (Abeam Abl08410) (1 :2000) diluted in modified blocking buffer for 45 mins at room temperature 15 μL , / well. Plates are then washed with 3 x PBS using a BioTek washer and sealed with black plate seals.
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 any data points that fall on the hook of the curve are masked before fitting. 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.
Reference: COMPOUNDS AND THEIR USE IN TREATING CANCER (patent)
Source database: TPDdb