Compound (SMILES): Cc1ncsc1-c1ccc([C@@H](C)NC(=O)C2C[C@H](O)CN2C(=O)[C@@H](NC(=O)CCNC(=O)c2nc(N3CCCc4c3nnc(Nc3nc5ccccc5s3)c4C)ccc2-c2cnn(CC34CC5(C)CC(C)(C3)CC(OCCN3CCCC3)(C5)C4)c2C)C(C)(C)C)cc1
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): BCL2L1 (UniProt Q07817)
POI sequence: MSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEMETPSAINGNPSWHLADSPAVNGATGHSSSLDAREVIPMAAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQERFNRWFLTGMTVAGVVLLGSLFSRK
E3 ligase: VHL (UniProt P40337)
Ligase sequence: MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPRPVLRSVNSREPSQVIFCNRSPRVVLPVWLNFDGEPQPYPTLPPGTGRRIHSYRGHLWLFRDAGTHDGLLVNQTELFVPSLNVDGQPIFANITLPVYTLKERCLQVVRSLVKPENYRRLDIVRSLYEDLEDHPNVQKDLERLTQERIAHQRMGD
Cell line: HCT 116 (Homo sapiens)
Cell line description: African=0%; Native American=0.08%; East Asian, North=1.16%; East Asian, South=0%; South Asian=0.97%; European, North=64.85%; European, South=32.93%.
Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC
A*01,02; B*18,45 A*01 A*01 A*01 A*01
Instable (MSI-high).
In situ; Colon; 
Caucasian.
Cancer cell line
Assay: Automated Capillary Electrophoresis Immunoassay
DC50: > 316 nM
Assay description: HCT116 cells were purchased from ATCC and cultivated in RPMI 1640 supplemented with 10% heat inactivated fetal bovine serum, penicillin (100 IU/ml), streptomycin (100 μg/ml) and L-glutamine (2 mM). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.

Cells were seeded in 96 microwell plates, 24 h later cells were treated with compounds (2-point 10/100 nM and/or 9-point serial dilution to determine DC50; quadruplicates) during 24 h and harvested in lysis buffer containing 10 mM Hepes (pH 7.5), 150 mM KCl, 5 mM MgCl2, 1 mM EDTA, 0.4% TritonX100, protease and phosphatase inhibitors cocktails (Calbiochem).

Protein concentration in the supernatants was determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific). Lysates (1.5 μg/μL) were analyzed using an automated capillary electrophoresis WES system with 2-40 kDa Wes Separation Module (25 capillary cartridges, Proteinsimple), in accordance with the manufacturer's protocols. Antibodies against the following proteins were used: Bcl-xL (#2764, Cell Signaling Technology, 1:50) and cofilin (#5175, Cell Signaling Technology, 1:1000). Signals were detected with an HRP-conjugated secondary anti-rabbit antibody (#042-206, Proteinsimple) and were quantified using Compass software (version 4.0, Proteinsimple).

Bcl-xL level was calculated by normalizing Bcl-xL values to cofilin and expressed as a percentage of the normalized value of the untreated cells. DC50 were calculated using standard four-parametric curve fitting. DC50 is defined as the compound concentration at which Bcl-xL is reduced to 50% of that measured for the control. Measured levels of Bcl-xL protein of HCT116 cells treated with the tested compounds at 2-point 10nM / 100nM and/or 9-point for DC50 determination are shown in Table 15.
Reference: SELECTIVE BCL-XL PROTAC COMPOUNDS AND METHODS OF USE (patent)
Source database: TPDdb