Compound (SMILES): Cc1ncsc1-c1ccc([C@H](C)NC(=O)[C@@H]2C[C@@H](O)CN2C(=O)[C@@H](NC(=O)CCCCCC(=O)N2CCN(CC[C@H](CSc3ccccc3)Nc3ccc(S(=O)(=O)NC(=O)c4ccc(N5CCN(CC6=C(c7ccc(Cl)cc7)CCC7(C6)CN(C)C7)CC5)cc4)cc3S(=O)(=O)C(F)(F)F)CC2)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: MOLT-4 (Homo sapiens)
Cell line description: African=0.38%; Native American=0%; East Asian, North=1.74%; East Asian, South=0%; South Asian=0.94%; European, North=68.08%; European, South=28.87%.
Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC
A*01 A*01 A*01
Instable (MSI-high).
In situ; Peripheral blood; 
Caucasian.
T-cell; CL=CL_0000084.
Cancer cell line
Assay: ELISA
DC50:  5.39 nM
Assay description: MOLT-4 cells were cultured in complete medium (RPMI1640 medium containing 10% FBS) and passaged 2-3 times a week at a passage ratio of 1:5. During passage, the cells were transferred to a centrifuge tube, centrifuged at 1200 rpm for 3 minutes, the supernatant medium was discarded, and the cells were resuspended in complete medium.
During the experiment, the cells were transferred to a centrifuge tube, centrifuged at 1200 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended in complete medium. The density was adjusted to 1.78×10? cells/mL after counting with a cell counter, and 225 μL/well was added to a 48-well cell culture plate (400,000 cells per well), and placed in a 37°C constant temperature 5% CO? incubator.
The sample to be tested was diluted to 0.1 mM with DMSO, and then diluted 3-fold to 8 concentrations in a U-bottom 96-well plate, and a blank well was set with DMSO. 5 μL of the compound solution prepared in gradient concentrations was added to 95 μL of complete culture medium and diluted 20 times. 25 μL of the above compound-containing culture medium was added to a 48-well cell culture plate and cultured in a 37°C constant temperature 5% CO? incubator for 16 hours.
Transfer the cells to a 96-well V-bottom plate, centrifuge at 2000 rpm for 3 minutes, discard the supernatant, add pre-cooled PBS to wash once, centrifuge at 2000 rpm for 3 minutes, discard the supernatant, then add 130 μL/well cell lysis buffer (1 mM EDTA, 0.5% Triton X-100 in PBS, pH 7.2-7.4), and place on ice for 20 minutes.
After lysis, centrifuge at 2000 g for 5 minutes at 4°C, and then determine the protein concentration by BCA method according to the instructions of BCA protein assay kit. Then take 20 μg protein from each well and add it to the ELISA plate for ELISA detection. The ELISA experiment was performed according to the instructions of the Human/Mouse Total Bcl-xL DuoSet IC ELISA kit. Finally, the OD450/OD540 program of the PHERAstar microplate reader was used to read the signal value, and the data was processed using Graphpad Prism 9 software.
Reference: CHIMERIC COMPOUND USED FOR TARGETED DEGRADATION OF BCL-2 PROTEIN, PREPARATION METHOD THEREFOR, AND PHARMACEUTICAL APPLICATION THEREOF (patent)
Source database: TPDdb