Compound (SMILES): Cc1ncsc1-c1ccc(CNC(=O)[C@@H]2C[C@@H](O)CN2C(=O)[C@@H](NC(=O)C2(F)CC2)C(C)(C)C)c(OCCCCCCCCCCCCN(C)CCOC23CC4(C)CC(C)(CC(Cn5ncc(-c6ccc(N7CCCc8c7nnc(Nc7nc9ccccc9s7)c8C)nc6C(=O)O)c5C)(C4)C2)C3)c1
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): BCL2L1 (UniProt Q07817)
POI sequence: MSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEMETPSAINGNPSWHLADSPAVNGATGHSSSLDAREVIPMAAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQERFNRWFLTGMTVAGVVLLGSLFSRK
E3 ligase: VHL (UniProt P40337)
Ligase sequence: MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPRPVLRSVNSREPSQVIFCNRSPRVVLPVWLNFDGEPQPYPTLPPGTGRRIHSYRGHLWLFRDAGTHDGLLVNQTELFVPSLNVDGQPIFANITLPVYTLKERCLQVVRSLVKPENYRRLDIVRSLYEDLEDHPNVQKDLERLTQERIAHQRMGD
Cell line: NCI-H1650 (Homo sapiens)
Cell line description: African=0.57%; Native American=0%; East Asian, North=3.27%; East Asian, South=0%; South Asian=0%; European, North=61.83%; European, South=34.34%.
Mutation; HGNC; HGNC Mutation; HGNC; HGNC
A*02
Stable (MSS) (Sanger).
Metastatic; Pleural effusion; 
Caucasian.
Cancer cell line
Assay: HiBiT
DC50:  3 nM
Assay description: The ability of the BCL-xL degrader low molecular weight compounds to inhibit cell proliferation and survival was assessed using the Promega CellTiter-Glo proliferation assay on the EBC-1 lung cancer cell line (RCB1965, obtained from RIKEN). The cells were cultured in media that is optimal for their growth at 5% CO2, 37°C in a tissue culture incubator.

Prior to seeding for the proliferation assay, the cells were split at least 2 days before the assay to ensure optimal growth density. On the day of seeding, cells were lifted off tissue culture flasks using 0.25% trypsin. Cell viability and cell density were determined using a cell counter (Vi-Cell XR Cell Viability Analyzer, Beckman Coulter). Cells with higher than 85% viability were seeded in white clear bottom 384-well plates (Greiner cat 781098) at a density of 1000 cells per well in 50 μL of standard growth media. Plates were incubated at 37°C overnight in a tissue culture incubator.

The BCL-xL degrader low molecular weight compounds were prepared to desired concentrations. A series of 10 dilutions were made for each prepared compound. The prepared compounds were then added to the cells resulting in final concentrations of 0.508 nM to 10 μM. An acoustic transfer device (Echo555, Beckman Coulter) was used to add the compounds to the cells. Each treatment was tested in triplicate assay plates. Plates were incubated at 37°C overnight or for 5 days in a tissue culture incubator.

The ability of the compounds to inhibit cell proliferation and survival was assessed using the Promega CellTiter-Glo proliferation assay. Plates were incubated at room temperature for 20 minutes to stabilize luminescent signals prior to reading using a multimode plate reader (PHERAstar FSX Microplate Reader, BMG Labtech).

To evaluate the effect of the compound treatments, luminescent counts of untreated cells were taken the day after seeding (Day 0 readings), and after 5 days of treatment (Day 5 readings). The Day 5 readings of the untreated cells were compared to the Day 0 readings. Assays with at least one cell doubling during the incubation period were considered valid. To evaluate the effect of the drug treatments, luminescent counts from wells containing untreated cells (100% viability) were used to normalize treated samples. The concentrations of treatment required to inhibit 50% of cell growth or survival (GI50) were calculated using a four parameter logistic regression equation.

The ability of the BCL-xL degrader low molecular weight compounds to degrade BCL-xL protein was assessed using the Promega Nano-Glo HiBiT Lytic Detection System. The NCI-H1650 (ATCC No. CRL-5883) was engineered to express HiBiT-tagged BCL-xL. The NCI-H1650 HiBiT cell line was cultured in media that is optimal for their growth at 5% CO2 37°C in a tissue culture incubator.

Prior to seeding for the protein level assay, the cells were split at least 2 days before the assay to ensure optimal growth density. On the day of seeding, cells were lifted off tissue culture flasks using 0.25% trypsin. Cell viability and cell density were determined using a cell counter (Vi-Cell XR Cell Viability Analyzer, Beckman Coulter). Cells with higher than 85% viability were seeded in white clear bottom 384-well plates (Greiner cat 781098) at a density of 5000 cells per well in 30 μL of standard growth media. Plates were incubated at 37°C overnight in a tissue culture incubator.

The BCL-xL degrader low molecular weight compounds were prepared to desired concentrations. A series of 10 dilutions were made for each prepared compound. The prepared compounds were then added to the cells resulting in final concentrations of 0.508 nM to 10 μM. An acoustic transfer device (Echo555, Beckman Coulter) was used to add the compounds to the cells. Each treatment was tested in triplicate assay plates. Plates were incubated at 37°C overnight in a tissue culture incubator.

BCL-xL degradation was assessed through the addition of 30 μL of Nano-Glo HiBiT Lytic Reagent (Promega, cat N3040), a reagent which lyses cells and contains a furimazine substrate and the large subunit (LgBiT) used in NanoLuc Binary Technology. The LgBiT subunit having high affinity to a HiBiT-tagged protein will bind HiBiT, if present, resulting in complex formation of a luminescent NanoBiT enzyme. This reaction will emit a luminescent signal proportional to the amount of HiBiT-tagged BCL-xL present in the cell lysate. Plates were incubated at room temperature for 20 minutes to stabilize luminescent signals prior to reading using a luminescence reader (PHERAstar FSX Microplate Reader, BMG Labtech).

To evaluate the effect of the drug treatments, luminescent counts from wells containing untreated cells (100% HiBiT-tagged BCL-xL) were used to normalize treated samples. The concentrations of treatment required to inhibit 50% were calculated using a four parameter logistic regression equation.
Reference: SELECTIVE BCL-XL PROTAC COMPOUNDS AND METHODS OF USE (patent)
Source database: TPDdb