Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(NCC5CCN(C6CCN(CCCSc7cccc8c7CN(C7CCC(=O)NC7=O)C8=O)CC6)CC5)c([N+](=O)[O-])c4)c(Oc4cnc5[nH]ccc5c4)c3)CC2)=C(c2ccc(C(F)(F)F)cc2)C1
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): BCL2L1 (UniProt Q07817)
POI sequence: MSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEMETPSAINGNPSWHLADSPAVNGATGHSSSLDAREVIPMAAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQERFNRWFLTGMTVAGVVLLGSLFSRK
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
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: Flow Cytometry
DC50: < 10 nM
Assay description: In vitro BCL-XL protein degradation assay in MOLT-4 cells

A dish of MOLT-4 cells in good exponential growth was collected and transferred to a centrifuge tube. The cells were centrifuged at 1500 rpm for 3 minutes at a low speed on a tabletop. The supernatant was discarded and the cells were resuspended in 5 mL of complete medium (RPMI basal medium with 10% FBS) using a pipette. The cells were counted using a cell counter and diluted with complete medium to adjust the cell density to 1 x 107 cells/mL. The cells were seeded onto a 96-well plate using a pipette at a rate of 100 μL/well. Compounds were added using a nanoliter pipette to a final concentration of 1000 nM to 1 nM. Two replicates were set up for each concentration and the cells were incubated at 37°C in a humidified cell culture incubator with 5% CO2.

After 24 hours of incubation, the cells were harvested for flow cytometry analysis. After washing with PBS containing 2% BSA, cells were first fixed with 80% methanol, then permeabilized with PBS containing 0.1% Tween 20, and blocked with PBS containing 10% BSA before antibody labeling. Cells were incubated with a BCL-XL rabbit mAb primary antibody for 0.5 h at room temperature. After washing with PBS containing 2% BSA, cells were incubated with anti-rabbit IgG (H+L) and F(ab')2 fragment for 0.5 h at room temperature. After incubation, cells were washed and resuspended in PBS containing 2% BSA and detected using an IQue3 assay.
The group labeled with the secondary antibody alone served as a background control, and cells without compound addition served as a negative control. Mean fluorescence intensity (MFI) was used to measure BCL-XL protein expression, and inhibition rates were calculated. Degradation rate (%) = (average MFI of negative control group - MFI of compound group) / (average MFI of negative control group - average MFI of background group) × 100%. The logarithm of compound concentration was used as the abscissa, and degradation rate was used as the ordinate. Four-parameter analysis was performed, and a dose-effect curve was fitted to calculate the DC50 (half-maximal degradation concentration). The results are shown in Table 3.
Reference: COMPOUND CONTAINING TRIFLUOROMETHYL GROUP (patent)
Source database: TPDdb