Compound (SMILES): Cc1ncsc1-c1ccc([C@H](C)NC(=O)[C@@H]2C[C@@H](O)CN2C(=O)[C@@H](NC(=O)C2CCN(c3ccc4c(n3)NCC43CCN(c4cc(-c5ccccc5O)nnc4N)CC3)CC2)C(C)C)cc1
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): SMARCA2 (UniProt P51531)
POI sequence: MSTPTDPGAMPHPGPSPGPGPSPGPILGPSPGPGPSPGSVHSMMGPSPGPPSVSHPMPTMGSTDFPQEGMHQMHKPIDGIHDKGIVEDIHCGSMKGTGMRPPHPGMGPPQSPMDQHSQGYMSPHPSPLGAPEHVSSPMSGGGPTPPQMPPSQPGALIPGDPQAMSQPNRGPSPFSPVQLHQLRAQILAYKMLARGQPLPETLQLAVQGKRTLPGLQQQQQQQQQQQQQQQQQQQQQQQPQQQPPQPQTQQQQQPALVNYNRPSGPGPELSGPSTPQKLPVPAPGGRPSPAPPAAAQPPAAAVPGPSVPQPAPGQPSPVLQLQQKQSRISPIQKPQGLDPVEILQEREYRLQARIAHRIQELENLPGSLPPDLRTKATVELKALRLLNFQRQLRQEVVACMRRDTTLETALNSKAYKRSKRQTLREARMTEKLEKQQKIEQERKRRQKHQEYLNSILQHAKDFKEYHRSVAGKIQKLSKAVATWHANTEREQKKETERIEKERMRRLMAEDEEGYRKLIDQKKDRRLAYLLQQTDEYVANLTNLVWEHKQAQAAKEKKKRRRRKKKAEENAEGGESALGPDGEPIDESSQMSDLPVKVTHTETGKVLFGPEAPKASQLDAWLEMNPGYEVAPRSDSEESDSDYEEEDEEEESSRQETEEKILLDPNSEEVSEKDAKQIIETAKQDVDDEYSMQYSARGSQSYYTVAHAISERVEKQSALLINGTLKHYQLQGLEWMVSLYNNNLNGILADEMGLGKTIQTIALITYLMEHKRLNGPYLIIVPLSTLSNWTYEFDKWAPSVVKISYKGTPAMRRSLVPQLRSGKFNVLLTTYEYIIKDKHILAKIRWKYMIVDEGHRMKNHHCKLTQVLNTHYVAPRRILLTGTPLQNKLPELWALLNFLLPTIFKSCSTFEQWFNAPFAMTGERVDLNEEETILIIRRLHKVLRPFLLRRLKKEVESQLPEKVEYVIKCDMSALQKILYRHMQAKGILLTDGSEKDKKGKGGAKTLMNTIMQLRKICNHPYMFQHIEESFAEHLGYSNGVINGAELYRASGKFELLDRILPKLRATNHRVLLFCQMTSLMTIMEDYFAFRNFLYLRLDGTTKSEDRAALLKKFNEPGSQYFIFLLSTRAGGLGLNLQAADTVVIFDSDWNPHQDLQAQDRAHRIGQQNEVRVLRLCTVNSVEEKILAAAKYKLNVDQKVIQAGMFDQKSSSHERRAFLQAILEHEEENEEEDEVPDDETLNQMIARREEEFDLFMRMDMDRRREDARNPKRKPRLMEEDELPSWIIKDDAEVERLTCEEEEEKIFGRGSRQRRDVDYSDALTEKQWLRAIEDGNLEEMEEEVRLKKRKRRRNVDKDPAKEDVEKAKKRRGRPPAEKLSPNPPKLTKQMNAIIDTVINYKDRCNVEKVPSNSQLEIEGNSSGRQLSEVFIQLPSRKELPEYYELIRKPVDFKKIKERIRNHKYRSLGDLEKDVMLLCHNAQTFNLEGSQIYEDSIVLQSVFKSARQKIAKEEESEDESNEEEEEEDEEESESEAKSVKVKIKLNKKDDKGRDKGKGKKRPNRGKAKPVVSDFDSDEEQDEREQSEGSGTDDE
E3 ligase: VHL (UniProt P40337)
Ligase sequence: MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPRPVLRSVNSREPSQVIFCNRSPRVVLPVWLNFDGEPQPYPTLPPGTGRRIHSYRGHLWLFRDAGTHDGLLVNQTELFVPSLNVDGQPIFANITLPVYTLKERCLQVVRSLVKPENYRRLDIVRSLYEDLEDHPNVQKDLERLTQERIAHQRMGD
Cell line: HT-1080 (Homo sapiens)
Cell line description: African=0.86%; Native American=0.35%; East Asian, North=1.36%; East Asian, South=0.29%; South Asian=0%; European, North=68.21%; European, South=28.94%.
This cell line is considered by some researchers to represent a dedifferentiated chondrosarcoma due to the presence of an IDH1 mutation.
Gene deletion; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC
A*31 A*31 A*31
Stable (MSS) (Sanger).
In situ; Bone, pelvis, acetabulum; 
Caucasian.
Cancer cell line
DC50:  2.64 nM
Assay description: 1. On the first day, cells were plated, 10,000 HT-1080 (ATCC-CCL-121) cells were added to each well of a 384-well plate (Corning-3764), and cultured overnight at 37°C and 5% CO? (volume fraction, the rest was air).
2. On the second day, the compound was dissolved in DMSO with an initial concentration of 10 mM. The compound was diluted with DMSO and culture medium (MEM culture medium (Gibco-11095080) containing 10 vol% FBS (Gibco-10099141C)) in turn, 3-fold dilution, 10 concentration gradients, 2 replicates. The prepared compound gradient dilutions were added to the 384-well plate plated the day before, with a final DMSO concentration of 0.1% or 0.3%. The wells containing 0.1% or 0.3% DMSO and no compound were set as DMSO control wells. The compound was treated for 18 h at 37°C and 5% CO?.
3. On the third day, a polyformaldehyde fixation solution (PFA, Thermo-28908) with a final concentration of 4 vol% was added to each well for fixation at room temperature for 1 h; the supernatant was discarded, and PBS (Cytiva-SH30256.01) containing 0.1 vol% Triton X-100 (Sigma-T8787) was added to each well for permeabilization, and the reaction was performed at room temperature for 10 min; then the supernatant was discarded, and a blocking solution (Li-Cor-927-50003) was added to each well for blocking at room temperature for 1 h; finally, the blocking solution was discarded, and a 1:1000 diluted SMACAR2 antibody (CST-11966P) or SMACAR4 antibody (CST-49360S) was added to each well, and a control well without primary antibody was set up, and incubated overnight at 4°C.
4. On the 4th day, discard the supernatant and wash the plate 4 times with PBS (Cytiva-SH30256.01) containing 0.1 vol% Tween-20 (Sigma-P9416); continue to discard the supernatant, add 1:1000 diluted secondary antibody (Li-Cor-926-32211) and 1:500 diluted CellTag 700 dye (Li-Cor-926-41090) to each well, and incubate at room temperature in the dark for 1 hour; then discard the supernatant, add PBS containing 0.1 vol% Tween-20 to each well, wash the plate 4 times; finally discard the supernatant, use Li-COR Odyssey CLx infrared laser scanner to read the fluorescence values of each well under 700 nm and 800 nm channels and image.
5. Data processing: First, the fluorescence value under the 800 nm channel was divided by the fluorescence value under the 700 nm channel to obtain the standardized value of the number of cells in each well, and then the standardized value of the number of cells in the sample well was subtracted from the standardized value of the number of cells in the well without primary antibody to obtain the standardized value of the background signal in the sample well, and the standardized value of the number of cells in the DMSO control well was subtracted from the standardized value of the number of cells in the well without primary antibody to obtain the standardized value of the background signal in the DMSO control well, and finally (1-standardized value of the background signal in the sample well/standardized value of the background signal in the DMSO control well) × 100% was used to calculate the percentage of the degradation rate of the target protein in each well of the sample well. Finally, GraphPad Prism software was used to plot the percentage of degradation rate at different concentrations under the formula of log (inhibitor) vs. response-Variable slope (four parameters) and calculate the relative half degradation concentration (DC50) and absolute half degradation concentration (DIC50) values. Selectivity data calculation: Selectivity (DC50)=DC50(SMARCA4)/DC50(SMARCA2); Selectivity (DIC50)=DIC50(SMARCA4)/DIC50(SMARCA2).
Reference: SUBSTITUTED TRICYCLIC DERIVATIVE, AND PHARMACEUTICAL COMPOSITION AND USE THEREOF (patent)
Source database: TPDdb