Compound (SMILES): Cc1ncsc1-c1ccc([C@H](C)NC(=O)[C@@H]2C[C@@H](O)CN2C(=O)C(c2cc(N3CCN(CC4CCC(c5cnc(N6CCC(n7nc(N)c8nnc(-c9ccccc9O)cc87)CC6)nc5)CC4)CC3)no2)C(C)C)cc1
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): SMARCA4 (UniProt P51532)
POI sequence: MSTPDPPLGGTPRPGPSPGPGPSPGAMLGPSPGPSPGSAHSMMGPSPGPPSAGHPIPTQGPGGYPQDNMHQMHKPMESMHEKGMSDDPRYNQMKGMGMRSGGHAGMGPPPSPMDQHSQGYPSPLGGSEHASSPVPASGPSSGPQMSSGPGGAPLDGADPQALGQQNRGPTPFNQNQLHQLRAQIMAYKMLARGQPLPDHLQMAVQGKRPMPGMQQQMPTLPPPSVSATGPGPGPGPGPGPGPGPAPPNYSRPHGMGGPNMPPPGPSGVPPGMPGQPPGGPPKPWPEGPMANAAAPTSTPQKLIPPQPTGRPSPAPPAVPPAASPVMPPQTQSPGQPAQPAPMVPLHQKQSRITPIQKPRGLDPVEILQEREYRLQARIAHRIQELENLPGSLAGDLRTKATIELKALRLLNFQRQLRQEVVVCMRRDTALETALNAKAYKRSKRQSLREARITEKLEKQQKIEQERKRRQKHQEYLNSILQHAKDFKEYHRSVTGKIQKLTKAVATYHANTEREQKKENERIEKERMRRLMAEDEEGYRKLIDQKKDKRLAYLLQQTDEYVANLTELVRQHKAAQVAKEKKKKKKKKKAENAEGQTPAIGPDGEPLDETSQMSDLPVKVIHVESGKILTGTDAPKAGQLEAWLEMNPGYEVAPRSDSEESGSEEEEEEEEEEQPQAAQPPTLPVEEKKKIPDPDSDDVSEVDARHIIENAKQDVDDEYGVSQALARGLQSYYAVAHAVTERVDKQSALMVNGVLKQYQIKGLEWLVSLYNNNLNGILADEMGLGKTIQTIALITYLMEHKRINGPFLIIVPLSTLSNWAYEFDKWAPSVVKVSYKGSPAARRAFVPQLRSGKFNVLLTTYEYIIKDKHILAKIRWKYMIVDEGHRMKNHHCKLTQVLNTHYVAPRRLLLTGTPLQNKLPELWALLNFLLPTIFKSCSTFEQWFNAPFAMTGEKVDLNEEETILIIRRLHKVLRPFLLRRLKKEVEAQLPEKVEYVIKCDMSALQRVLYRHMQAKGVLLTDGSEKDKKGKGGTKTLMNTIMQLRKICNHPYMFQHIEESFSEHLGFTGGIVQGLDLYRASGKFELLDRILPKLRATNHKVLLFCQMTSLMTIMEDYFAYRGFKYLRLDGTTKAEDRGMLLKTFNEPGSEYFIFLLSTRAGGLGLNLQSADTVIIFDSDWNPHQDLQAQDRAHRIGQQNEVRVLRLCTVNSVEEKILAAAKYKLNVDQKVIQAGMFDQKSSSHERRAFLQAILEHEEQDESRHCSTGSGSASFAHTAPPPAGVNPDLEEPPLKEEDEVPDDETVNQMIARHEEEFDLFMRMDLDRRREEARNPKRKPRLMEEDELPSWIIKDDAEVERLTCEEEEEKMFGRGSRHRKEVDYSDSLTEKQWLKAIEEGTLEEIEEEVRQKKSSRKRKRDSDAGSSTPTTSTRSRDKDDESKKQKKRGRPPAEKLSPNPPNLTKKMKKIVDAVIKYKDSSSGRQLSEVFIQLPSRKELPEYYELIRKPVDFKKIKERIRNHKYRSLNDLEKDVMLLCQNAQTFNLEGSLIYEDSIVLQSVFTSVRQKIEKEDDSEGEESEEEEEGEEEGSESESRSVKVKIKLGRKEKAQDRLKGGRRRPSRGSRAKPVVSDDDSEEEQEEDRSGSGSEED
E3 ligase: VHL (UniProt P40337)
Ligase sequence: MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPRPVLRSVNSREPSQVIFCNRSPRVVLPVWLNFDGEPQPYPTLPPGTGRRIHSYRGHLWLFRDAGTHDGLLVNQTELFVPSLNVDGQPIFANITLPVYTLKERCLQVVRSLVKPENYRRLDIVRSLYEDLEDHPNVQKDLERLTQERIAHQRMGD
Cell line: SW1573 (Homo sapiens)
Cell line description: African=0.36%; Native American=0.18%; East Asian, North=0.45%; East Asian, South=0%; South Asian=0%; European, North=66.92%; European, South=32.09%.
Gene deletion; HGNC; HGNC Gene deletion; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC
A*02
Stable (MSS) (Sanger).
In situ; Lung; 
Caucasian.
Cancer cell line
Assay: HiBiT
DC50: > 1000 nM
Assay description: Detecting Compounds' BRM and BRG1 Protein Degradation Activity Using HiBiT Detection Technology
1. Cell Line Construction:
The effects of compounds on BRM and BRG1 protein degradation were detected using HiBiT detection technology. A HiBiT tag was inserted after the start codon or before the stop codon of the BRM and BRG1 proteins. When compounds degrade BRM or BRG1, the expression of the HiBiT tag can be used to quantify BRM and BRG1 protein degradation. Design sgRNAs and donor DNA for BRM and BRG1 based on the HiBiT insertion site. Prepare Cas9 transfection reagent, calculate the required amounts of various reagents, sgRNA, and donor DNA, and transfect SW1573 cells according to the Cas9 transfection protocol. After 24 hours, switch to normal culture medium. Continue culturing for three days after transfection. Expand each cell group and measure HiBiT tag expression in the pool using the Nano Glo HiBiT Lytic Detection System. Cell pools with high HiBiT tag expression are seeded as single clones in a 96-well plate and assayed for HiBiT tag expression in each clone using the Nano Glo HiBiT Lytic Detection System. If the fluorescence value of the HiBiT tag amplified from a single clone is high, it indicates that the cell group has likely successfully attached the HiBiT tag. Expand and sequence these single clones to confirm successful HiBiT sequence attachment and homozygosity, and then use them in subsequent degradation experiments. 2. Cell Plating:
Cells were seeded at an adjusted concentration (SW1573 BRM/BRG1 knock-in cell line: 9,000 cells/well) in 384-well plates and incubated overnight at 37°C, 5% CO?. Compounds were dissolved in DMSO and diluted three-fold with DMSO and culture medium, then transferred to the plate to a final concentration of 1 μM. The cells were incubated at 37°C, 5% CO? for 16-18 hours.
3. Protein Degradation Assay:
15 μL of the reaction solution (Nano Glo HiBiT Lytic Detection System configured according to the protocol) was added to each well of the 384-well plate. The cells were incubated at room temperature for 10 minutes and analyzed using an Envision multi-function microplate reader.
4. Degradation DC??? Calculation: The final drug concentration was 1000 nM. Three-fold dilutions were performed at 10 concentration points. Protein degradation rates were calculated using Excel XLfit 5.4.0. Each concentration point was replicated in duplicate. Protein degradation rate (%) = (High control - corresponding well reading) / (High control - Low control) * 100
High control = 0.1% DMSO, defined as 0% inhibition
Low control = PBS (no cells), defined as 100% inhibition
A degradation curve was generated using the degradation rates at 10 concentrations, and the degradation DC50 and Dmax values were calculated.
Reference: BRM SELECTIVE DEGRADATION AGENT COMPOUND AND USE THEREOF (patent)
Source database: TPDdb