Compound (SMILES): Cn1cc(-c2sc(C(C)(C)O)cc2N2CCc3ccccc32)c2cc(C(=O)NCCOCCNC(=O)COc3cccc4c3C(=O)N(C3CCC(=O)NC3=O)C4=O)[nH]c2c1=O
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): BRD4 (UniProt O60885)
POI sequence: MSAESGPGTRLRNLPVMGDGLETSQMSTTQAQAQPQPANAASTNPPPPETSNPNKPKRQTNQLQYLLRVVLKTLWKHQFAWPFQQPVDAVKLNLPDYYKIIKTPMDMGTIKKRLENNYYWNAQECIQDFNTMFTNCYIYNKPGDDIVLMAEALEKLFLQKINELPTEETEIMIVQAKGRGRGRKETGTAKPGVSTVPNTTQASTPPQTQTPQPNPPPVQATPHPFPAVTPDLIVQTPVMTVVPPQPLQTPPPVPPQPQPPPAPAPQPVQSHPPIIAATPQPVKTKKGVKRKADTTTPTTIDPIHEPPSLPPEPKTTKLGQRRESSRPVKPPKKDVPDSQQHPAPEKSSKVSEQLKCCSGILKEMFAKKHAAYAWPFYKPVDVEALGLHDYCDIIKHPMDMSTIKSKLEAREYRDAQEFGADVRLMFSNCYKYNPPDHEVVAMARKLQDVFEMRFAKMPDEPEEPVVAVSSPAVPPPTKVVAPPSSSDSSSDSSSDSDSSTDDSEEERAQRLAELQEQLKAVHEQLAALSQPQQNKPKKKEKDKKEKKKEKHKRKEEVEENKKSKAKEPPPKKTKKNNSSNSNVSKKEPAPMKSKPPPTYESEEEDKCKPMSYEEKRQLSLDINKLPGEKLGRVVHIIQSREPSLKNSNPDEIEIDFETLKPSTLRELERYVTSCLRKKRKPQAEKVDVIAGSSKMKGFSSSESESSSESSSSDSEDSETEMAPKSKKKGHPGREQKKHHHHHHQQMQQAPAPVPQQPPPPPQQPPPPPPPQQQQQPPPPPPPPSMPQQAAPAMKSSPPPFIATQVPVLEPQLPGSVFDPIGHFTQPILHLPQPELPPHLPQPPEHSTPPHLNQHAVVSPPALHNALPQQPSRPSNRAAALPPKPARPPAVSPALTQTPLLPQPPMAQPPQVLLEDEEPPAPPLTSMQMQLYLQQLQKVQPPTPLLPSVKVQSQPPPPLPPPPHPSVQQQLQQQPPPPPPPQPQPPPQQQHQPPPRPVHLQPMQFSTHIQQPPPPQGQQPPHPPPGQQPPPPQPAKPQQVIQHHHSPRHHKSDPYSTGHLREAPSPLMIHSPQMSQFQSLTHQSPPQQNVQPKKQELRAASVVQPQPLVVVKEEKIHSPIIRSEPFSPSLRPEPPKHPESIKAPVHLPQRPEMKPVDVGRPVIRPPEQNAPPPGAPDKDKQKQEPKTPVAPKKDLKIKNMGSWASLVQKHPTTPSSTAKSSSDSFEQFRRAAREKEEREKALKAQAEHAEKEKERLRQERMRSREDEDALEQARRAHEEARRRQEQQQQQRQEQQQQQQQQAAAVAAAATPQAQSSQPQSMLDQQRELARKREQERRRREAMAATIDMNFQSDLLSIFEENLF
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: MV4-11 (Homo sapiens)
Cell line description: African=0.59%; Native American=0%; East Asian, North=0.34%; East Asian, South=0%; South Asian=2.57%; European, North=67.58%; European, South=28.91%.
48,XY,t(4;11)(q21;q23),+8,+19 (ATCC=CRL-9591).
Gene fusion; HGNC; HGNC Mutation; HGNC; HGNC
CSF2 and IL3 dependent.
A*03 A*03
Stable (MSS) (Sanger).
In situ; Peripheral blood; 
Caucasian.
Cancer cell line
Assay: Western Blot
DC50:  0.6 nM
Assay description: Protein degradation assay
After incubating MV411 cells with the compound for 24 hours, the cells were harvested and centrifuged at 3000 rpm for 2 minutes in a 1.5 mL centrifuge tube. The supernatant was removed, the pellet was resuspended in PBS, and the supernatant was removed again. 60 μL of pre-chilled cell lysis buffer was added to the cell pellet, resuspended by pipetting, and lysed on ice for 10 minutes. Vortex every 2 minutes. After complete lysis, the pellet was centrifuged at 14,000 g at 4°C for 15 minutes, and the supernatant was collected. After protein quantification, 8 μL of the protein was mixed with 2 μL of 5X loading buffer (prepared with SDS and bromophenol blue solution) and electrophoresed at 100 V for 1 hour, stopping before the blue color reached the bottom edge. The separation gel was removed and placed on a PVDF membrane, and the membrane was electroporated at a constant current of 250 mA for 90 minutes. After the membrane transfer was completed, the PVDF membrane was removed and blocked with 5% BSA solution for 1 hour. The primary antibody of BRD4 protein (1:2000) was added and incubated at 4°C with shaking overnight. The membrane was washed 3 times with TBST solution for 5 minutes each time. The HRP-labeled secondary antibody was added and incubated at room temperature with shaking for 1 hour. The membrane was washed 6 times with TBST for 5 minutes each time. The mixed developer was added and developed in a Bio-Rad imaging system. The grayscale value of the development was scanned and the fitted protein degradation DC50 was calculated.
Reference: NEW TYPE BRD4 BROMODOMAIN PROTAC PROTEIN DEGRADATION AGENT, PREPARATION METHOD THEREFOR AND MEDICAL USE THEREOF (patent)
Source database: TPDdb