Compound (SMILES): Nc1ncnc2c1c(-c1ccc(Oc3ccccc3)cc1F)nn2C1CCN(C(=O)C2CCN(CCNc3ccc4c(c3)C(=O)N(C3CCC(=O)NC3=O)C4=O)CC2)CC1
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): BTK (UniProt Q06187)
POI sequence: MAAVILESIFLKRSQQKKKTSPLNFKKRLFLLTVHKLSYYEYDFERGRRGSKKGSIDVEKITCVETVVPEKNPPPERQIPRRGEESSEMEQISIIERFPYPFQVVYDEGPLYVFSPTEELRKRWIHQLKNVIRYNSDLVQKYHPCFWIDGQYLCCSQTAKNAMGCQILENRNGSLKPGSSHRKTKKPLPPTPEEDQILKKPLPPEPAAAPVSTSELKKVVALYDYMPMNANDLQLRKGDEYFILEESNLPWWRARDKNGQEGYIPSNYVTEAEDSIEMYEWYSKHMTRSQAEQLLKQEGKEGGFIVRDSSKAGKYTVSVFAKSTGDPQGVIRHYVVCSTPQSQYYLAEKHLFSTIPELINYHQHNSAGLISRLKYPVSQQNKNAPSTAGLGYGSWEIDPKDLTFLKELGTGQFGVVKYGKWRGQYDVAIKMIKEGSMSEDEFIEEAKVMMNLSHEKLVQLYGVCTKQRPIFIITEYMANGCLLNYLREMRHRFQTQQLLEMCKDVCEAMEYLESKQFLHRDLAARNCLVNDQGVVKVSDFGLSRYVLDDEYTSSVGSKFPVRWSPPEVLMYSKFSSKSDIWAFGVLMWEIYSLGKMPYERFTNSETAEHIAQGLRLYRPHLASEKVYTIMYSCWHEKADERPTFKILLSNILDVMDEES
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: DoHH2 (Homo sapiens)
Cell line description: African=0.28%; Native American=1.29%; East Asian, North=0%; East Asian, South=0%; South Asian=1.29%; European, North=69.22%; European, South=27.92%.
A TP53 mutation is indicated as being at p.Ala138Pro (c.412G>C) in PubMed=17363600 but has not been confirmed by CCLE and Cosmic-CLP.
A*01 A*01 A*01
Stable (MSS) (Sanger).
In situ; Pleural effusion; 
Caucasian.
NCBI_TaxID; 10376; Epstein-Barr virus (EBV).
Cancer cell line
Assay: Western Blot
DC50: < 10 nM
Assay description: Degradation of BTK and IKZF3 targets (Western blot)
Cells (DOHH2, THP1, HBL1 C481S, Ramos cells) were suspended in 1.5 mL of culture medium and placed in 12-well cell culture plates (5x10 5 to 1x10 6 cells/well). They were treated with different concentrations of the present compound or control compound, where control 1 was a physical mixture of L18I (a known BTK protein degrader, Leukemia, 2019, 33, 2105-2110) and pomalidomide (a small molecule known to induce IKZF1 and IKZF3 degradation, British Journal of Haematology, 2014, 164(6), 811-821).
After incubation for the specified time, the cells were collected, the supernatant medium was removed by centrifugation, and then washed twice with PBS. Cell samples were lysed on ice using RIPA for 20 minutes. 2x protein loading buffer (50 mM Tris-HCl (pH 6.8), 2% (w/v) sodium dodecyl sulfate, 0.1% (w/v) bromophenol blue, 10% (v/v) glycerol, and 10% (v/v) β-mercaptoethanol in ultrapure water) was added and heated in a 100°C metal bath for 20 minutes before cooling to obtain protein samples. Ten μL of protein sample was then separated by 10% SDS-PAGE gel electrophoresis. After electrophoresis, the membrane was wet-transferred to a PVDF membrane (100 V for 1.5 hours at 4°C). After transfer, the membrane was blocked with 5% milk for one hour. Following blocking, the membrane was washed three times with PBST for 5 minutes each, followed by incubation with the primary antibody at 4°C overnight. After incubation, the antibody was removed, and the membrane was washed three times with PBST for 5 minutes each. The membrane was then incubated with the corresponding rabbit/mouse secondary antibody for one hour at room temperature. The secondary antibody was removed and the membrane was washed three times with PBST for 10 minutes each wash.
The membrane was developed using a chemiluminescent solution on a Tianneng imaging instrument, as shown in Figure 1. The grayscale analysis of the development results was performed using Image J, and the degradation ratio was obtained by normalization, as shown in Figures 1-7 and Table 2.
Reference: BIFUNCTIONAL COMPOUND FOR SIMULTANEOUSLY REGULATING BTK AND IKZF3 (patent)
Source database: TPDdb