Compound (SMILES): Nc1ncnc2c1c(-c1ccc(Oc3ccccc3)cc1)nn2C1CC2CN(C3CN(C4CCN(c5ccc(NC6CCC(=O)NC6=O)cc5F)C4)C3)CC2C1
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): BTK (UniProt Q06187)
POI sequence: MAAVILESIFLKRSQQKKKTSPLNFKKRLFLLTVHKLSYYEYDFERGRRGSKKGSIDVEKITCVETVVPEKNPPPERQIPRRGEESSEMEQISIIERFPYPFQVVYDEGPLYVFSPTEELRKRWIHQLKNVIRYNSDLVQKYHPCFWIDGQYLCCSQTAKNAMGCQILENRNGSLKPGSSHRKTKKPLPPTPEEDQILKKPLPPEPAAAPVSTSELKKVVALYDYMPMNANDLQLRKGDEYFILEESNLPWWRARDKNGQEGYIPSNYVTEAEDSIEMYEWYSKHMTRSQAEQLLKQEGKEGGFIVRDSSKAGKYTVSVFAKSTGDPQGVIRHYVVCSTPQSQYYLAEKHLFSTIPELINYHQHNSAGLISRLKYPVSQQNKNAPSTAGLGYGSWEIDPKDLTFLKELGTGQFGVVKYGKWRGQYDVAIKMIKEGSMSEDEFIEEAKVMMNLSHEKLVQLYGVCTKQRPIFIITEYMANGCLLNYLREMRHRFQTQQLLEMCKDVCEAMEYLESKQFLHRDLAARNCLVNDQGVVKVSDFGLSRYVLDDEYTSSVGSKFPVRWSPPEVLMYSKFSSKSDIWAFGVLMWEIYSLGKMPYERFTNSETAEHIAQGLRLYRPHLASEKVYTIMYSCWHEKADERPTFKILLSNILDVMDEES
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: Mino (Homo sapiens)
Cell line description: African=0.85%; Native American=0%; East Asian, North=1.5%; East Asian, South=0%; South Asian=6.1%; European, North=39.56%; European, South=51.98%.
Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC
A*11 A*11,68; B*35,39; C*04,12. A*11
In situ; Peripheral blood; 
Caucasian.
Cancer cell line
Assay: Western Blot
DC50:  505 nM
Reported range: 10 to 1000
Assay description: Use Mino and HEK293 transfection BTK (C481S) cells to detect the degradation ability of the compound on BTK (WT) and BTK (C481S) protein. Mino human mantle cell lymphoma cell line was purchased from ATCC, cultured in RPMI-1640 medium with 15% FBS and 1% antibiotics. HEK293 OE BTK (C481S) human embryonic kidney cells 293 were transfected with BTK C481S plasmid to form overexpressed BTK C481S cells, cultured in DMEM with 10% FBS and 1% antibiotics.

Mino cells and HEK293 OE BTK (C481S) stably transfected cell line suspension were added to 6-well plates and left at 37°C for 0.5 hours. Compounds of different concentrations were added to the cell suspension, and treated at 37°C, 5% CO2, and saturated humidity for 24 hours. After culture completion, cells were collected and 1×loading buffer (Biorad, Cat. 1610747) was added.

The expression of BTK (CST, Cat. 8547S) and β-actin (CST, Cat. 4970S) was detected by Western Blot, and the expression of BTK relative to the internal reference β-actin was calculated. The experimental results are shown in Table 2. The degradation rate (BTK%) of the BTK protein in the treatment group relative to the vehicle group was calculated according to the following formula:

BTK% = (1 - (BTK treatment/β-actin)/(BTK vehicle/β-actin)) × 100%

Where BTK treatment is the expression level of BTK in Mino cells at different doses of the compound, and BTK vehicle is the expression level of BTK in Mino cells in the control group. The maximum concentration of the compound in this experimental system is 1000 nM, and Dmax is the maximum degradation value in the experimental concentration system. Graphpad 5.0 software was used to fit the DC50 value according to the formula Y = 100/(1 + 10^((LogIC50 - X)*HillSlope)).
Reference: PROTAC COMPOUND, PHARMACEUTICAL COMPOSITION COMPRISING SAME, PREPARATION METHOD THEREFOR, AND USE THEREOF (patent)
Source database: TPDdb