Compound (SMILES): CN1CCN([C@@H]2CCCN(c3c(F)cc(C(N)=O)c4[nH]c(-c5ccc(N6CCN(CC7CCN(c8ccc(NC9CCC(=O)NC9=O)cc8F)CC7)CC6)nc5)cc34)C2)C1=O
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: < 1 nM
Assay description: Degradation experiments primarily use Western blotting to detect the intensity of target protein bands to assess compound activity. The basic principle is to stain cells or tissue samples after gel electrophoresis using specific antibodies. Analysis of the location and depth of the staining provides information on the expression of a specific protein in the analyzed cells or tissues. Addition of the compound degrades the target protein. After exposure, the grayscale of the band represents the signal intensity, with lower grayscale indicating lower signal. Signal intensity is used to assess changes in protein expression, and the degradation rate is calculated by comparing the grayscale intensity with the intensity of the control band.

The degradation ability of compounds against BTK (WT) protein was tested using Mino cells. The Mino human mantle cell lymphoma cell line was purchased from ATCC and maintained in RPMI-1640 culture medium supplemented with 15% FBS and 1% dual-antibody. Add Mino cell suspension to a 6-well plate and incubate at 37°C for 0.5 h. Dilute the compound to the desired concentration (1000 nM, 100 nM, 10 nM, 1 nM). Add the compound to the cell suspension and shake at 300 rpm for 1 min at 37°C, 5% CO2, and saturated humidity for 24 h. Transfer the plate to an ice bath, resuspend on ice, transfer the cells to an EP tube, centrifuge and discard the supernatant. Wash the wells and combine the cell suspension, then centrifuge at 4°C. Carefully aspirate the supernatant, add 100 μL of 1× Loading Buffer (Biorad, Cat. 1610747) while vortexing, and lyse the cells on ice for 30 min. The membranes were placed in a 95°C metal bath for 15 minutes, pre-cooled on ice for 30 seconds, and then centrifuged at 12,000 g for 1 minute. The membranes were then stored at -20°C or electrophoresed directly. After electrophoresis, the membranes were rapidly transferred to a membrane and incubated with antibodies overnight at 4°C before exposure (the maximum compound concentration in this experimental system was 1000 nM, and the Dmax values are within the experimental concentration range).
The "exposure" mentioned above refers to the Western blot analysis of BTK (CST, Cat. 8547S) and β-actin (CST, Cat. 4970S) expression. BTK expression was calculated relative to the internal control β-actin. The results are shown in Table 1. The degradation rate of BTK protein in the drug-treated group relative to the vehicle-treated group (BTK%) was calculated using the following formula: BTK% = (1 - (BTK treatment/β-actin) / (BTK vehicle/β-actin)) × 100%. BTK dose represents the expression of BTK in Mino cells at different compound doses, and BTK vehicle represents the expression of BTK in Mino cells in the control group (DMSO). The maximum compound concentration in this experimental system was 1000 nM, and Dmax represents the maximum degradation value within the experimental concentration range. Graphpad 5.0 software was used to fit the DC50 value using the formula Y = 100/(1 + 10^((LogIC50 - X) * HillSlope)).
Reference: PROTAC COMPOUND, PHARMACEUTICAL COMPOSITION CONTAINING SAME, AND PREPARATION METHOD THEREFOR AND USE THEREOF (patent)
Source database: TPDdb