Compound (SMILES): C=C1c2cc3c(n2CCN1c1nccc(-c2cc(Nc4ccc(N5CCC6(CC5)CCN(c5ccc7c(c5)C(=O)N(C5CCC(=O)NC5=O)C7=O)CC6)cn4)c(=O)n(C)c2)c1CO)CC(C)(C)C3
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): BTK (UniProt Q06187)
POI sequence: MAAVILESIFLKRSQQKKKTSPLNFKKRLFLLTVHKLSYYEYDFERGRRGSKKGSIDVEKITCVETVVPEKNPPPERQIPRRGEESSEMEQISIIERFPYPFQVVYDEGPLYVFSPTEELRKRWIHQLKNVIRYNSDLVQKYHPCFWIDGQYLCCSQTAKNAMGCQILENRNGSLKPGSSHRKTKKPLPPTPEEDQILKKPLPPEPAAAPVSTSELKKVVALYDYMPMNANDLQLRKGDEYFILEESNLPWWRARDKNGQEGYIPSNYVTEAEDSIEMYEWYSKHMTRSQAEQLLKQEGKEGGFIVRDSSKAGKYTVSVFAKSTGDPQGVIRHYVVCSTPQSQYYLAEKHLFSTIPELINYHQHNSAGLISRLKYPVSQQNKNAPSTAGLGYGSWEIDPKDLTFLKELGTGQFGVVKYGKWRGQYDVAIKMIKEGSMSEDEFIEEAKVMMNLSHEKLVQLYGVCTKQRPIFIITEYMANGCLLNYLREMRHRFQTQQLLEMCKDVCEAMEYLESKQFLHRDLAARNCLVNDQGVVKVSDFGLSRYVLDDEYTSSVGSKFPVRWSPPEVLMYSKFSSKSDIWAFGVLMWEIYSLGKMPYERFTNSETAEHIAQGLRLYRPHLASEKVYTIMYSCWHEKADERPTFKILLSNILDVMDEES
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: Ramos (Homo sapiens)
Cell line description: EBV-negative.
Gene fusion; HGNC; HGNC Mutation; HGNC; HGNC
A*03 A*03 A*03 A*03
Stable (MSS).
In situ; Ascites; 
Caucasian.
B-cell; CL=CL_0000236.
Cancer cell line
Assay: HTRF
DC50:  1.292 nM
Assay description: Evaluation of Compounds' BTK Protein Degradation Activity in Ramos Cells
Experimental Methods:
1. RAMOS cells were seeded in 96-well U-bottom cell culture plates, with 80 μL of cell suspension per well (50,000 RAMOS cells per well). The plates were placed in a CO2 incubator and incubated overnight at 37°C.
2. The test compound was diluted with 100% DMSO to a first concentration of 0.2 mM. The compound was then pipetted three-fold to an eighth concentration, from 0.2 mM to 0.091 μM. Add 2 μL of compound to 78 μL of cell starvation medium and mix thoroughly. Then, add 20 μL of compound solution to the corresponding well of the cell plate. Return the cell plate to the CO2 incubator and continue incubating for 24 hours. At this point, the compound concentration ranges from 1 μM to 0.46 nM, and the DMSO concentration is 0.5%.
3. After the incubation period, discard 60 μL of cell supernatant and add 10 μL of stock cell lysis buffer to each well. Incubate at room temperature for 60 minutes with shaking.
4. Dilute Eu-Total BTK 52-fold and Acc-Total BTK 13-fold using 1X Detection Buffer.
5. Transfer 15 μL of cell lysate supernatant to each well of a new 384-well white microplate. Add 2.5 μL of Eu-Total BTK dilution buffer and 2.5 μL of Acc-Total BTK dilution buffer. Incubate at room temperature for 4 hours.
6. After the incubation period, read the HTRF using a multilabel analyzer. Excitation: 320 nm, emission: 615 nm, 665 nm.
Data Analysis:
Raw data were converted to degradation rates using the equation (Sample-Min)/(Max-Min)*100%. DC50 values were then derived using a four-parameter curve fit (using the log(inhibitor) vs. response - Variable slope mode in GraphPad Prism). The degradation effects of the compounds of the present invention on total BTK are shown in Table 4.
Reference: NITROGEN-CONTAINING TRICYCLIC BIFUNCTIONAL COMPOUND, PREPARATION METHOD THEREFOR, AND APPLICATION THEREOF (patent)
Source database: TPDdb